Advances in Applied Microbiology, Volume 3

ADVANCES IN

Applied Micro biology VOLUME 3

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ADVANCES IN

Applied Microbiology Edit.ed by WAYNE W. UMBREIT Department of Bacteriology Rutgers, The State University New Brunswick, New Jersey

VOLUME 3

@

1961

ACADEMIC PRESS, New York and London

Copyright

0, 1961, by

Academic Press Inc.

ALL RIGHTS RESERVED

NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS INC. 111 FIFTHAVENUE NEW YORK 3, N. Y.

United Kingdom Edition Published by ACADEMIC PRESS INC. (LONDON)LTD. 17 OLD QUEENSTREET,LONDON S.W. 1

Libray of Congress Catalog Card Number 59-13823

PRINTED IN THE UNITED STATES O F AMERICA

CONTRIBUTORS ELIOBALDACCI, Institute of Plant Pathology, University of Milan, Ztaly

J. D. BU’LOCK, Department of Chemistry, The University, Manchester, England

MARK A. CHATIGNY, Naval Biological Laboratoy, School of Public Health, University of California, Berkeley, California NORMANC. DONDERO, Department of Sanitation, Rutgers University, New Brunswick, New Jersey C. RICHARDEIDAM,Department of Bacteriology, The University of Michigan, Ann Arbor, Michigan

ROBERTJ. HECKLY, Naval Biological Laboratory, School of Public Health, University of California, Berkeley, California

A. C. HULME,Dition Laboratory, Agricultural Research Council, Larkfield, Maidstone, Kent, England DONALDJ. MERCHANT,Department of Bacteriology, The Uniuersity of Michigan, Ann Arbor, Michigan ROBERT F. PITTZLLO, Kettering-Meyer Laboratory, Southern Research Institute, Bimhingham, Alabama

MARTINH. ROGOFF,United States Bureau of Mines, Pittsburgh Coal Research Center, Pittsburgh, Pennsylvania1

FRANK M. SCHABEL, JR., Kettering-Meyer Laboratory, Southern Research Iwtitute, Birmingham, Alabama ELWOOD TITUS,Laboratoy of Chemical Pharmacology, National Heart Institute, National Institutes of Health, Bethesda, Maryland Present address: Bioferm Corporation, Wasco, California.

V

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PREFACE With the third volume of Advances in Applied Microbiology, it is time to reply to certain critics of the earlier volumes, not because this is the place to do it, but because there is no other. In the first year of the publication of Advances there appeared a new edition of Prescott and Dunn’s “Industrial Microbiology” and the first volume of a series Progress in Industrial Microbiology, and shortly thereafter a further volume, Developments in Industrial Microbiology, as part of a continuing series. To certain reviewers, this seemed to be evidence of some kind of collusion. It seemed to violate some kind of scientific antitrust law, and whatever a particular reviewer disliked in one volume, he attributed to all the rest. This attitude seemed to be based upon a complete misunderstanding of the basis of scientific publication, its nature, its purpose, and its evolution across the years. Some felt that these volumes competed with well-established and valuable reviews-as Annual Review of Microbiology or Bacteriological Reviews. Others deplored the growth of secondary publication. And still others berated one editor for publishing in one area and another editor for omitting the same area, or condemned the series for not being textbooks and the textbooks for not being a series. But such a reaction is based upon a complete misconception of the nature and role of the essay (not the review) in modern science, So long as we continue to insist upon publishing scientific communications as if they were to be read, or, to put it less bluntly, to be read as a novel is read, so long as this is done we shall have to have reviews. But the essayist is not restricted nor is he expected to merely ‘mention all of the papers of the year or the decade, nor is he limited in what he can say about them, nor indeed should one go to an essay for the information one would expect in a review. Advances in Applied Microbiology is devoted to competent essays in the field of applied microbiology. As such, we have and we intend to continue to publish reasoned thoughtful essays (almost 18th century essays, if you like the term) on the contemporary problems of this field, written by men of competence, with some literary skill, who can bring to bear, upon an area of knowledge, their judgment and experience. We are interested in the soundest, and not necessarily in the latest, information. As such, this represents a someVii

viii

PREFACE

what different kind of publication than the journal of communication or the review, and it should be so recognized. Further, in today’s scientific communication, the essayist plays an important role, not solely as a voice of wisdom as in the past, but as a seasoned guide through the chatter of primary publication, who thoughtfully sifts that which is fundamental and enduring from that which is transient and doubtful.

W. W. UMBREIT New Brunswick, New Jersey September, 1961

CONTENTS ................................................... .............................

PREFACE..........................

v vii

Preservation of Bacteria by lyophilization ROBERTJ. HECKLY

I. Introduction . . ............... 1 11. General Proced .................. .. 2 111. Procedures and ................... 3 IV. Factors Affecting Survival of Organisms on zation and Storage.. . 23 V. Comparison of Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 VI. Safety Aspects . . . . . . . . . . . . . . . . .......................... VII. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 References ..................... ........................

Sphaerotilus, Its Nature and Economic Significance NORMANC. DONDERO I. Introduction . . . . . . .. ....................... 11. Technical Problems . . . . . . . . . . . . . . . . .................. 111. Identification and Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . IV. Causes of and Factors Affecting Stream Infestations.. . . . . . . . . . . . . . . . . . V. Nutrition and Physiology of Sphaerotilus ................ VI. Conclusion . . ...................... ................ References . . . . . . . . . . . . . . ...........................

78 93 97

104

large-Scale Use of Animal Cell Cultures DONALDJ. MERCHANT AND C. RICHARD EDAM I. Introduction . ............................... 11. Current Areas ge-Scale Use of Tissue Cultures . . 111. Some Biochemical Activities of Animal Cells Grown in Vftro IV. Some Trends in Animal Cell and Organ Culture Research V. Speculation on Applications of Cell and Organ Culture. References . . . . . .......................................... I

125

Protection against Infection in the Microbiological Laboratory: Devices and Procedures MARKA. CHATIGNY

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Recent History . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 111. Routes and Sources of Infection . ............................. IV. Safety Devices and Techniques . . . . . . . . . . . . . . . .

ix

131

137

X

CONTENTS

V. Decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI Laboratory Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Safety Programs and Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX . Commercially Available Safety Devices .............................. X . Motion Pictures and Filmstrips on Laboratory Safety Devices and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......................

.

173 178 182 183 185 186 187

Oxidation of Aromatic Compounds by Bacteria MARTINH . ROCOFF I. Introduction

....................................................

I1. A Central Metabolic Pathway for the Degradation of Aromatic Rings .... I11. Oxidative Metabolism of Polynuclear Aromatic Compounds by Bacteria . . . IV . Oxidative Metabolism of Benzenoid Compounds by Bacteria . . . . . . . . . . . V . Degradation of Miscellaneous Aromatic Compounds . . . . . . . . . . . . . . . . . .

VI . Future Experimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .....................................................

193 195 201 208 212 217 218 219

Screening for and Biological Characterization of Antitumor Agents Using Microorganisms FRANK M. SCHABEL.JR., AND ROBERT F. PITTILLO I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Microbial Screening Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Microbial Assay of Antitumor Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Biological Characterization of Antitumor Agents ...................... V. Correlative Observations in Microbial and Mammalian Systems . . . . . . . . . VI . Discussion and Future Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .....................................................

223 229 232 234 243 251 253

The Classification of Actinomycetes in Relation to Their Antibiotic Activity ELIO BALDACCI

I. Introduction

....................................................

257

IV. General Lines for Classifying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

270 276 277

I1. The “Formation of Antibiotics” as a Taxonomic Characteristic ......... 258 I11. Rules of Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

The Metabolism of Cardiac Lactones by Microorganisms ELWOODTITUS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

279 281

xi

CONTENTS

111. Conclusion References

..................... .....................

............ . . . . . . 290 ......................... 290

Intermediary Metabolism and Antibiotic Synthesis J. D. BU’LOCK Antibiotics as Secondary Metabolites . . . . . . . . A Classification of Secondary Metabolites . . Connections between General and Secondary The Interaction of General and Secondary M The Functions of Secondary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . 335 VI. A Postscript ...... . . . . . . . . . . . . . . . . . . References . . . . . . . .................. ...........

I. 11. 111. IV. V.

Methods for the Determination of Organic Acids A. C. HULME I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 11. List of Acids to Be Considered .................... .... . 345 111. Preparation of Material for the Analysis of Acids. General Principles . . . . 346 IV. Acids of the Citric Acid Cycle and Other Organic Acids (Groups 1 and 2) 349 V. Sugar Acids (Group 3) .......................................... 385 VI. Keto Acids (Group 4) . . VII. Fatty Acids (Group 5) . .......................... 374 VIII. Conclusion ...................................... References ..................................................... 391

AUTHORINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJECTINDEX .......................................................

395 413

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Preservation of Bacteria by Lyophilization ROBERTJ. HECKLY Naval Biological Laboratory, School of Public Health, University of California, Berkeley, California

A. B. C. D.

Freezing of Culture Preparations Design Considerations for Dr Pressure Cages . . . . . . Moisture Determinatio

.

...............

1

............... ...............

2 3

........................ .................... .................... .....................

...............

IV. Factors Affecting Survival of Organisms on Lyophilization and Storage. ... A. Type of Organism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Nutrition and Age . . . . . . C. Cell Concentration . . . . . . D. Suspending Medium ....... E. Rate and Method of Freezing . . . . . . . . . . . F. Method of Drying . . . . . . . . . . . . . . . . . . . . . . . G . Extent of Drying . . . . . . . . . . H. Storage Conditions ...................... ................. I. Method of Reconstitution . J. Assay Methods . . . . . . . . . . ................ V. Comparison of Organisms . . . VI. Safety Aspects . . . . . . . . . . . . . A. Hazards of Lyophilization B. Hazards of Opening Ampu VII. Discussion ...... ........... ............................ A. Loss of Viability ........ B. Importance of Keeping Losses to a Minimum . . . . . . . . . . . . . . . . . . . . . . C. A Suggested Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Future of Lyophilization . ...................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11 13 17 20 23 24

45 54

65 69 70 71 72

I. Introduction Lyophilization, or freeze-drying, is a term applied to the procedure of freezing and subliming water from frozen preparations. The precise origin of the term “lyophilization” is somewhat obscure. Flosdorf and Mudd (1935) and Reichel (1936) described the preparation of what they termed “lyophil” forms of biological substances. Originally, the 1

2

ROBERT J. HECKLY

“lyophil method” specifically applied to the condensation of water, sublimed from frozen preparations, on a cold surface as distinct from the use of desiccants. Since most biological materials dried from the frozen state were lyophilic, regardless of how water was removed, the term “lyophilization” was adopted by a number of workers to describe any process involving the sublimation of water from frozen preparations. British publications, however, have used and continue to use the more descriptive term “freeze-drying.” Shackell (1909) was probably the first person to apply lyophilizaticin to biological substances although others have been credited with the early development of the method prior to 1909 (Strumia et al., 1941). Probably the first application of freezing and drying to bacteria was by Hammer ( 1911) who demonstrated that lyophilized cultures survived much longer than did comparable cultures that were dried from the liquid state. Although the basic principles governing or associated with the preservation of bacteria by lyophilization were described and shown to be applicable over 50 years ago, it was only with the improvement of vacuum pumps, and the availability of dry ice and low-temperature refrigeration equipment that there was any promise of widespread use of the method. Before 1930 physicists used high-vacuum systems and had the required refrigeration equipment available to perform the task, but such equipment was not generally available to the biologist until later. A more complete account of the historical developments, especially the commercial aspects, of lyophilization is given by Flosdorf et nl. ( 1945; Flosdorf, 1949, 1954). Historical information more specifically applicable to the drying of bacteria has been summarized by Fry (1954). Additional information on lyophilization and related subjects that may be of considerable interest can be found in the recent publications of Harris ( 1954), Meryman (1960a), and Parkes and Smith (1960). This paper is not intended to describe or summarize all work pertaining to the lyophilization of bacteria; rather it is hoped that the information and views presented will be helpful in the lyophilization of microorganisms, particularly bacteria.

II. General Procedure The initial step in the process, but one which has received only limited attention, is the preparation of the culture. Usually one employs a culture of the desired organisms in the stationary phase of growth wherein the maximum cell yield is obtained. The highest possible cell concentration is used because it seems reasonable to assume that the number of cells

PRESERVATION OF BACTERIA BY LYOPHILIZATION

3

surviving will be proportional to the number of viable cells prior to lyophilization. Either solid or liquid culture media may be used, and, if the latter is used, the cells may or may not be separated from the culture fluid to be resuspended in specified diluents. Protective substances such as milk, serum, sugar, or sodium glutamate are usually added to the bacterial culture immediately before the next step, that of freezing the preparations. The nature of the suspending menstrua for optimal survival is dictated by the species or strain of the organism, the rate of freezing, and the extent of drying and storage conditions. Ampules or bottles containing the suspension are either placed in a chamber or attached to a manifold so that a high vacuum can be applied, permitting the water to sublime at a temperature below the eutectic point. Moisture is usually condensed on a cold surface or absorbed by a desiccant. The heat necessary to vaporize water from the culture preparation is usually supplied by conduction and radiation from the environment. After the cultures are dry the ampules are sealed by fusing the glass stems or, for certain applications, containers can be closed with a rubber stopper. It is difficult to establish criteria for successful drying, for reasons to be discussed later, but if more than 0.1% of the original cells remain viable after the required storage period it may be assumed that the lyophilization has been satisfactory for the maintenance of stock cultures. Some investigators consider processing to have been successful if, upon reconstitution, a sufficient number of viable cells remain to produce a culture having the characteristics of the original culture. Obviously every effort should be made so that the highest number of viable cells in the reconstituted preparation is obtained, and that the biochemical and serological specificity, virulence, and morphology of the original culture be preserved. 111. Procedures and Equipment A. FREEZING OF CULTURE PREPARATIONS The rate of freezing is largely determined by the method employed, within wide limits. 1. Plug Freezing

The simplest method of freezing a preparation is to immerse the container in a freezing bath which results in the formation of a “‘plug” of solidified material in the bottom of the container. This is a convenient method and extremely slow freezing rates can be achieved easily. How-

4

ROBERT J. HECKLY

ever, plug freezing is suitable only for lyophilizing relatively small amounts of material. 2. Shell Freezing

A frozen shell of material is obtained by rotating the bottle or ampule, with its axis horizontal, or at a small angle from horizontal in a freezing bath. Most commonly a dry-ice bath is used for this process but “shelling machines,” as described by Flosdorf et al. (1945) using mechanical refrigeration, have been used extensively, particularly for such products as serum and plasma. Spin freezing, which is high-speed rotation of the container on a vertical axis in a room at -2OOC. (Greaves, 1941, 1954), also produces a thin shell of material inside the bottle, Both processes are designed to maximize the area-to-volume ratio of frozen material and hence decrease the time required for drying. 3. Evaporative Freezing The freezing of preparations without the use of refrigerants was first described by Greaves and Adair (1936) and Flosdorf and Mudd (1938). They showed that the application of a vacuum increased the rate of evaporation, which in turn reduced the temperature of the remaining culture. Thus, when the pressure was reduced sufficiently the culture froze. This is commonly called snap-freezing because freezing of such preparations, which are frequently supercooled, occurs suddenly when the bottles are snapped or tapped. In the system of centrifugal vacuum freezing described by Greaves ( 1944), which is similar to snap-freezing, the ampules are held in a rotor and inclined inward at about 5 degrees from the vertical. By subjecting the preparation to low-speed centrifugation (about 1000 r.p.m.) foaming, which is frequently encountered during the degassing stage of snap-freezing, is suppressed and pressure in the system can be reduced rapidly without danger of the contents bubbling out of the containers. Centrifugal freeze-drying appears to be the most common method for lyophilization in England.

4. Pelletizing A method described by Graham et al. (1958), and Maister et al. (195813) is particularly useful for processing large quantities of material, The method consists of placing drops of a slurry on or in a Freon bath held at about -5O”C., the size of the drops being governed by the size of the needle used and the feed rate. Spherical pellets of about 6 mm. diameter were found to be a convenient size. The size of the drops, as well as the temperature of the bath, determine the freezing rate. A sig-

PRESERVATION OF BACTERIA BY LYOPHILIZATION

5

nificant advantage of pelletized preparations is that they are conveniently handled for weighing or repacking since the transfer of ordinary lyophilized preparations, particularly of those dried from a dilute suspension, is an extremely di5cult operation. FOR DRYING EQUIPMENT B. DESIGNCONSIDERATIONS

The various aspects of designing lyophilization equipment will be discussed only in general terms. A more complete discussion of the basic physical and engineering aspects of designing lyophilization equipment is given by Flosdorf (1949), Barrett and Beckett (1951), Harris (1954), Rowe (1960), and Stephenson (1960b).

1. Vacuum Requirements For practical purposes lyophilization will be effective only if the total pressure within the system is less than the vapor pressure of the preparation being dried. However, as was confirmed by Meryman (1959) it is the partial pressure of water, not the total pressure in the system, that governs the rate of drying. Obviously, since the vapor pressure can be no greater than the total pressure in the lyophilizing system, it is generally assumed that the total pressure in a lyophilizer is approximately equivalent to the water vapor pressure. The direct correlation between vapor pressure and temperature of ice is shown in Fig. 1. Since the vapor pressure of water at OOC. is about 4.6 mm. Hg, ice will sublime at any pressure below 4.6 mm. Hg and thus one could lyophilize at relatively high pressures. However, in practice lower pressures are required for two reasons, The first is that, generally, the material is in a botttle with a restricting orifice, and the pressure drop from the sample to condenser may be appreciable. The other reason is that the presence of solutes in bacterial suspensions depresses both the freezing point and the vapor pressure, so that pressures considerably below 4.6 mm. Hg are actually required. In most bacterial suspensions, the presence of proteins and other colloids modifies this effect by keeping the ice separated from the salts, and hence the maximum temperature allowable during lyophilization of a pure salt solution is lower than for one containing added colloids. Serum proteins are frequently used to provide the familiar “plug” structure and to accomplish this stabilizing effect. An observation using Stokes exhaust tube stoppers1 will be cited to illustrate how a restrictive orifice can determine the ice temperature with 60-ml. vaccine bottles. The stoppers had a relatively small opening ( 6 Obtained from F. J. Stokes Machine Company, Philadelphia, Pennsylvania.

6

ROBERT J. HECKLY

mm.) which restricted the flow of vapor out of the bottle. Although the lyophilization apparatus and manifold was evacuated to 20 p Hg, the temperature of the drying culture was not below -18OC. (Heckly et al., 1958), yet at a pressure of 200 p the temperature of the cultures was also -18OC. Early workers lyophilized their preparations using relatively high pressures, for instance Hammer (1911) evacuated to 18 mm. Hg and Swift (1921) used a pressure of 2 to 3 mm. Hg, but both workers kept

10-5

5 -20 0 -00

-60

-40

TEMPERATURE,

OC

FIG.1. Vapor pressure of ice as'a function of temperature. Data obtained from the Handbook of Chemistry and Physics ( Hodgman, 1949).

the cultures frozen by external cooling, In practice most, if not all, of those who use vacuum lyophilization systems use the best obtainable vacuum with their pumping system. With modern equipment a pressure of 20 to 100 p Hg is considered to be a good vacuum and quite satisfactory for most lyophilization work. However, at -4OoC., which is usually the temperature obtained with mechanical refrigeration, the pressure can be reduced to no less than 100 p Hg without contaminating the pump oil because, as is indicated in Fig. 1, the vapor pressure of water at -4OOC. is approximately 100 p Hg. A pressure of 200 p Hg is satisfactory for most such applications and provides a margin of safety in regards to pump contamination.

PRESERVATION OF BACTERIA BY LYOPHILIZATION

7

The capacity of the pump is usually such that the entire apparatus can be evacuated to about 500 p Hg in 5 to 15 minutes. Room temperature and the nature and thickness of the material in the ampules or bottles are the major factors which determine how rapidly the system must be evacuated to prevent thawing of materials. If small ampules containing 0.2 ml. or less cannot be evacuated in less than 5 minutes, provision must be made to keep the suspension from melting during the time required to reduce the pressure in the system to 500 p Hg. On larger systems it is useful and convenient to install both a large and a small pump. With a reasonably tight system a small pump can maintain the vacuum, although it would be inadequate for initial evacuation. Hence it is desirable to provide a large pump to be used for the initial evacuation and to ensure satisfactory completion of drying should a leak develop. Another advantage of a dual pumping system is that, since the larger pump removes the bulk of the air with its relatively high moisture content during the initial evacuation, less water enters the small pump. Thus, the efficiency of the small pump is not lost by contamination and low final drying pressures can be attained consistently. Graphs, tables, and formulas may be obtained from the major manufacturers of vacuum pumps, which are useful for selecting the size and type of pump suited to specific needs. Using the appropriate formula one can select the proper pump to evacuate a system to a given or desired vacuum in a specdied time. At the Naval Biological Laboratory, a 250 liter/minute pump has been quite satisfactory for the lyophilization units which have a total volume of between 8 and 20 liters. A pressure of less than 300 p Hg has been attained regularly in less than 15 minutes and a 33 liter/minute pump has been adequate for maintaining the vacuum at 10 to 20 p Hg. If one uses only the snap-freezing method, a single small pump may be adequate since it is desirable to evacuate the system slowly. Rowe (1960) described the operation of various kinds of pumps and indicated that a vapor booster pump is applicable to general lyophilization work, since it operates efficiently between 1 p and 1 mm. Hg and can operate at a backing pressure of up to 5 mm. Hg. Only in those instances where one wishes to study the effect of lyophilization at temperatures below -40°C. as did GreiE and Pinkerton (1954) and Greiff et al. (1954) is it essential that a diffusion pump be used. A mercury diffusion pump should never be used without an adequate cold trap, since without such precautions mercury vapor can migrate to the dried specimens, and possibly adversely affect the viability. Except in instances where one wishes to obtain extremely low pressures, oil diffusion pumps are generally not applicable.

8

ROBERT J. HECKLY

2. Duct Sine The size of the connections between the sample being dried and the condenser or desiccant often limits the rate of drying. At the usual lyophilization pressures, 50 to 1,000 p Hg, the resistance to flow through a tube is directly proportional to tube length but inversely proportional to the fourth power of the diameter of the tube. An approximation of the pressure drop along a straight pipe can be obtained by the formula Ps = 0.346(D 4 F / L ) ,where Pd = pressure drop in microns of Hg, D = diameter in centimeters, L = length of pipe in centimeters, and F = flow rate in liters per second. Rearrangement of the terms permits the capacity of the pipe to be estimated for a given pressure drop. The equation shows that the diameter rather than length of the manifold or connecting tubing is more important. For instance doubling the length of a connecting tube would decrease the flow rate by a factor of 2, but doubling the diameter would increase the flow rate by a factor of approximately 16. In small laboratory-scale apparatus the size of the connection between the pump and the condenser is not critical; 1/4-in. tubing usually is quite adequate. However, in larger installations in which the volume of the condenser or lyophilization chamber is over a few liters, undersized pump connections can seriously increase the time required to reach operating pressures, The time required to evacuate a chamber is also a function of the pumping speed and pump characteristics. For large-scale laboratory or industrial applications, or bulk drying on a tray, it is most efficient to place the condenser coils in the same chamber as the trays. The centrifugal vacuum-freeze device described by Greaves (1944) also has the condenser coils in the same chamber as the material, but because the material is in bottles or ampules, the opening of the container limits the flow rate of the water vapor. 3. Removal of Water Vapor At the reduced pressures usually used in lyophilization, 100 p Hg, the volume of 1 ml. of water is about 9500 liters. Therefore, water is usually removed by a desiccant or by condensation rather than by pumping which requires a special system to pump water vapor directly. a. Desiccants. Early lyophilization methods used desiccants exclusively, in spite of severe limitations on the amount of moisture that could be handled conveniently, because the method is simple and, in certain respects, economical. The Cryochem apparatus described by Flosdorf and Mudd (1938) seems rather large and cumbersome but it has been widely applied because the desiccant, calcium sulfate, could be re-

PRESERVATION OF BACTERLA BY LYOPHILIZATION

9

generated by heating and thus no dry ice or refrigeration equipment was required. The Adtevac process described by Hill and Pfeiffer (1940) used silica gel as the desiccant. Hill and Pfeiffer claimed that silica gel can adsorb up to 30% of its weight in water and that it can be regenerated repeatedly. Since heat is given off during the adsorption of water by silica gel it was refrigerated to increase the capacity of the gel. Unfortunately, it is difficult to obtain very dry products using silica gel without a secondary drying because the vapor pressure rises markedly as moisture is adsorbed. Holden (1958) mentioned the use of calcium carbide to remove water vapor in his lyophilization apparatus but no mention was made of problems caused by the production of acetylene as water was taken up. Recently Meryman (1959) described a novel system for lyophilizing small blocks of tissue at atmospheric pressure. By recirculating air, first over calcium sulfate and then over the specimen, the vapor pressure near the ice surface was so low that evaporation was sufficiently rapid to keep the specimen frozen. Another method of lyophilizing at atmospheric pressure was developed by Graham et al. (1958, 1959). Briefly, their method consisted of mixing pellets of culture with successive batches of dry silica gel at -3OOC. to remove most of the ice, The pellets, which retained their shape, were separated from the desiccant by sieving at each step and, after addition of the last batch of desiccant, drying was completed at room temperature. The heat to vaporize the water from the pellets was provided largely by the heat generated as the desiccant adsorbed the moisture. Therefore, because the pellets of culture were in intimate contact with the desiccant, the process was rapid and efficient. Although desiccants are no longer generally used for lyophilizing bacterial cultures, pellet preparations may find wide application, particularly as used by Loewus et al. ( 1960). b. Condensation. By 1935 condensers cooled by dry ice were used in several laboratories (Flosdorf. and Mudd, 1935; Elser et al., 1935), and today condensation of water on cold surfaces is perhaps the most popular method for removing water vapor. Dry ice is widely used as the refrigerant because of the low initial cost and high capacity of small lyophilizing equipment. Another advantage of using dry ice or liquid nitrogen to cool condensers, if they are designed properly, is that at temperatures below -6OOC. practically no water passes into the pumping system. However, industrial or large-scale laboratory equipment generally is cooled by mechanical refrigeration because direct refrigeration is economical and convenient in operation. In some laboratory instruments, such as described by Warren et al. (1951), provision has been made to use either or both methods of cooling the condenser. The installation of

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ROBERT J. HECKLY

a refrigeration system capable of attaining low temperatures is relatively expensive, particularly for the small units. Rowe (1960) has discussed at considerable length the problems involved in mechanical refrigeration systems and the factors involved in their operation. Flosdorf et al. (1945) mentioned that condensers have been operated successfully at -2OOC. but it is now generally accepted that satisfactory operation can be obtained only by maintaining the condenser at or below -4OOC. Unfortunately the expense of maintaining a condenser below -4OOC. increases rapidly as the temperature is lowered because the capacity of most mechanical refrigeration systems decreases markedly at temperatures below -4OOC. As is shown in Fig. 1, the vapor pressure of ice at temperatures above -4OOC. is so high that oil in pumps operating at less than 100 p Hg is rapidly contaminated. Greaves (1946, 1954) described a heating system to strip water from the oil of pumps operating at such low pressures. However, a pump2 with an air ballast feature, which prevents the condensation of water in the oil, is simpler than the system devised by Greaves. Ice is believed to be an extremely poor conductor of heat, especially as condensed under the usual lyophilizing conditions, although the specific conductivity of heat of such ice has not been measured. Thus the benefits of a dry-ice-cooled condenser are lost if the condenser is being overloaded or if the ice layer thickens. Obviously it is essential that a condenser be of such size that the heat of condensation can be absorbed rapidly enough to keep the surface temperature near that of the condenser itself. Liquid nitrogen has been used to achieve extremely low vapor pressures (10-13 p Hg is possible), but condensation of water with liquid nitrogen is uneconomical; the heat of vaporization is low, about one-half that of dry ice, and the cost is several times that of dry ice. The size of the condenser is usually dictated by the particular needs of the moment, and, since a high capacity is not compatible with high efficiency,two condensers in series are particularly effective. The purpose of the secondary condenser, which is usually smaller than the main condenser, is to protect the pump by removing traces of moisture not collected initially. c. Direct Pumping. Direct pumping of water vapor is generally inefficient because, as mentioned before, the volume of even small amounts of water is large at the reduced pressures. The limitations of direct pumping are illustrated by the specifications of a pumping unit manufactured by NRC Equipment Corporation. On using a gas ballast pump, rated at

' Such as is manufactured by NRC Equipment Corporation, Newton, Massachusetts.

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11

6 cu. ft./minute when driven by a X-hp motor, approximately 20 hours is required to sublime 40 ml. of water at -22OC. A small dry-ice-cooled condenser with a small pump could achieve the same results in less time. The Desivac system described by Flosdorf et al. (1940) was a direct pumping system using a conventional vacuum pump in which most of the moisture condensed in the pump oil, A continuous centrifuge was employed to separate the water from the pump oil. Steam ejection pumps have also been used for direct pumping because of their high capacity but these seem to be economical only for certain industrial applications, and a good supply of cheap steam and cooling water is necessary.

4. Heat Znput Generally energy input in the form of heat is not considered important since the heat required to sublime the water from frozen preparations is usually supplied by the environment. However, at -25OC. the heat of evaporation of ice is about 670 cal./gm., which is significantly greater than the heat required to vaporize 1 gm. of water at 100OC. For drying large volumes rapidly, or for special studies, the application of heat to frozen preparations is essential. Heat is usually applied by infrared radiation or by conduction from a heated plate. Greaves (1954) cited some studies which indicated that infrared radiation from heaters at 400° to 7OOOC. was most suitable and effective for drying frozen plasma. Greaves also mentioned that it might be possible to use dielectric heating or short-wave diathermy but at present technical difficulties make this form of heating impractical. More recently, Greaves (1960a) has discussed some special problems concerned with the application of heat in large-scale lyophilization systems, and presented other means of solving the problem, such as removing ice by continuously scraping the condenser surface. C. PRESSURE GAGES The simplest gage is the familiar closed-end mercury manometer, which is not sufficiently sensitive to measure the pressures usually used for lyophilization. Devices have been described ( Dushman, 1949) using mechanical or optical levers to increase manometer sensitivity but these, too, are not suitable for routine lyophilization work. The McLeod gage, which is perhaps the most widely used instrument for measuring low pressures, operates on the principle that if a large, but known, volume of gas is compressed into the small volume of a capillary tube, the pressure can be measured by a column of mercury. The gage is less expensive than most of the other types of gages, which reliably measure pressures in the 10 to 500 p Hg range.

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ROBERT J. HECKLY

The major disadvantage of the McLeod gage for lyophilization work is that it is not a continuously indicating gage. Therefore, the rate at which a vacuum is being attained is difficult to observe. Malfunctions of a system are not easily traced by means of a McLeod gage because of the intermittant checking and rechecking of pressure that is required. Another disadvantage of the McLeod gage is that it measures only noncondensable gases accurately. If there is moisture in the gas some of the water will condense and as a result the observed reading will be low. Insertion of an absorbent in the line to remove water vapor, and hence protect the gage, is also unsatisfactory. A third factor to consider is that the mercury in a McLeod gage has an appreciable vapor pressure. Unless an adequate cold trap is placed between the gage and the samples being dried, viability of the organisms may be affected by diffusion of some of the mercury into the culture, A more complete discussion of the McLeod gage and how one can obtain more meaningful and accurate measurements is given by Flosdorf (1949) and Dushman (1949). Greaves (1954) recently discussed at considerable length the limitations and advantages of this gage. High-frequency spark coils have been employed to show the presence of low pressure in sealed ampules, and some claim to have achieved a quantitative measurement by this method, but it hardly seems applicable because on changing the pressure in the glass tube to which the spark coil is applied from 20 mm. to 20 $I Hg little change can be observed. Furthermore it had been shown that the spark can actually produce small holes in glass; thus it should be used cautiously in testing ampules. Even a Plucker tube, as Rogers (1949) used, will give only approximate pressures in terms of the nature of the discharge. The Pirani gage is based on the fact that thermal conductivity of a gas is a function of its pressure. Thus, the resistance of a heated wire in the vacuum system changes as the pressure changes. Although the calibration curve is not the same for all gases it measures what is effectively total pressure, The Pirani gage is most useful for locating leaks in a system because it responds rapidly, and a leak can be located by the sudden change in the apparent pressure if acetone is applied to the source of a leak. The useful range of the Pirani gage is rather limited, about 1 to 300 p Hg, but with advances in technology the instruments now available possess expanded ranges and are less subject to change of calibration by fouling of the filaments than were those available 10 to 20 years ago. The thermocouple gage is basically similar to the Pirani gage except that a thermocouple is used to measure the temperature of a heated junction. The conventional thermocouple gage is undesirable because it

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responds more slowly than does the Pirani gage, because of the mass of the junction. Thermocouples have the advantage of being unaffected by atmospheric pressure and of being somewhat resistant to fouling. Modern thermocouple gages, such as those produced by Hastings-Raydist, Inc., Hampton, Virginia, are more responsive than were the earlier types available, yet they have apparently retained ruggedness and reliability. Ionization gages, either of the hot filament, or cold-cathode (Phillips) type, are applicable only to pressures below 100 p Hg and hence will not be discussed here. A gage using alpha-particle ionization, produced by the NRC Equipment Corporation, has a wide range. By using a selector switch the gage is able to measure pressures from 10 to mm. Hg in 4 ranges. The molecular vacuum gage3 is satisfactory for monitoring pressures in lyophilization systems because its operation is simple, and a continuous indication of pressure is provided. The gage measures pressures up to 20 mm. Hg but pressures as low as 10 p Hg can be measured accurately. The wide range is useful for snap-freezing in which 3 to 5 mm. Hg is a critical pressure range. The General Electric gage consists of a springrestrained cylinder which is coaxially aligned with an electrically driven rotor. The rotor is driven at a constant speed, and the displacement of the restrained cylinder is a function of the pressure in the system. A rather different method of measuring pressure is based on the fact that the vapor pressure of ice is dependent upon its temperature. Pressure can be estimated by imbedding a thermocouple in a block of ice which’ is suspended in the vacuum system. The response would be slow, but for measuring pressures at a number of points in a system this might be an economical method. A more complete review of all types of gages is given by Dushman ( 1949) and Roach ( 1954). D. MOISTURE DETERMINATION Moisture has been shown to be an important factor influencing the survival of lyophilized bacteria (Proom, 1951; Hutton et al., 1951; Fry and Greaves, 1951; Fry, 1954; Leach and Scott, 1959). Although the moisture content of dried cultures is frequently reported, many authors fail to give much thought to the assay method as evidenced by their failure to indicate the methods employed. For some materials, very good agreement is obtained between various methods but for many biological preparations, especially lyophilized cultures, the exact conditions under which the determination was made are extremely important. Recently made available commercially by General Electric Company, West Lynn, Massachusetts.

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ROBERT J. HECKLY

The general problem of determining moisture was the subject of a symposium held by the American Chemical Society (Stillman, 1951).

I. Co-distillation Fetzer (1951) described a number of modifications of a co-distillation method which employed organic solvents to extract the water. The sample is added to an immiscible liquid less dense than water and with a boiling point above 1 0 0 O C . The liquid is then distilled from the sample, condensed in an appropriate manner so that the water can be separated, and the water is then measured in a calibrated tube. Such distillation methods are entirely unsuitable for lyophilized preparations because the amount of water in the lyophilized preparations is too small.

2. Chemical The Karl Fisher iodometric method is sensitive and well suited for measuring the small amounts of water in dried bacteria. Hutton et al. (1951) employed this method for determining moisture in dried Brucella abortus. Since the method is titrimetric it can be made fairly rapid and semiautomatic, but it has three inherent disadvantages. The most serious one is that some substances interfere with the reaction. Ascorbic acid, for example, interfered by reacting with iodine, Cultures prepared according to Naylor and Smith (1946) would have a relatively high blank because the recommended suspending medium contains 0.5%ascorbic acid before drying. Ketones and aldehydes also interfere. A second disadvantage of the Karl Fisher method is that, since it is a destructive analysis, additional specimens must be prepared. A lesser problem is that the test is very sensitive and the reagents must be kept dry at all times. The article by Mitchell (1951) describing the various modifications of the Karl Fisher method and problems concerned with its use is well worth reading if this method is to be used.

3. Weight Loss The classic method of drying to a constant weight, oven drying, was reviewed by Willits (1951) who discussed the problems of determining the true moisture content of biological materials. The most serious problem seems to be that at temperatures above 5OOC. substances other than water are lost ( Flosdorf and Webster, 1937). Nevertheless, vacuum ovens have become standard pieces of equipment for such determinations and fortunately the newer automatic or semiautomatic balances have removed much of the tedium of repeated weighings. Moisture testers, which are balances with built-in heating devices to

PRESERVATION OF BACTERIA BY LYOPHILIZATION

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dry a sample on a pan, are not highly sensitive. An analytical balance having an enclosed infrared lamp might be suitable, albeit costly. Stephenson et ul. (1957) described a system which automatically records both weight and temperature of a sample. This system is suitable for vacuum sublimation studies. With suitable modifications such a system might be applicable to lyophilized preparations. Measuring the increase in weight of a chemical absorbent, specific for water, provides a more accurate measurement of the moisture content of samples containing volatile organic substances. Potter ( 1951) described such a system in which the sample was suspended in methanol to remove the water. The water was absorbed by calcium sulfate and, after washing with dry ether, the calcium sulfate was dried to remove the ether and weighed.

4. Manometric Beckett (1954) and Robson (1954) presented some of the historical developments in manometric methods for estimating moisture content of dried substances, and described an apparatus manufactured by W. Edwards and Company of London, England. An apparatus based on the same principles has also been described by Heckly (1955) and is illustrated in Fig. 2. Moisture from the sample is distilled under high vacuum and collected on a condenser cooled with dry ice. The condensed moisture is then confined in a known volume by closing the stopcocks, the dry ice is removed, and the condenser is warmed to revaporize condensed moisture. The pressure of the water vapor is then measured on a sliding scale attached to an oil-filled manometer. Since the volume of the confined water vapor and the density of the oil is known, the weight of water corresponding to a given pressure (manometer reading) can easily be calculated. The usable range of the instrument, illustrated in Fig. 2, is from about 0.05 to about 20 mg. of moisture per determination. Heckly et al. (1958) showed that the manometric method was suitable for determining the moisture content of lyophilized cultures of Pasteurellu pestis, and other workers, both in the United States and England, routinely use this method. When working with small amounts of extremely dry material it is particularly important to avoid opening the container to the atmosphere. It has been observed that removal of the stopper from a bottle containing 100 mg. of a lyophilized culture for 10 to 15 seconds, in a room at about 50% relative humidity, increased the moisture content from 0.3%to over 3%(Heckly, 1953). Figure 2 shows a unit with an infusion needle adapter which has been employed to measure moisture in rubber-stoppered bottles. For measuring the moisture in glass-sealed ampules, also without

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ROBERT J. HECKLY

FIG. 2. Appnratus for measuring small amounts of water manometrically (Heckly, 1955). Photo courtesy of California Laboratory Equipment Company, Berkeley. California.

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exposing the contents to the atmosphere, other systems have been devised ( Beckett, 1954; Robson, 1954; Heckly, 1955) . The manometric method is more rapid than the conventional gravimetric methods because the water is removed under high vacuum thus eliminating tedious cooling and weighing procedures. The specimen can be held at any desired temperature as moisture is removed; therefore results are obtained comparable to those obtained by vacuum oven methods. If the temperature is controlled, generally not exceeding 6OoC., viability of the lyophilized culture is not reduced and a single sample can thus be assayed for both moisture and viability. The design of the manometer on the apparatus shown in Fig. 2 offers two significant advantages over an ordinary U-tube manometer. The most important one is that the manometer fluid can be dried and degassed in situ by heating and circulating the manometer fluid. Once degassed and evacuated the manometer can serve to check the operation of the instrument and pump, obviating the necessity of a separate vacuum gage.

E. LABORATORY FABRICATED LYOPHILIZERS 1 . Glass Equipment

It is hoped that a review of equipment described by various investigators will provide a basis for selecting the apparatus best suited for a specific operation. As will be seen, some of the equipment described require the services of an experienced glass blower or machinist whereas others may be assembled with only moderate facilities. Most of the early lyophilization equipment was simply an adaptation of available apparatus. Hammer (1911), Rogers (1914), Swift (1937), Greaves (1956), and Elser et al. ( 1935) have described modifications of a method originally reported by Shackell (1909) who used an ordinary laboratory desiccator. The modifications were concerned primarily with the method of freezing the samples, the choice of desiccant, or the techniques of operation. They employed concentrated sulfuric acid, phosphorus pentoxide, or calcium sulfate, which was placed in the bottom of a desiccator to absorb moisture, and various methods were utilized to freeze the samples and to keep them frozen during the operation. Leifson (1936) described the use of small museum jars instead of desiccators. The covers were so arranged that after the jar was evacuated it could be sealed and stored as a unit. The most serious disadvantage of systems using desiccators, or comparable containers, is that after the culture is dry the ampules must be re-evacuated on a manifold if they are to be sealed under vacuum. This should be done because, as is shown in part H of Section IV, viability

18

ROBERT J. HECKLY

can be maintained more consistently for extended periods only when sealed in glass ampules. Therefore a manifold system, in which the water vapor is either condensed or absorbed, is to be preferred for the preservation of stock cultures. The “simple, inexpensive apparatus” described by Hornibrook ( 1949) requires only moderate skill in glass blowing. It consisted of a 2-in.diameter glass tube with 6 outlets attached along each side of the tube which was supported in a horizontal position and partly filled with sulfuric acid. Since the efficiency of the acid was rapidly reduced by accumulation of moisture on the acid surface, only very small volumes could be processed; otherwise, the apparatus had to be tilted occasionally to mix the sulfuric acid. One essential factor pointed out by Hornibrook (1949) is that the acid used to absorb moisture must be kept clean and especially free of salt, since liberated hydrochloric acid can be very toxic in the closed system. The modified Holzman (1950) apparatus described by Pratt and Yamaguchi (1950) is another relatively easily constructed glass lyophil apparatus of the manifold type. It consisted of 6 outer “ 5 standard taper joints interconnected centrally, each at right angles to the others and in the form of a symmetrical “cross.” The condenser was an 800-ml. Kjeldahl flask immersed in dry ice and attached to the joint pointing downward. Manifolds or other flasks with material to be dried were attached to the horizontal joints, The system was evacuated through a 10-mm. glass tube, attached by a rubber stopper inserted in the upper joint. The tube extended downward into a condenser. Since, under normal operation, a small amount of water vapor would pass into the pump a small secondary trap should be provided. Flosdorf and Mudd (1935) also described a small glass apparatus which consisted of a main condenser cooled with dry ice with an 8-outlet manifold attached at right angles. A smaller secondary condenser was attached to the main condenser through a rubber stopper. The unit was small enough so that both condensers could be placed into a l-gallon Dewar flask. Heckly (1947) described a lyophilizing apparatus which was similar in some respects to Flosdorfs glass unit but it required less glass blowing skill. The condenser was a Pyrex serum bottle into which was fitted a standard taper joint, through a rubber stopper so that various manifolds could be conveniently interchanged. A tube for evacuating the systems was sealed through the side of the standard joint since the insertion of two tubes through a rubber stopper distorted the stopper sufficiently to cause leaks. A vertical manifold system requires no separate support but a horizontal manifold must be supported; a standard tee adapter will serve as a support to accommodate 2 horizontal manifolds. A 2-liter con-

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denser which can lyophilize up to 800 ml. at a time can be cooled in a 1-gallon thermos jar. The apparatus described by Hays and Koch (1942) was designed primarily for drying large quantities (up to 600 ml.) in a single flask but a manifold could be attached to permit the use of multiple chambers. A more complex arrangement described by Stull and Herreid (1949) included a mercury diffusion pump and certainly required the services of an expert glass blower to assemble it. On the basis of the data presented the performance of their apparatus did not seem to be significantly superior to that of other simpler designs. The apparatus described by Campbell and Pressman (1944) requires the services of a professional glass blower but it has the advantage of not requiring a separate insulated container for the refrigerant. His apparatus was a Dewar-type container with standard taper joints attached around the bottom and an evacuation port near the top. Moisture was condensed on the inner portion of the apparatus which was cooled by dry ice. Thus, instead of placing the condenser in a dry ice bath the condenser itself serves as an insulated container for the dry ice. Up to 400 ml. of water could be condensed in the unit. A similar apparatus, which looked like any ordinary widemouthed Dewar flask with tubulatures in the outer wall was described by Jacobs ( 1947).

2. Metal Lyophilixers In general, metal units are desirable because they are safer and more durable than glass lyophilizers. Considerable machine shop facilities were required to build some of the equipment, particularly the larger units, described by Flosdorf and Mudd ( 1935). The apparatus described by Strumia et al. (1941) was a modification of Flosdorf‘s metal condenser system. The Strumia apparatus, which was able to process bottles up to 20-liter capacity, was designed primarily for processing serum and plasma and seems not particularly well suited to the lyophilization of bacterial cultures. Wyckoff and Lagsdin (1944) described “a simple outfit for drying plasma from the frozen state” which had several useful features, some of which are evident in equipment now commercially available. Essentially it consists of a vertical metal cylinder with a number of outlets, for attaching bottles or ampules, which in effect was a vertical manifold, and often referred to as a “pig.” Moisture was condensed on the walls of the center well which extended down into the cylinder, a situation comparable to that described by Campbell and Pressman (1944) or Jacobs (1947). Wyckoff and Lagsdin made the condenser removable so that it could be rapidly defrosted.

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ROBERT J. HECKLY

Van Rooyen and Janes (1954) described a small unit made of brass which weighed 35 lb. Despite elaborate arrangements, they apparently seem to have failed to provide for a secondary moisture trap in their system. They did mention that the pump oil should be changed for each operation, which indicates that presumably the oil was easily contaminated with water, Rhian et al. (1957) described “a continuous freeze drier for laboratory studies.” The apparatus was designed to dry organisms which had been frozen in the form of pellets about % in. in diameter and it would be applicable only to the mass preservation of organisms such as yeast or lactic acid bacteria used commercially. Moisture was collected on a mechanically refrigerated condenser.

3. Do-It-Yourself Type A simplified design which can be assembled without any glass blowing or other special skills is shown in Fig. 3. The manifold was assembled from %-in. copper tubing and %- to %-in. reducing tees. Short sections of %-in. tubing are fitted into the tees to facilitate attaching the %-in. gum rubber tubing. As illustrated, the main condenser was a 500-ml. Pyrex filter flask. The %-in. tubing was extended into the flask so that most of the water vapor would be directed down and condensed near the bottom of the flask. If it extends too far below the level of the refrigerant and is cooled below OOC. the %-in. tube may become plugged before drying is completed. The secondary trap, which effectively prevented contamination of the pump oil with water, consisted of a loop of tygon tubing submerged in the same ethanol-dry-ice bath as the flask. This apparatus, though not as ‘efficient as the more elaborate systems, is effective and can truly be said to be a do-it-yourself apparatus. Care must be exercised to select glassware that will withstand vacuum. Pyrex spherical or cylindrical flasks should always be used, although the shape is not too important for small condensers. Heavy-wall amber gum tubing in, i.d., with %-in, wall) has been found to be more convenient for making connections than the heavy, less elastic pressure tubing sold for vacuum connections. Unless a large amount of tubing is in the system pure gum rubber is not significantly more permeable to air and moisture than is the special vacuum tubing.

(x

F. COMMERCIALLY AVAILABLEEQUIPMENT Stokes Machine Company was perhaps the first to make lyophilization equipment available, of which the most widely known is the Cryochem apparatus described by Flosdorf and Mudd (1938). The desiccant,

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calcium sulfate, was contained in a large drum. The inherent disadvantage of bulkiness and the time-consuming process of regenerating the desiccant have made the Cryochem apparatus virtually obsolete. The advent of mechanical refrigeration has offset the former advantage of the Cryochem apparatus, that of not requiring dry ice.

FIG. 3. A simplified but effective lyophilization apparatus, Naval Biological Laboratory.

The VirTis Company, Inc., Gardiner, New York, makes a number of laboratory scale lyophilization instruments which have considerable utility. Figure 4 shows a drying unit with a small condenser of the “pig” type on the upper shelf of the cart and a secondary condenser below it. The central well is not removable, as was that described by Wyckoff and

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ROBERT J. HECKLY

Lagsdin (1944),but this is no serious disadvantage. VirTis also manufactures mechanically refrigerated units in which the condenser is separate from the drying chamber or manifold. The American Instrument Company, Inc., Silver Spring, Maryland, manufactures a similar line of laboratory equipment. One of their units, which uses either dry ice or mechanical refrigeration, is similar to that described by Warren et al. (1951). No personal recommendation can be made because the writer

FIG.4. A portable manifold-type lyophilization apparatus. Courtesy of the VirTis Company, Inc., Gardiner, New York.

has not used any of the commercially available lyophilization equipment. However, the McLeod gage which is furnished as standard equipment by the VirTis Company is not satisfactory for lyophilization work for reasons discussed in Section 111, B. The American Sterilizer Company, Erie, Pennsylvania also manufactures lyophilization equipment designed primarily for tray drying and it can be used for pilot plant production. With their laboratory model

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they do provide a small manifold which can be attached to the vacuum chamber. The above discussion has included only some instruments manufactured in the United States though there are probably many other manufacturers especially in Europe and elsewhere who have not been mentioned. For example, W. Edwards Company, London, is a wellknown manufacturer of lyophilization equipment. This company makes small laboratory units as well as larger plant production equipment and their centrifugal freeze-drying equipment seems to be the most widely used type of drier in England. As indicated in Section 111, A, the centrifugal freeze-drier presents the advantages of snap-freezing without the troublesome degassing and frothing that is associated with snap-freezing in the ordinary manifold type of apparatus. Recently RePP Industries, Inc., Gardiner, New York announced the production of a Centri-freeze which is, in principle, the same as the centrifugal freeze-driers manufactured by the W. Edwards Company. The drying chamber of the RePP instrument is a Lucite cylinder wherein heat is applied by an infrared lamp as ampules are rotated in it at 3 r.p.m. None of the centrifugal drying equipment has facilities for sealing containers; therefore ampules must be attached to a manifold for sealing under vacuum and bottles must be processed through some sort of a vacuum-sealing apparatus. Large production equipment is not widely applicable to lyophilization of bacteria and hence will be mentioned only briefly. RePP Industries, Inc. produces a pilot plant scale tray-drying apparatus with no provisions for attaching a manifold. Such equipment should be useful for lyophilizing large amounts of culture in bulk. The continuous vacuum-drying systems, such as that manufactured by Chain Belt Company, Milwaukee, Wisconsin are designed to dry from the liquid state, but it would seem feasible to adapt such equipment to process pelletized material as described by Rhian et al. (1957).

IV. Factors Affecting Survival of Organisms on lyophilization and Storage Under favorable conditions the percentage of organisms that die during the process of lyophilization is usually greater than the percentage that fail to survive subsequent storage. Because of this, and other reasons, the bulk of the studies have been concerned with the lyophilization process rather than with factors affecting survival on storage. The percentage of cells surviving lyophilization is, in general, indicative of the survival that can be expected on subsequent storage, but, unfortunately, the two properties are not always correlated. There are numerous factors which determine the survival of organisms and certain of these factors

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ROBERT J. HECKLY

are so interrelated that it is difficult to evaluate them independently. However, an attempt will be made to delineate those factors which have received the greatest amount of attention,

A. TYPEOF ORGANISM In view of the vast differences in morphology and physiology between the various microorganisms one would expect that there would be gross differences in their behavior upon being lyophilized. One of the most important factors in determining the percentage survival on drying is undoubtedly the nature of the organism. On the basis of information available today it would be difficult to list the organisms in order of decreasing resistance to lyophilization. A general impression of the relative resistance of various organisms to drying can be obtained from the results of Stamp (1947), Proom and Hemmons (1949), Rhodes and Fisher (1950), Rhoades (1958), Haynes et al. ( 1955), Wickerham and Flickinger (1946), and Kirsop (1955), who have reported data on drying and storing relatively large collections of bacteria and yeasts. Rhodes and Fisher (1950) tested the lyophilized preparations by the “growth or no growth method but since several cultures of each strain were tested some differences were shown in terms of a percentage of cultures which were viable. Despite the crude assay procedure the report of Rhodes and Fisher was a significant contribution because it included over 2724 strains. Spores of the sporeforming bacilli are so readily preserved by drying that they are seldom considered in discussions on lyophilization. It is of interest however, that Haynes et al. (1955) were not able to preserve anaerobic sporeformers by lyophilization. Of the nonsporeformers the gram-positive cocci are perhaps the most , ~ and Greaves ( 1951 ) resistant, especially Streptococcus p y ~ g e n e s Fry showed 36 and 100%survival of Staphylococcus aureus and a hemolytic streptococcus, respectively. Proom and Hemmons ( 1949) indicated that S. aurew was extremely resistant to drying. Proom (1951) stated that they were resistant because of the strength of the cell wall. The majority of the genera studied, Salmonella, Shigellu, Brucella, Pasteureh, Mycobacterium, Sewatla, Lactobacillua, etc., are only moderately resistant to lyophilization. It is in this group that the various investigators differ in their classification of the organisms as to resistance to lyophilization. For instance, Naylor and Smith (1946) stated that they selected Serrutia marescens as the test organism because it was very sen-

‘ The nomenclature of organisms presented in Bergey’s Manual (Breed et al., 1957) was used rather than the original author’s terminology, except where otherwisc indicated.

PRESERVATION OF BACTERIA BY LYOPHILIZATION

25

sitive to dehydration. Yet there are many organisms more sensitive to drying under comparable conditions. Perhaps some of the differences in opinion arise because conditions best suited for one species or genus are inadequate, or at least less than optimal, for another. Fry and Greaves (1951), however, stated that they believed that optimal conditions for one organism would be optimal for all others. There does seem to be rather general agreement that the Neisseria and Vibrio are among the bacteria least resistant to lyophilization. Stamp (1947) reported satisfactory survival of a large number of organisms but the survival rates of Neisseriu meningitidis and Vibrio comma were unsatisfactory. Similarly Proom and Hemmons ( 1949) showed that Neisseriu was the most sensitive of the large number of genera tested and Vibrio was nearly as sensitive. Others, including Hornibrook ( l950), Rhodes and Fisher (1950), and Annear (1956a), found that Neisseria was significantly more resistant to drying than Vibrio. Leptospiru have received relatively little attention but every indication is that they are even more difficult to preserve by lyophilization than the Vibrio or Neisseriu. Proom and Hemmons (1949) stated that leptospirae failed to survive drying, although all other bacteria in their collection survived. Annear ( 1 9 5 6 ~ first ) applied his peptone plug technique to the preservation of Leptospira and more recently he published the results after 2 years storage at room temperature and at 4OC. (Annear, 1958a). Very few of the preparations stored at room temperature yielded a culture and those obtained from cultures stored at 4OC. developed slowly. By comparison, between 1 and 15%of the Vibrio he processed in the same manner remained viable after 9 to 12 months storage at room temperature. From the above discussion it seems that the differences among bacteria are as great or greater than the differences between bacteria and yeasts. Viruses are, as a whole, more sensitive to certain drying procedures, but they are more resistant to cold shock than are bacteria. Most rickettsiae are relatively difficult to maintain as dry stock cultures although Topping (1940) and Jackson and Smadel (1951) were successful in maintaining infectivity by lyophilization. About 1%of the original infectivity of Rickettsia tsutsugamushi was retained by lyophilization (Jackson and Smadel, 1951). The commercial production of active dry yeasts and the extensive collections of Wickerham and Flickinger (1946), Haynes et al. (1955), Kirsop (1955), and Brady (1960) show that practically all yeasts can be preserved. There is a marked difference in the resistance to lyophilization among the yeasts, just as there is among bacteria, and as might be expected the various investigators are not in agreement as to which yeast

26

ROBERT J. HECKLY

is the least resistant. The common Saccharomyces cerevisiae is generally rated as having moderate resistance to lyophilization. B. NUTRITION AND AGE Aeration and nutrition during growth are as much a function or part of physiological cell age as is chronological age. Too frequently the age of the culture has been established without considering other factors that are involved. In fact, relatively few studies on lyophilization of bacteria have considered, as a variable, the conditions under which the organisms were produced. However, most of the investigators described in detail the exact conditions employed in a particular study, such as stock culture maintenance, growth medium, size of flasks, etc., implying, at least, that they considered these factors to have some influence on survival. Although a marked difference between cells grown on agar surface and submerged liquid culture might be expected this factor has scarcely been studied. Solid cultures, obtained from agar slants or plates, have been used generally for aerobic organims by those interested in preserving large collections (Proom and Hemmons, 1949; Rhodes and Fisher, 1950) because it was convenient. Liquid cultures were used more frequently by those interested in studying the effect of other variables, but these studies were confined to a limited number of organisms (Benedict et al., 1958; Lemcke, 1959; Christian and Stockton, 1956; Naylor and Smith, 1946; Heckly et al., (1958). Verwey (1945) and Verwey and Scheidy (1946) used solid medium because that was the prescribed method for producing Brucella vaccine, Van Drimmelen (1956) is one of the few who have compared the survival rate of organisms grown on solid versus liquid medium. His results indicated that there was little difference between the survival rate of Bmccella abortus on drying or after storage for 1 month that could be attributed to the method of culture. Muggleton (1960) reported on the results of an experiment in which the Bacillus of Calmette and Guerin (BCG) was grown on 4 different media. The number of viable cells obtained on the 4 culture media varied considerably, from 24 to 183 million, but the percentage surviving after lyophilization was essentially the same for each of the preparations. No storage data were given. It has long been recognized that the age of a culture is a factor in determining the resistance to any deleterious treatment, whether it be heat, chemical, or simply drying. There is general agreement that mature cells are the most resistant to lyophilization but Proom and Hemmons (1949) using Shigella dysenteriae found in two experiments that 12-hour cultures were more resistant to lyophilization than either 3- or &day-old

PRESERVATION OF BACTERIA BY LYOPHILIZATION

27

cultures, and that extremely young (7 hour) cultures were the most resistant. They indicated that similar, but less marked, results were obtained with Escherichiu coli. However, extensive studies by Lemcke (1959) showed a direct correlation between the age of a broth culture and the percentage of E. coli surviving drying, The percentage surviving increased from 0.02% for a 1.25hour-old culture to 12.9% for an 18-hour-old culture. The contradictory findings may be partly due to differences in strain but one should consider carefully the methods employed by Proom and Hemmons. They grew the organism on agar, as opposed to broth used by Lemcke, and they used a turbidimetric method to determine the total cell count after lyophilization which was compared with the viable cell count to obtain a per cent survival; the assumption that turbidity was directly proportional should not have been made. Not only is it possible that the “old” agar culture contained more dead cells than the “young” cultures before drying but the relation of cell numbers to turbidity is not necessarily constant for cultures of all ages. Percentage survival should properly be obtained only by comparing the number of viable cells before and after lyophilization. There now seems to be ample evidence that mature cultures are more resistant to lyophilization. Fry and Greaves (1951) showed that very young ( 4 to 6 hour) cultures of their paracolon bacillus seemed to be far more sensitive to drying than older (18 to 25 hour) cultures. Naylor and Smith (1946) found that S. mrcescens grown at 30° to 34OC. for 18 to 24 hours was more resistant to drying than cultures grown for longer or shorter periods. Benedict et al. (1958) not only confirmed the general observations of Naylor and Smith, but showed that, with constant aeration, cultures incubated at 29O or 3OOC. were significantly more resistant to lyophilization than those cultured at 21° or 25OC. although the total viable cell counts were comparable. Further, at 29OC., the highest aeration rate yielded the most resistant cultures. Annear (1956b) studying the survival of organisms as a function of the growth curves found that Vibria comma and the paracolon bacillus were significantly more resistant in the stationary phase than in the growth phase but Staphylococcus showed no change in resistance. Some observations brought out in a discussion on the maintenance of cultures by freeze-drying (Thornton, 1954) also may be of interest. Chromobacterium vwlaceum grown on ordinary nutrient agar and harvested and lyophilized after 24 hours exhibited a low survival rate. Survival was higher if the cultures were harvested after the pigment was fully developed. It was suggested that the pigment had nothing to do with survival but that pigmentation was merely a property of older cultures and that young cultures were sensitive to lyophilization.

28

ROBERT J. HECKLY

Thus it appears that the most resistant cell is one that is well aerated, well nourished, and mature, i.e., the culture is at the maximum stationary phase. C. CELLCONCENTRATION The highest concentration of cells which can be conveniently prepared and dispensed is ideal since the objective is to obtain dry cells and any unnecessary water is avoided. For various reasons a number of people have considered the effect of cell concentration on the percentage of cells surviving lyophilization (Otten, 1930; Fry and Greaves, 1951; Miller and Goodner, 1953; Benedict et al., 1958 and others). Fry (1954) stated that when cultures are dried with adequate protective colloid the percentage survival probably is independent of the initial cell concentration. However, it is not easy to be certain that the protective colloid is adequate and hence the cell concentration may indeed be important. Otten (1930) showed that with typhoid bacillus the concentration of the suspension was most important, On drying an undiluted culture he observed that 2.8%of the cells survived but when the culture was diluted tenfold only 0.03 to 0.08%of the cells remained viable after lyophilization. However his observations may have been obscured by the fact that as soon as the culture was diluted the cells began to grow, and, hence, Otten dried young cells which, as was shown in Section IV, B, are more sensitive to drying. Since Otten also showed that dead cells were protective it seems logical to expect a higher percentage of survivors from the more concentrated suspensions, particularly if no protective colloids are added, For instance, if one starts with a concentrated cell suspension of 1OI1 cells ' cells could die, and perhaps thus act as a protective colloid, up to 1OO yet one would observe 90%survival. Obviously if the starting concentration were only 1O1O then the concentration of protective colloid liberated would never equal that obtained in the first instance. Since numerous workers have obtained appreciable survival on drying cell suspensions of various initial concentrations it appears that if any correlation exists between cell concentration and survival it is not linear. Stamp (1947) found an increased survival ratio on drying from the more dilute suspensions but his conclusions may be in error because his technique of drying was such that some growth could occur before drying. Furthermore since streptococci are such resistant organisms (Frobisher et al., 1947) it is difficult to understand why he failed to obtain 100%survival in all instances. Some of the results of Benedict d. al. (1958) are of interest in that they demonstrated both a direct and inverse relationship between cell concentration and percentage of cells remaining viable after drying.

PRESERVATION OF BACTERIA BY LYOPHILIZATION

29

In undiluted Naylor and Smith (1946) stabilizer, Benedict showed that the percentage of S. mrcescens surviving increased as the cell concentration increased; and if the same culture were suspended in a 1:lO dilution of the Naylor and Smith stabilizer, the percentage survival decreased as the cell concentration was increased, On the basis of other experiments, in which they varied only the concentration of the stabilizer, they concluded, as did Miller and Goodner (1953), that there was an optimal cell-to-stabilizer ratio for maximal survival. Obviously this ratio, if it is critical, would have to be determined for every system.

MEDIUM D. SUSPENDING The suspending medium may be the most important single factor in determining the survival of an organism and certainly it seems to have received more attention, as indicated by the literature, than any of the other variables associated with lyophilization of bacteria. One of the reasons why additives have been studied so extensively is that the experimental design is simple and that positive results are so readily obtained. Rogers (1914) was primarily interested in drying the lactic acid bacteria, thus he logically tlsed skim milk as the suspending fluid and obtained excellent results. Since that time numerous investigators ( Hornibrook, 1950; Verwey, 1945; Heckly et al., 1958; van Drimmelen, 1956; and others) have used skim milk as a standard for comparison in developing substitutes. It is of interest that Hornibrook (1950) found a lactose-salts solution to be superior to skim milk in protecting a number of organisms, including the very sensitive Neisseria and Vibrio. Serum has also been rather widely used, not only for bacteria but for yeasts as well (Wickerham and Flickinger, 1946; Rhoades, 1958). Weiser and Hennum (1947) found serum to be slightly superior to skim milk for preserving E . coli and the famous “mist. desiccans,” recommended by Fry and Greaves (1951) contains 3 parts serum and 1 part broth with 7.5%glucose added. It is now generally agreed that a protective colloid is necessary to protect cells during freezing and drying and since the report of Naylor and Smith (1946) many workers believe that an antioxidant is also essential. In most of the studies on the effect of the suspending medium, cells were either washed off solid agar or were centrifuged out of liquid cultures and washed free of culture medium. Some workers, such as Heckly et al. (1958), merely mixed broth cultures with double strength additive. The effect of sugars, etc., as reported by Heckly et al. (1958) was probably masked by the presence of 50% of the growth medium, because

30

ROBERT J. H E C n Y

Benedict d al. (1958) showed that S. murcescens survived lyophilization about as well in spent growth medium as in any of the other materials tested. From the practical point of view additional steps, such as centrifugation, are to be avoided. Furthermore it seems likely that the presence of at least some spent medium, if it is not too acid or alkaline, is desirable for the preservation of all organisms, Although it has been generally believed that death caused by freezing is a result of puncture of the cell wall by ice crystal formation, there is little evidence that this is actually the cause of death. As a result of their studies on the drying of tubercle bacilli Miller and Goodner (1953) concluded that the additives exert their protective effect at a metabolic rather than at a structural level. A metabolic effect was suggested because there seemed to be no common basis, such as cell permeability or osmotic pressure effects, to account for the action of the various adjuvants they found to be effective in the preservation of tubercle bacilli. Heller ( 1941) suggested that dissimilable crystalline compounds best protected bacterial cells. In support of his statement he showed that xylose and tryptophan, which are dissimilated by E . coli, protected that organism more than did salicin. Conversely Streptococcus pyogenes was protected more by salicin than by tryptophan or xylose which are not dissimilated by S. pyogenes. Those who are interested in maintaining a large variety of organisms, such as stock culture collections, would welcome a universal menstrum but if the protection is concerned with metabolic processes, as has been indicated above, it would seem unlikely that one medium can be optimal for all species, Splittstoesser and Foster (1957) did indicate that the medium developed by Naylor and Smith (1946) was not universally applicable. They found that the Naylor and Smith solution protected S. murcescens, E , coli, and Brevibacterium linens but it did not protect Micrococcus ureae or Streptococczls lactis. Another theory on the mechanism of action of additives, suggested by Scott (1958) and by Webb (1960) and others, is concerned with structure at the molecular level rather than at the cellular level, Webb suggested that water may be an integral part of protein structures, thus protective substances may act by bridging bonds that would otherwise be broken by the removal of water. Sometimes the protective effect of an additive can be demonstrated only under certain conditions. For instance, Heckly et al. (1958) showed that when cultures of Pasteurella pestis were slowly frozen to -5OC. before lyophilization the control preparations (distilled water added) survived almost as well as the preparation containing skim milk. However if the preparations were shell frozen at -78OC. the percentage pf cells surviving in the skim milk preparation was 10 times as high as the control.

PRESERVATION OF BACTERIA BY LYOPHILIZATION

31

Although the pH of the medium is known to be important in the growth of microorganisms the importance of pH in the lyophilization of bacteria has scarcely been considered. One small series of experiments with P. pestis (HecMy et al., 1958) showed that an initial pH of 7.6 was significantly superior to pH 7.0 or 7.2 in terms of per cent survival immediately after lyophilization. One point that is generally not considered is the change of pH on cooling of the suspensions and the change in pH produced by removal of CO, under vacuum. Topping (1940) indicated that the reason skim milk is superior to serum for the preservation of rickettsia is that under vacuum the pH of serum rises appreciably as a result of CO, evolution. The protective effect of high cell concentrations or even dead cells, as Otten (1930) reported, is perhaps due to the liberation of certain substances, probably colloids, Record and Taylor (1953) showed that, on being dried and reconstituted, E . coli liberated a protective substance which not only protected E. coli but also S. marcescem on lyophilization. Similarly Hutton and Shirey (1951) reported that cell-free extracts of Bmcella abortus protected B. abortus on lyophilization. More recently Bergman et al. (1957) isolated a factor which protected the viability of lyophilized B . abortus, The relation of this factor from B. abortus to the active principle described by Hutton and Shirey (1951) was not determined. It was suggested by Bergman et al. that since the factor was a lecithin it probably protected the cells because of its antioxidant properties. Obviously the question of which suspending fluid is superior is far from settled and since so many factors are interrelated it is not surprising that so many different menstrua have been described as being superior to another. From Table I, in which some of the suspending fluids used for BCG are listed, it can be seen that even though considerable effort has been expended to standardize this organism there is little agreement on how the cultures should be prepared for lypohilization.

E. RATEAND METHOD OF FREEZING Disagreement between authors as to the superiority of rapid freezing over slow freezing is probably a result of the differences in organisms, and/or suspending menstrua. If one believes, as do Proom and Hemmons ( 1949), Luyet ( 1951), and others, that cells are killed by penetration of the cell wall by ice crystals, then few if any cells would be killed by ultrarapid freezing. It is well established (Luyet, 1960a, b ) that the faster the rate of cooling the smaller the size of ice crystals formed and at sufficiently high freezing rates (several hundred degrees per second) there are no crystals formed, Methods of achieving rapid freezing, and the problems of controlling and measuring the rate of cooling, have been

32

ROBERT J. HECKLY

TABLE I A SUMMARY OF SUSPENDING FLUIDS USEDFOR LYOPHILIZING VARIOUS ORGANISMS AND SURVIVAL AFTER STORAGE AT VARIOUS TEMPERATURES ~

~~

~~

Storage

Organism and suspending fluid Temperature Brucella 5 % Lactose, 0.5% ascorbic acid, 0.25 % thiourea, and 0.05% carhoxymethyl cellulose Skim milk

5% Lactose plus salts

58 53 50 25

0 1 Month

48 21

0°C. 20OC. 37°C.

0 7 Months 7 Months 2 Months

50 50 0.02 0.0001

-

0

4°C. 37°C. 37°C.

-

___~__

2 % Dextrin, 0.5 % ascorbic acid, (Room 0.5% NHICl, temperature) 0.25% NaCI, RT 0.1 % tryptose RT

RT Serum

RT

0.5 % Ascorbic acid and 0.25% thiourea

4°C.

50% Skim milk and phosphate buffer

2"-4"C.

Equal volumes of cell suspension and skim milk Escherichia coli 1% Sucrose

1 % Glucose Broth

Per cent viability"

0 1 Month 1 Month 2 Months

-

:, Equal volume of cell suspension and 6% sucrose

-

Time

0

3-72 69

7 Days 30 Days 90 Days

50 60 8.7

10 Years

Growth

-

References

__

Alexander and van Drimmelen (1956) and van Drimmelen (1956)

Heckly et al. (1960)

Hornibrook (1950)

-

Hutton et al. (1951)

Rhoades (1958)

26 Weeks

4gL

van Drimmelen and Steyn (1958)

50 45 17

Verwey (1945)

Rr

0 19 Months 19 Months

RT

3 Years

-

0 20 Days 84 Days 84 Days 56 Days 84 Days

__

3°C. 3°C. 3°C. 3°C. 3°C.

Usable Vaccine

Verwey and Scheidy (1946)

23 0.6 0.03 0.00015 100

Heller (1941)

50

33

PRESERVATION OF BACTERIA BY LYOPHILIZATION

TABLE I (Continued) Storage Organism and suspending fluid Temperature Time 5 % Lactose plus salts Mist,. desiccansc

-

-

RT RT Serum Peptone Serum Skim milk

Neisseria 10% Glucose and 10% peptone on starch

0 0 8-14 Months 24-30 Months

RT

10 Years

-

0 0 0 40 Days 10 Days

-

10°C. RT

RT

0 8 Months

Culture added to dried plugs of peptone, dextran, and glucose

RT

18 Months

Mist. desiccansc

RT RT

3 4 Months 1 Year

Phy$iological saline

RT

18 Years

-

Per cent viability"

References

30-70

Hornibrook (1950)

55 14 7

Fry and Greaves (1951)

Growth

Rhoades (1958)

10 43 26 13 13

Weiser and Hennum (1947)

61 41

Annear (1956a)

25-30

Annear (1956b)

1 0.02

Fry and Greaves (1951) and Fry (1954)

_______---

5 % Lactose plus salts Paracolonbacillus Broth containing 7.5% glucose Serum Mist. desiccansc Pasteurella Equal volumes of culture and 6 % lact>ow

-

0

RT RT RT RT RT

1 8-12 22-31 20-22 3

4°C. nT

RT

Growth 6-180

Day Months Months Months Years

0 3 Years 10 Months 3 Tears

50 47 55 10

Elser et al. (1935) Hornibrook (1950)

Fry and Greaves (1951) and Fry (1954)

10 70-80 70-80 7-8 0.2

Heckly et al. (1958)

34

ROBERT J. HECKLY

TABLE I (Continued) Storage Organism and suspending fluid Temperature Time ~~

Per cent viabilityu

References

~

RT

4 Years

Growth

Miller (1946)

Serum

RT

10 Years

Growth

Rhoades (1958)

Skim milk

RT

18 Years

Growth

Beef and rabbit infusion with cystine ~~

~

Stein el al. (ltJ3-9) _ _ ~

Rickettsia 0.2 M Sucrose plus butter salts

- 20°C.

0

-

1 Month

1 0.1

Jackson and Sinadel (1951)

5 Months

Infective

Topping (1940)

~

Skim milk plus infected tissue

nT

Salmonella 10% Peptone and 10 % glucose on cellulose or alginate 10% Peptone and 10% glucose

RT

0 12 Months

Broth with 7.5% glucose Broth with 7.5% lactose

RT RT RT RT

4-7 Months 18 Months 4-7 Months 18 Months

Serum

ItT

10 Years

Growth

Rhoades (1958)

Skim milk

Iw

5 Years

Growth

Schoening et d.

-

-

0

90-100

Annear (1954,1957, 1958a, b)

92-97 45-99 35 19 21 26

Fry and Greaves (1951)

(1949)

Papain digest broth

25°C.

83 Weeks

10

Scott (1958)

-

0 12 Months

92 81-89

Annear (19.58~~)

0 12 Months

81 10-44

.~

Serratia marcescens 10 % Peptone plus 10 % glucose on cotton 10% Peptone pluli 10 % glucose only

RT -

RT

35

PRESERVATION OF BACTERIA BY LYOPHILIZATION

TABLE I (Continued) Storage Organism and suspending fluid Temperature Time Growth medium (5% skim milk solids, 3% prot opeptone and 2% glucose) 0.5% Ascorbic acid, 0.5% thiourea, 0.5% NHIC1, and 2 % dextrin

Naylor-Smith solution without dextrin

-

RT

0

Per cent viability"

References Renedict et al. (1958)

70

Naylor and Smith (1946)

0 100 2 Months 100

1 Year 100 6 Months 90 6 Weeks 50 12 Hours 50

Maister et al. (1958a)

4 Years

60

Stamp (1947)

0

21-60

Hornibrook (1950)

RT RT

1 Month 8 Months

93 82

Annear (1954, 1958a)

-

RT

0 12 Months

RT RT

1 Month 8 Months

80 63

Serum-water

RT

8-14 Months

100

Mist. desiccansc

RT RT RT

1 Month 8-14 Months 24-30 Months

100 72 20

5°C. 20°C. 50°C. 70°C.

Nutrient gelatin with 0 . 2 5 4 5 % ascorbic acid

RT

5% Lactose plus salts

-

~

Shigell a 5% Glucose on

dried peptonestarch plugs 10% Ulucose and 10% peptone Streptococcus 5 % Glucose on peptone-starch Plugs

77-86 69-90

Annear (1954)

Fry and Greaves (1951)

~

1%Sucrose 1% Glucose

3°C. 3°C.

84 Days 84 Days

31 0.005

Heller (1941)

36

ROBERT J. HECKLY

TABLE I (Continued) Storage Organism and suspending fluid Temperature Time Slap hy lococcus 10% Peptone and 10% glucose Mist. desiccansc

20°C. POOC. -

rvr

RT Papain digest broth

0 2-3 Months 24-30 Months

96 49-109 100 68 36

References

Annear (1958a)

Fry and Greaves (1951)

8'2 Weeks

20

Scott (1958)

2 Days 3 Months 0 Months 12 Months

17 16 15 14

Birkhaug (1951)

4°C.

0 6 Months

37 12

B@eand Evjen (1952)

5°C. 5°C. RT RT

3-4 Months 6 Months 1 Month 5 Months

100

5°C. 37°C. 37°C. 5°C. 37°C. 37°C.

0 12 Months 2 Months 12 Months 0 12 Months 2 Months 12 Months

10 3

1 % Sodium glutamate 1 % Sucrose

5°C. 37°C. 5°C. 37°C.

6 Months 6 Months 6 Months 6 Months

10% Sucrose and 1% gelatin

-

0

Mycobacterium tuberculosis (BCG) 50% Glucose

Milk whey plus 3 % glucose 50 % Glucose

1% Sucrose

1 % Sodium glutamate

25°C.

6 Months 12 Months

l'er cent viability"

P"4OC. 2"-4"C. 2'4°C. 2"-4"C. -

van Ileinse (1951)

90 80 60

0.01 0.001 10-30 12 1.5 0.5 Retained immunogenicity Decreased immunogenicity 40

Cho and Obayashi (1956) and Obayashi and Cho (1957)

Cho et al. (1956)

Kurylowicz el al. (1958)

37

PRESERVATION OF BACTERIA BY LYOPHILIZATION

TABLE I (Continued) Storage Organism and suspending fluid Temperature Time 50% Glucose

18 Months

Retained immunogenicity

4°C. 4°C. 38°C. 38°C. 4°C. 38°C. 38°C. 38°C.

0 3 Months 10 Months 3 Months 10 Months 0 10 Months 2 Months 3 Months 10 Months

1 1 0.1 0.001 0.00001 10 10 10 1 1

Miller and Goodner (1953)

29

Muggleton (1960)

0.1 to 0.25% Sodium glutamate

8% Dextran and 7.5 yo sucrose 5 % Dextran and 7.5% sucrose 5 % Dextran, 7.5% sucrosc, and 2% glutamate

-

0

37°C.

1 Month

2-3

37OC. 37°C.

1 Month 2 Months

10-15 4-7

0

~

1 % Glutamate or 1yo sucrose 1%Glutamate

37°C.

1%Sucrose

37°C.

15% Lactose

Undiluted serum 8.3% Dextran and 7.5% glucose

References

-30" to 25°C. (outdoor temp.)

5 % Sucrose

5.75 % Lactose and 0.05% Tween 80

Per cent viabilitya

lo7 cells per mg.

9 Months No loss of potency 9 Months No loss of potency 9 Months Lost potency

5°C.

4°C. 12-20 Months 10 RT 12-20 Months Marked loss 37°C. 12-20 Months Poor survival 0

?

4°C. 4°C. 20°C. 20°C. 37°C. 37OC.

12 Months '

5 Weeks 16 Weeks 5 Weeks 16 Weeks 5 Weeks 16 Weeks

Lesc hinskay a (1946)

North and Newman (1951) Obayashi et al. (1957)

Rosen thal (1952a, b)

60 60

Ungar (1949)

90b

Ungar et al. (1956)

9Ob

65b 90b 6.5b 0 . 03b

38

ROBERT J. HECKLY

TABLE I (Continued) Storage Organism and suspcnding fluid Temperature Time

Per cent. viabilitya

References

Vibrio

40

Annear (1956b, 19584

I tT

18 Months

-

RT

0 12 Months

Mist. desiccansc

RT RT

1 Month 2 Years

5 3

Fry and Greaves (1951)

5% Lactose plus salts

-

0

7

Hornibrook (1950)

Dried plugs of peptone, dextran, and glucose 10% Glucosc and 10% peptone

Skim milk

Yeast Mist. desic*ransc

4°C.

-

0.5"C.

I3ovine serum

9-42 0.2-15

22 Months Growth

0 9 Months

8.6 7.6 (1-27)

5"-10°C. "Remained alive for long periods " .-

Bovine serum

4°C.

2 Years

Stockton and Newman (1950) Kirsop (1955)

Haynes et al. (1 955)

__

Growth in 98% of

Wickerham and Flickinger (1946)

tubes Commercial yeast at 5 % moisture Commercial yeast at 7.8% moisture

46°C.

16 Weeks

46°C.

16 Weeks

10% Sucrose

-

0

Unacceptable bread Acceptable bread

Mitchell and Enright (1957)

Large number of cells

Guibert and Brbchot (1955)

Approximate values, especially those obtained by estimation from data in figures. Percentage based on the number of viable cells immediately after lyophilization, rather than the viable cell count before drying. Mist. desiccans is a mixture of 3 parts serum and 1 part broth with 7.5%glucose added. a

PRESERVATION OF BACTERIA BY LYOPHILIZATION

39

discussed by a number of investigators (Stephenson, 1960a, b; Luyet, 1960a, b). The question of rapid versus slow freezing is essentially the controversy between the theory of mechanical injury versus chemical injury as a cause of cell death due to freezing. On the basis of evidence cited by Meryman (1956) and Meryman (1960b, c ) there is probably an optimal rate of freezing. Also, in the presence of colloids or other protective substances the optimal rate of cooling is altered. Meryman has indicated that either glycerol, lactose, or glucose protects the cells by inhibiting the freezing of some water and thus excessive concentration of electrolytes is prevented. Lovelock and Bishop (1959) and Luyet and Keane (1952) presented evidence that protection afforded erythrocytes is due to penetration of protective substances. Lovelock and Bishop believed that dimethylsulfoxide protected erythrocytes more than did glycerol because the cells were more permeable to dimethylsulfoxide than to glycerol. Cells were frozen at -79O and thawed at 35OC. Rates of temperature change were not given but they appear to have been relatively slow and good survival was obtained. Practically all those who have studied the effect of rate of freezing of bacterial suspensions have come to the conclusion that in general a high percentage of cells survive when frozen slowly (Mazur et al., 1957a, b; Meryman, 1956; Heckly et al., 1958). Nei (1960) studied the effects of freezing rate and lyophilization on a variety of microorganisms by measuring physiological activities of recovered cells. He found that between 0.017O and 3°C./second the higher cooling rates resulted in the most cell injury. Tanguay (1959) stated that shell freezing was unsuccessful and that best survival was obtained in 15%glycerol by slow freezing (15 to 30 minutes when placed in a -4OOC. deep freeze) as recommended by Meryman (1956). Some of the results reported by Mazur et al. (1957a) are difficult to reconcile or explain but their studies showed that slower freezing generally resulted in a higher survival of Pmteurella tulurensis. Heckly et al. (1958) compared snap-freezing with rapid and slow freezing of P . pestis in a number of menstrua. In all instances a smaller percentage of bacteria shell frozen at -78°C. survived drying than those either snap-frozen or frozen slowly at -5OC. When P . pestis was suspended in skim milk, casein, or lactose solutions, survival after lyophilization of preparations frozen slowly was the same as of snap-frozen preparations. However, in the absence of additives, the percentage surviving after being snap-frozen was about 4 times greater that the percentage surviving when frozen rapidly. Survival of other organisms may be less affected by the rate of freezing since it has been observed (Heckly, 1953) that varying the method of freezing without additives had no significant effect on the survival of either S. marcescens or Klebsielln

40

ROBERT J. HECKLY

pneuntoniae. These organisms were grown in the same medium as was the P. pestis referred to above (Heckly et al., 1958). In studies on the preservation of tissue cultures it has been observed that slow freezing is markedly superior to rapid freezing, Scherer (1960), for example, using two types of mammalian cells cultured in vitro and suspended in glycerol solution, found that cells frozen slowly (1 to 1.5 hours) survived better than those frozen rapidly ( 3 to 5 minutes). Viruses, on the other hand, survive as well, or better, when frozen rapidly. Greiff (1960) showed that survival of influenza virus after slow cooling was approximately equivalent to survival after rapid cooling and others have indicated that infectivity of viruses was best maintained by rapid freezing. A rationalization of why slow freezing is superior to fast freezing for the preservation of bacteria and other cells is that slow freezing allows time for the cells to adapt to the new environment produced by freezing of the preparation. Viruses, being smaller and possibly more like an inert organic molecule than a complex cell, may not need to adapt to the new environment and thus are not adversely affected by rapid freezing. Much of the confusion concerning the protective effect of additives may be a result of the fact that the mechanisms of protection during slow freezing may be quite different from those of rapid freezing (Meryman, 1960b). Substances that protect against slow freezing are ineffective during rapid freezing, and conversely, substances that protect cells on rapid freezing are ineffective when the cell suspensions are frozen slowly.

F. METHODOF DRYING 1. From the Frozen State Some authors state that a specific temperature during drying or a rate of dehydration is optimal for the survival of bacteria; others maintain the opposite view. Actually there are few studies in which the rate of drying was not obscured by the fact that, to achieve the different drying rates, the temperature of the sample was changed. Conversely, those who studied various drying temperatures failed to maintain constant drying rates. Muggleton (1960) cited evidence that the lowest drying temperatures resulted in the highest survival of BCG. At -loo to -15OC. he reported 8%survival whereas at -31OC. the survival was 51%;he did not indicate whether this was the temperature of the preparation or of the bath, The results of a study by Schmidt et al. (1958) on the effect of freezing and drying rates, and drying temperatures, indicate that generally the

PRESERVATION OF BACTERIA BY LYOPHILIZATION

41

greatest loss of viability of a variety of organisms was sustained by those preparations which were frozen rapidly and dried at -3OOC. Snap-freezing and drying at higher temperatures resulted in significantly higher survival of E . coli and S . aureus. Since Schmidt et al. (1958) did not include the tubercle bacillus in their studies, and because organisms may differ in their response to various drying temperatures and rates, it is difficult to say whether those authors’ results were actually at variance with the data of Muggleton (1960). Annear (1958b) reported that -8OC. was the optimal temperature for lyophilizing Salmonella ndolo, but in view of his statements about frothing, etc., there is some doubt as to whether the preparations had remained frozen throughout the drying process. He obtained the highest survival using a centrifugal freeze-drier in which the temperature was estimated, but not determined, to be -7OC. Hutton et al. (1951) studied the effect of temperature and rate of drying on the survival of Brucella abortus. Ice film temperature was varied by adjusting the total pressure in the system and the rate of drying was varied by adjusting the temperature of the surrounding metal surfaces. There was some indication that an optimal drying rate might exist, but, because of the inconsistencies between experiments, the differences in the observed survival may not have been significant. Temperature seemed to be relatively unimportant because Hutton et al. (1951) showed little difference in the percentage of cells surviving in preparations dried either at -26O or at -34OC. The results of one experiment on the influence of drying temperature on the survival of Brucellu melitensis (Heckly et al., 1960) show that although there was no apparent difference immediately after lyophilization, samples dried at -18OC. survived 4 months’ storage better than those cultures dried at -28OC. This difference in storage stability is probably not a function of the final drying temperature because the lower temperature (-28OC.) was obtained by increasing the size of the connection to the manifold. In all instances bottles were hanging free in air at about 20°C. for the first 4 to 6 hours and at 32OC. for about 18 hours. Cultures appeared to be dry within 4 hours. Some unpublished data (Heckly, 1953) indicated that conditions obtained by drying at -18OC. also resulted in a higher percentage survival of P. pestis when tested immediately after drying than when the preparations were dried at -3OOC.

2. %-package” Desiccant The inclusion of a package of desiccant in the same container as dried yeast was the subject of a patent issued to Ohlhaver in 1912 (Mitchell

42

ROBERT J. HECKLY

and Enright, 1957). The use of an “in-package” desiccant (calcium oxide) has been further explored by Mitchell and Enright ( 1957)In a method described by Barratt and Tatum (1950) phosphorus pentoxide was placed in the bottom of a large tube and then covered with cotton. A smaller tube containing a culture to be preserved was then inserted and the entire unit was evacuated and sealed. A process described by Graham et al. (1958, 1959), mentioned in Section 111, B, represents the use of “in-package” desiccation for large scale production. However this method differs from others in that the organisms, frozen in pellet form, were mixed with the desiccant rather than having provided a physical barrier to keep the organisms separated from the desiccant. A high percentage of the cells survived drying by this method.

3. From the Liquid State Drying of unfrozen preparations should be mentioned for comparative purposes and, also, the behavior of cultures dried in this manner may furnish data pertinent to problems which arise in some lyophilization procedures. Fry (1954) discussed at considerable length how difficult it is to avoid drying from the liquid state and implied that viability would be lost if one did not prefreeze the preparation or if it were allowed to thaw in the process. Greaves (1944), in his discussion of centrifugal vacuum freezing, also indicated that it was mandatory that adquate vacuum be attained rapidly if one is to avoid drying from the liquid state. On the basis of such information it is generally assumed that a higher percentage of cells survive when dried from the frozen state than when dried from the liquid state. Many organisms may be injured if dried from the liquid state but experiments in which comparisons have been made indicate that this is not applicable to all bacteria. Weiser and Hennum (1947) found that E . coli survived slightly better (50 versus 35%) on being dried from the liquid state at 20OC. than when lyophilized at -15OC. Annear (1957, 195813) found survival of S. ndolo, when dried from a liquid suspension, was comparable to that obtained by freezing and drying; the immediate recovery varied between 53 and 95%.Schmidt et al. (1958) compared the effect of simple drying and lyophilization at various rates on a number of different organisms. They concluded that generally removal of water directly from liquids was as satisfactory as lyophilization. A possible exception was that both S. aweus and E . coli survived better when snap-frozen and dried than when dried from the liquid state. Stamp (1947) compared drying from a 10%gelatin at ambient temperature over

PRESERVATION OF BACTERIA BY LYOPHILIZATION

43

phosphorus pentoxide with lyophilization using S. murcescens. He observed that a significantly higher percentage, 60 to 90%,survived the former treatment whereas 24 to 34% survived in lyophilized preparations. In Stamp’s technique organisms are suspended in 10%gelatin and dried without freezing. Although his method is not exactly comparable to the usual concept of drying from the liquid state it is also not freezing and drying from the frozen state. If one compares his results with those of other workers who lyophilized cultures in other suspending menstrua one is impressed by the high percentage survival of certain organisms. For instance, after 4 years of storage at room temperature or slightly below, Stamp (1947) reported 17.3%of dried Brucella melitensis to be viable. In comparison, Heckly et al. (1960) found that only 0.0% survived about 6 months’ storage at 2OOC. However it should be pointed out that since there was essentially no loss of viability in those cultures stored at OOC., storage temperature may be very critical. Stamp’s method may be less applicable to other organisms such as Neisseria meningitidis and Vibrio comma since he observed that less than 0.2%of V. comma survived the drying procedure and none were recovered after 2 years’ storage. Fry and Greaves (1951) on the other hand, reported 1%survival of Neisseria gonorrhoea after 1 year’s storage and 3%survival of V. comma after 2 years’ storage at room temperature. Two disadvantages of Stamp’s method might be mentioned, one is that dried gelatin is not easily dispersible in water and the other is that the method cannot be employed to process appreciable amounts of material, as for the production of vaccine. Despite the fact that some cells remain viable if dried from the liquid state it would seem that lyophilization is the best method for general application. Fry (1954), after discussing various aspects of the influence of pressure on temperature also concluded that drying from the frozen state is most universally applicable to all organisms.

G. EXTENTOF DRYING Most researchers agree that residual moisture content is a factor influencing the survival of organisms but there are diverse opinions as to what the optimal moisture content should be for optimal survival. It has been repeatedly stated, mostly without any evidence, that cultures should be as dry as possible to preserve viability, the reasoning being that only in the absence of water is the metabolism of the organism completely arrested. Others believe, however, that it is possible to dry organisms excessively and that one should allow a certain amount of moisture to remain. Stamp ( 1947), Fry and Greaves (1951), and others have added glucose (which is hygroscopic) to culture suspensions immediately before drying to assure some residual moisture. Muggleton (1960) contended that com-

44

ROBERT J. HECKLY

plete removal of water is incompatible with life, at least with respect to the tubercle bacillus. He also added glucose to retain moisture but he did not show that the beneficial effect was due to increased moisture rather than to the presence of glucose per se. In an attempt to differentiate between the effect of moisture and added glucose, Fry and Greaves (1951) dried a paracolon bacillus suspended in glucose-free serum solutions and found, for example, that prolonged drying, 336 versus 18 hours, decreased the percentage survival of the bacillus from about 8 to 0.2%. Although the results cited by Fry and Greaves seem to be definitive, other studies indicate that glucose may have effects other than the regulation of moisture content. Sugar or other additives may influence the osmotic balance or the metabolism in ways which are perhaps more significant than the regulation of the residual moisture content. The results of Heller (1941), Heckly et al. (1958), and Scott ( 1958) tend to prove that the nature of the sugar can be critical and that, as indicated in Section IV, D, sugars may exert their effect metabolically as well as physically. Studies on the acid production by dissimilation, either before freezing or after reconstitution of the cultures, may help to explain observed differences between the effect of various sugars. Heller (1941) indicated that when sucrose was added to either E . coli or S. pyogenes before lyophilization, more bacilli seemed to survive than when glucose was present, although the latter markedly increased survival above that obtained in the absence of sugars. Survival during storage of lyophilized cultures of P. pestis (Heckly et al., 1958) containing glucose was no better than the control containing no additive, whereas, in the presence of either lactose or sucrose, organisms survived 20 months' storage at room temperature. Similarly Scott ( 1958); after testing cultures of Salmonella newport lyophilized in sucrose, glucose, and arabinose, found the latter to be the least effective. A point that is not generally considered is that the effect of moisture may be influenced markedly by the atmosphere in the ampule. Scott (1958) showed that when bacteria were stored in vucuo those with the least moisture survived as well or better than those with appreciable amounts of moisture. However, when cultures of S . newport dried in papain digest broth were stored in air at atmospheric pressure optimal survival was obtained at an AW6of 0.22 and minimal survival at an A, of 0.00 (stored over phosphorus pentoxide). Some of the problems encountered by those who believe that complete 'A, = thermodynamic activity of water in a solution which is in equilibrium with the dried culture.

PRESERVATION OF BACTERIA BY LYOPHILIZATION

45

removal of water is lethal may be a result of inadequate reconstitution procedures. For instance, it has been observed (Mitchell and Enright, 1957) that the more yeast was dried, the more it lost its leavening power when rehydrated by simply adding water. However, the yeast could be activated by subjecting thin layers of dried cells to a humid atmosphere for about 4 hours, The problems concerned with rehydration are discussed in Section IV, I. Obviously considerably more work of this nature needs to be done to separate lethtal or protective effects of sugars from the effects of increased moisture content which may result from the presence of these or other additives. H. STORAGE CONDITIONS

1. Atmosphere Rogers (1914) was one of the first workers to compare storage in vacuo with storage in atmospheres of various common gases. He found that survival was the highest in cultures stored under vacuum and the lowest in those stored in air or oxygen. Atmospheres of nitrogen, hydrogen, and carbon dioxide yielded intermediate results. The observations of Naylor and Smith (1946) are in agreement with those of Rogers ( 1914). After 49 days’ storage in air of S. murcescens only 9% of cells remained viable as compared with 99%of those stored in vacuum. Under nitrogen, either treated to remove residual oxygen, carbon dioxide, and moisture, or untreated, only 26 to 28%survived. Studies of Maister et al. (1958a) showed that survival of S . marcescens in oxygen and air was lower than when stored in vacuo but they found that organisms dried in the form of pellets survived as well in atmospheres of nitrogen or helium as in a vacuum. Christian and Stockton (1956) studied the influence of various amounts of air on the viability of S. marcescens and S. aureus at room temperature though no storage period was noted. When sealed under 60 to 70 p Hg pressure, 3% of S. marcescens and 41%of S. aurem were viable when tested but these values dropped sharply to about 1 and 7%,respectively, when the cultures were sealed under 100 to 150 p Hg. However, losses sustained when cultures were stored under 700 to 750 p Hg were only slightly greater than those at 100 to 150 p Hg. Heckly et al. (1960) found that B. melitensis survived storage equally as well in nitrogen as under vacuum, but unpublished preliminary trials with P . pestis to intercompare dry air, nitrogen, and vacuum indicated that the viability of P. pestis was maintained best in 2racuo. The work of Scott (1958) indicated that the effect of the atmosphere is dependent upon the nature of the suspending medium and moisture

46

ROBERT J. HECKLY

content. He compared the effect of various humidities and additives (suspending fluids) on the survival of Salmonellu newport in vacuum and in air. Only under the very driest conditions and in the absence of sugars was there a marked difference in survival between cells stored in air and vacuum. Survival obtained in oacuo was much higher than that obtained in air. On the basis of the available information it seems that the safest procedure, in terms of preserving viability, is to store the cultures under vacuum.

2. Temperature Most workers, as a matter of convenience or without due consideration, store their lyophilized cultures at room temperature but there is every indication that most organisms survive better at 4OC. than at room temperature or above. Rogers (1914) showed that the higher the temperature of storage the lower was the survival rate of lactic acid bacteria dried in skim milk. Maister et al. (1958a) presented proof that as the storage temperature of lyophilized S . mrcescms was decreased from 8OOC. a marked increase in survival rate occurred. At 5°C. no loss of viability was noted after more than 1 year's storage. Although elevated temperatures have often been used to accelerate aging, these investigators found that survival at 80OC. was not correlated with survival at lower temperatures and hence could not be used as a rapid test method. Meanwhile, Proom and Hemmons (1949) had presented evidence that heating at 60" to 80OC. for 1 hour provided a simple measure of the capacity of a particular batch of culture to remain viable on storage at room temperature. No attempt will be made to reconcile their conclusions with those of Maister et al. (1958a) because of the numerous factors involved. Proom and Hemmons also showed that survival of N . meningitidis and E . coli was better when stored at 4OC. than when stored at 37OC. For instance, after E . coli had been stored for 6 months at 4 O and 37OC. they observed 28% and 4% survival, respectively. Weiser and Hennum (1947) showed that the halflife of the decay of viability of lyophilized E . coli at 10°C. was about 4 times that noted at room temperature. Obviously the magnitude of the effect of temperature reported by Weiser and Hennum on the viability of E . coli was greater than that reported by Proom and Hemmons but the reason for this is not readily apparent. Figure 5 shows that although there was virtually no loss of viability of P . pestis when stored at 4OC., there was a marked loss of viability in comparable cultures stored at room temperature. This difference in stability, however, was not as marked as that observed with lyophilized B. melitensis (Heckly et al., 1960) in which the number surviving after

PRESERVATION OF BACXFXUA BY LYOPHILIZATION

47

6 months' storage at OOC. was 1000 times that observed after the same storage period at 2OOC. Ungar et al. (1956) showed that there was essentially no change in the number of viable cells in one of his BCG preparations stored for 16 weeks at 4O, 20°, or 27OC. but that at 37OC. less than 0.04%survived 6 weeks. Verwey (1945) studied the eEect of temperature on lyophilized B . abortus vaccine using as a criterion the number of months elapsing before

-?

9 . 10

r' a W a

v)

f

g lo8 0

a

0

STORED AT 4 O C

- \'g

'

a

W

-1

m

a

7 . 10

61 lo

I

0

I

16

I

STORED AT ROOM TEMP. (ABOUT 2OoC)

32

I

I

48 64 MONTHS OF STORAGE

'

1

80

FIG. 5. Survival of Pasteurelkz pestis strain A-1122 stored at 4°C. and at room temperature. Equal volumes of 6%lactose and culture were mixed immediately before lyophilization. All containers were sealed under vacuum after lyophilization.

the viable cell counts were below lo6.The differences may not be significant but he indicated that if buffered saline were used to suspend the cells, survival at 37OC. was comparable to that at room temperature, whereas at approximately 4OC. survival was 2 times that obtained at room temperature. If either skim milk or bovine serum were used as the suspending menstrum survival at room temperature was comparable to that at 4OC. The addition of skim milk did not increase the survival at 37OC. appreciably over that obtained when organisms were suspended in buffered saline. However, when stored at 4OC. the addition of skim milk increased survival of cells several times over that obtained in buffered saline.

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ROBERT J. HECKLY

There are many other studies in which the effect of storage temperature has been considered but there seems to be no evidence to indicate that cultures should not be stored in the cold, It is reasonable to suggest, therefore, that lyophilized stock cultures be stored at Oo to 10OC.

3. Light There is very little comparative work showing the effect of irradiation on lyophilized bacteria. Since it is well known that ultraviolet energy is lethal to organisms, lyophilized cultures are usually stored in the dark. Fry and Greaves (1951) cited, as an incidental observation, that a collection of paracolon bacillus cultures which had been mounted on a board for demonstration, 7 months after they were dried, did not appear to have been harmed by 6 months' exposure to sunlight, It would be of interest to know how deeply these lethal rays can penetrate into a lyophilized preparation. Since the visible light is so effectively blocked by the outermost layer of material, relatively few cells may have been affected. Miller and Schad (1944b) described a more extensive study of the effect of daylight on dried meningococci. The organisms were not lyophilized but were dried on glass beads or gauze (Miller and Schad, 1944a). At room temperature the organisms survived 9 to 10 days in the dark, but when exposed to daylight through 2 thicknesses of window glass and 1 thickness of Pyrex for 30 hours the organisms were killed. In diffuse room light the organisms survived considerably longer than when subjected to direct sunlight and it was further determined, by using colored cellophane, that the killing was approximately proportional to the amount of blue light transmitted. Although short-wave radiation (260 to 280 mp) is usually considered to be the most germicidal portion of the spectrum, near-ultraviolet light (380 mp) which passes through ordinary window glass is also lethal. Researchers usually pay little attention to the reduction or elimination of the amount of light incident on the ampules during the drying process. Perhaps one should reduce the light intensity in the room or provide some shielding of the containers, particularly if erratic results are obtained. 4. Type of Container

Usually lyophilized stock cultures are sealed in glass ampules or tubes, and the viability of organisms stored in rubber-stoppered bottles has seldom been compared with storage in glass-sealed ampules. However, rubber-stoppered bottles are sometimes employed because they are more easily filled, sealed, and opened than are glass ampules. Heckly d al. (1958, 1960) demonstrated that a higher percentage of both P. pestk and

PRESERVATION OF BACTERIA BY LYOPHILIZATION

49

B. melitensis survived when stored in glass-sealed ampules than in rubber-stoppered bottles. After P. pestis had been stored 20 months at room temperature there was an average of 10 viable organisms per milliliter of original culture, contrasted to about 188per milliliter in the allglass ampules. Moisture was probably the cause of this rapid loss of viability of cells stored in rubber-stoppered bottles, but since it was shown that after the 7 months’ storage the pressure also had increased from 0.020 mm. to an average of about 136 mm. Hg it is probable that the presence of air was also a factor. Unfortunately, suitable control vials containing various amounts of dry air were not included. Flosdorf and Webster ( 1938) studied the comparative permeability of rubber and neoprene to air and water and concluded that a wellcompounded rubber was as satisfactory for sealing dry biological specimens as was neoprene. Although neoprene was less permeable to moisture it had the disadvantage of being less elastic, Flosdorf et (11. (1945) and Flosdorf ( 1945, 1949), using lyophilized horse serum diphtherial antitoxin, showed that rubber stoppers were, indeed, permeable to moisture. At room temperature and at ambient relative humidity the moisture content of the antitoxin increased from less than 1%to over 4% in a year. At 35OC. and at a high relative humidity the moisture content in rubberstoppered bottles reached about 171%. Flosdorf and Webster (1938) mentioned the use of a picein coating to minimize the passage of water through the rubber stoppers and Heller (1941) coated his stoppers with a resin for the same purpose. The coating did reduce the diffusion of moisture, but in practice it has not been entirely satisfactory. Reichel (1943) and Flosdorf et al. ( 1945) described a glass container which combined the advantages of a sealed all-glass container, obtained by sealing the ampule over the stopper, with the advantages, such as ease of dispensing, afforded by a rubber-stoppered bottle. In studies employing bottles fitted with Stokes exhaust tube stoppers, it was found that during 7 months’ storage at room temperature the moisture content of the samples increased from approximately 0.2%to an average of about 8% (Heckly et al., 1958). Water vapor probably accumulated more rapidly than air because it may enter by dissolution and diffusion whereas air enters by diffusion only (Flosdorf and Webster, 1938). I. METHODOF RECONSTITUTION 1. Effect of Temperature of Reconstitution Fluid Hiscox (1945) showed that significantly higher numbers of viable cells were demonstrable in spray-dried milk if the powder was reconstituted

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ROBERT J. HECMY

at 50OC. than when reconstituted with water at room temperature. She did not identify the species, but presumably these were those bacteria ordinarily found in milk. She also showed that the apparent number of viable cells was greater when the plates were incubated at 30° than at 37OC. The results of Speck and Meyers (1946) were essentially identical to those of Hiscox except that they used cultures of Lactobacillus bulguricus. They found that the observed viable cell count was greater when spray-dried cultures were reconstituted at 5OoC. than when reconstituted at 21OC. However, lyophilized cultures were not activated by reconstitution at 50OC. and in fact 5OOC. was lethal to many of the lyophilized cells. This suggested that there is a fundamental physiological difference between lyophilized and spray-dried L. bulguricus. Wasserman and Hopkins ( 1957), studying various factors affecting the recovery of lyophilized S. marcescem, found that 95% of the cells which developed into colonies when rehydrated at 3OOC. failed to grow when rehydrated with the same solution at 5OC. More recently Leach and Scott (1959) confirmed the observation of Wasserman and Hopkins, but they extended the studies to show that there is a significant difference between organisms with respect to the optimal temperature of reconstitution. On using distilled water for rehydration the highest temperature used (37OC.) yielded the highest number of viable S. murcescens and L. bulguricus which confirmed the observation of previous investigators. However, Leach and Scott found that the lowest temperature, OOC., was vastly superior to 37OC. for rehydration of Vibrio metschnikovii. The optimal temperature for rehydration of E . coli with distilled water was 2OOC. The results were significantly different when saline solutions were used for rehydration, in some instances the temperature effect being eliminated or obscured. Anyone interested in these details should consult Leach and Scott (1959). Strangely, in view of various reports by Mitchell and Enright (1957) and others, Leach and Scott found that the temperature of rehydration had essentially no effect on the viability of lyophilized yeast, Sacchuromyces cerevisiae. According to their data nearly 100% survival was obtained under all conditions of rehydration. Perhaps the temperature effect can be demonstrated only if the yeast has been appreciably damaged. It is of interest that Sant and Peterson (1958), using loss of nitrogen as a measure of cell damage, found that commercial active dry yeast was damaged more when reconstituted at 4 . 5 O than at 37OC. Twice as much nitrogen was lost when yeast was reconstituted at 4.5OC. They also showed that, when compared with samples rehydrated at 37OC., rehydration with water at 4.5OC. killed about 95% of the cells and reduced the C0,-producing capacity by about 90%.

PRESERVATION OF BACTERIA BY LYOPHILIZATION

51

A similar temperature effect in the rehydration of S. aureus was reported by Leach and Scott (1959), using absorbancy of the supernatant fluid at 256 mp as a measure of cell leakage. When reconstituted with water at OOC., 4 to 5 times as much material leaked out of the cells as when reconstituted with water at 22OC. A suitable explanation of why temperature of the reconstituting fluid should influence the viability or integrity of lyophilized cells has not been developed. Hiscox (1945) suggested that the viable cell count was increased by treatment with water at 50OC.because it shocked the cells, an action comparable to the development of spores after heat shock. This idea however would fail to explain why the highest viable cell count of V. metschnikovii was obtained by reconstitution with cold water (Leach and Scott, 1959). Until a better understanding of the phenomenon is obtained it appears that optimal temperature should be determined for each condition. 2. Effect of Composition of Reconstitution Fluid Lyophilized preparations are commonly reconstituted by the simple addition of distilled water on the basis that only water is removed in the lyophilization process. The rehydration procedures used by the early workers has not always been specified and investigators have used various nutrient broths without investigating the effect of their composition. Weiss ( 1957) and The American Type Culture Collection (Catalog of Cultures, 1958) recommend reconstitution in several volumes of the appropriate growth medium. Stamp (1947), Proom and Hemmons (1949), and Rhodes and Fisher (1950) also have used broth of a volume usually several times that of the original culture before drying. There are few reports on the effect of the composition of the reconstitution fluid. Wasserman and Hopkins (1957) compared the effect of various salt solutions, and water for the reconstitution of lyophilized S . murcescens. They found 0.05 M sodium malate at pH 6.0 to be particularly effective. Upon reconstitution of a lyophilized S. murcescens preparation with this malate solution, the observed viable cell count was 7.3 x loo, but when reconstituted with distilled water only 2.8 x lo3 cells were viable. Such large differences were observed only in the preparation in which less than 0.1% of the viable cells originally present remained viable. Thus, it would seem that no benefit would be derived from using the malate solution if the lyophilization procedure and storage survival for S . marcescens) . conditions were adequate (at least 10% Leach and Scott (1959) studied the effect of various rehydrating solutions on the viability of V. metschnikouii. In general they found that the viability was increased by increasing the concentrations of salt mixture, sucrose, glycerol, or sodium sulfate, but the optimal concentration

52

ROBERT J. HECKLY

was dependent upon the particular solute. The highest percentage of viable cells recovered was obtained by reconstruction with a brain plus heart broth and at a relatively high concentration of each constituent. The thermodynamic activity of water (A,) in the optimal concentration of brain plus heart broth was 0.97. Possibly this is in part a function of osmotic pressure similar to that described by Bretz and Hartsell (1959). They showed that the viable cell counts of E. coZi, which had been injured by repeated freezing and thawing, were higher if the frozen preparations were diluted with 20%sucrose instead of the usual dilute buffer solution. The number of viable cells observed when the preparations were diluted with 20%sucrose was about 100 times that of the control diluted in 0.067 M phosphate buffer. Those cells which failed to survive were termed osmosensitive. Wachter and Gilman (1960) observed a relatively large loss in infectivity when lyophilized Rickettsia Tickettsii was reconstituted with distilled water. However, if aliquots of the preparation, reconstituted in distilled water, were incubated for 3 hours in the presence of 0.014% diphosphopyridine nucleotide, an average of 82%of the infectivity lost on lyophilization was restored. This phenomenon is perhaps a reactivation and not strictly related to minimizing the damage caused by rehydration, but it is worth considering in terms of demonstrating maximal survival. The results of Leach and Scott (1959) and Bretz and Hartsell (1959) would indicate that a high osmotic pressure is generally conducive to obtaining maximal recovery of lyophilized preparations. However, because high osmotic pressures adversely affect some organisms and because the growth requirements of various organisms differ considerably, a universal rehydration medium may not be possible. 3. Effect of Volumes and Rate of Reconstitution Lyophilized preparations are most commonly reconstituted by the simplified procedure of adding water or a solution directly with a pipette or syringe. Alternatively, the pellet is transferred or dropped into a tube of the culture medium as suggested by Weiss (1957). In many instances volumes of solution have not been specified. It has been known for some time (Ohlhaver, 1912, cited by Mitchell and Enright, 1957) that dried yeast could be activated by exposing it in thin layers to a humid atmosphere. Studies on yeast have been extended by Mitchell and Enright (1957) who confirmed the fact that a vapor rehydration to the extent of 8 to 10%moisture was necessary to restore activity of yeast that had been dried to 2%or less moisture. Sant and Peterson (1958) found that vapor rehumidification of commercial active dry yeast to about 25%was essential for maximal survival

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53

when it was reconstituted at 4.5OC., but rehumidification was not necessary when the yeast was reconstituted at 37OC. When the cells regained a moisture content of 20-25% they were apparently completely reorganized and were then independent of former low-temperature effects. Without rehumidification, 95%of the cells were killed by reconstitution at 4.5OC. They also showed that cell damage, as indicated by a loss of nitrogen, was roughly correlated with loss of viability. The beneficial effects of slow vapor rehydration are not restricted to yeasts since King (1959) has shown similar effects on the viability of pine pollen. Some of his observations relative to the effect of vapor rehydration as a function of dryness and temperature of storage may be of interest. When pollen was cultured immediately after drying or after storage at 5°C. for 74 days approximately 70 to 90% germination was observed with or without any vapor treatment. In contrast to this, pollen which had been stored in nitrogen at room temperature presented an entirely different picture. When tested immediately after opening the ampule, no viability was demonstrated in those preparations dried for 1to 7 hours but 60%of the pollen was viable in the preparation dried only 5 minutes. On the other hand there was essentially no difference in the observed viability of the various preparations, whether dried 5 minutes or 7 hours, when the pollen was cultured after 48 hours at 5°C. and 60% relative humidity. Viability ranged from 40 to 90%. Leach and Scott (1959) reported that the addition of moisture as a vapor to V. mdschnikovii was highly lethal but by rehydration with mists sprayed at rates of from 0.1 to 0.01 mg. water/sec./mg. dry matter resulted in recoveries several fold higher than with more rapid addition of water. They indicated that the lethal effect of rehydration with water vapor was due to the extreme slowness of the method. They estimated the highest rate attained by this method to be mg. water/sec./mg. dry matter. In view of the highly beneficial effects of vapor rehydration cited above, it may be advisable to explore further the possibilities of applying the method to bacteria. The rate of rehydration can probably be increased by evacuating the container before introducing the water vapor. It has been observed in connection with measurements of moisture content by the manometric method (Heckly, 1955) that, in a vacuum, dried cultures would absorb moisture rapidly. The rate of absorption could be increased further by cooling the bottle to 4OC. Leach and Scott (1959) also studied the effect of volume of the reconstitution fluid and concluded that smaller volumes resulted in the highest observed viability. They added various amounts of water or solutions to 0.2 m].of a dried V. metschnikovii suspension and showed that in general

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ROBERT J. HECKLY

the addition of 0.25 ml. fluid resulted in a higher number of viable cells than if either 2 or 16 ml. were added. On the basis of the above discussion no single method can be recommended, but it seems that if 100%survival of lyophilized organisms is not obtained one should investigate the rehydration procedures.

J. ASSAYMETHODS Most bacteriologists think of the usual dilution and plating methods when one mentions assaying a culture, but there are in fact several other methods which have been used, depending to some extent upon the objectives.

1. Viability a. Qualitutiue. The growth or no growth method yields relatively little information about the successfulness of the processing. Unfortunately, a considerable amount of work has been reported only in terms of a percentage of those ampules tested which yielded a culture, or, whether growth was obtained after a given storage period. Frobisher et al. (1947) described a simple method of preserving cells on sand stored in Mason jars under only moderate vacuum, They claimed relatively long survival, up to 18 years for Steptococcus and Staphylococcus, but since they observed only growth or no growth their results are not too easily compared with studies of death rates. Appleman and Sears (1946), Stein (1954), and Stockton and Newman (1950) are also among those who did not count the number of viable cells. Rhoades (1958) claimed on the basis of 10 years’ storage of 111 strains that there was no difference between room temperature storage and refrigerated storage, since he obtained cultures from all ampules, but had he made a count of the number of cells remaining viable after each storage period he probably would have observed a difference. The study of 1500 strains by Proom and Hemmons (1949) would also have been more meaningful if numbers of viable cells had been reported instead of only the percentage of ampules yielding a culture. Obviously, if a single cell remained viable under the imposed conditions, such a test would not distinguish these cultures from those in which no cells had died. b. Quantitative. As indicated above, it is desirable to have an actual count of the number of cells surviving. This may be estimated by determining the highest dilution in which growth can be obtained in liquid medium, or, as is more commonly done, by diluting and plating on a suitable solid medium. Seldom has much thought been given to selecting the proper assay medium, but in view of the report by Straka and Stokes

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(1959), media adequate for growing ordinary cultures may not be adequate for growing lyophilized cells. They found that freezing produced some metabolic injury to a variety of species so that the apparent number of viable cells obtained was a function of the composition of the plating medium. Cells apparently injured by freezing were no longer able to grow on the usual minimal medium but were able to form colonies when plated on a richer and more complete medium. A difference of as much as 40%between frozen and unfrozen cultures was noted. In their system they showed that peptides in enzyme-digested casein may be the active substance required by injured cells for resynthesis of essential proteins. The tubercle bacillus has always been a difficult organism with which to work. The introduction of oleic acid and albumin in growth media by Dubos and Middlebrook (1947) was a significant development and has become almost standard in this field. Thow (1956) compared a number of different media for estimating numbers of viable cells in lyophilized BCG vaccine and found that growth was significantly improved by the incorporation of 5% whole blood in the oleic acid albumin medium. This medium was superior to all others tested in that the colony count was highest and the colonies appeared most rapidly. Blood-enriched medium only slightly increased the viable cell count of fresh cultures, but the viable cell count of lyophilized cultures was increased from 2.5 to over 36 times that obtained in ordinary media. Rosenthal (1952b) observed that after BCG was dried, growth was slower in liquid media and was inhibited on solid media, but did not mention whether other media had been examined. He interpreted the slower and more erratic growth as a state of partial dormancy. Wasserman and Hopkins (1957) observed that the apparent number of viable cells can be increased by transferring the cells, for a period prior to plating, to a different medium. They found that the greatest increase of viable S. marcescens bacilli was obtained if the culture was rehydrated with malate. The apparent increase in number of viable cells obtained on richer media may be a “reactivation” similar to that described by Heinmetz et nl. (1954), or it might be related to the lethal effects of certain diluents as described by Straka and Stokes (1957). An increase in virulence without growth of P . pestis (Heckly et al., 1958) may also be a “reactivation” phenomenon, but for the moment the mechanisms involved are obscure and more work is needed along these lines. 2. Biochemical Activity In some instances the activity of a dried preparation is of greater practical importance than is the number of viable cells. For example, commercial dry yeast must be “active”; the effectiveness of preservation

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being evaluated on the basis of the quality of the bread produced under standard baking conditions (Mitchell and Enright, 1957). This test requires that not only must a sufficient number of cells be viable but that these cells be fully capable of carrying on their normal metabolism. Mitchell and Enright demonstrated that the leavening ability of dried yeast was destroyed by certain procedures which did not impair viability. Fermentation tests using subcultures [such as those applied by Kirsop (1955) to lyophilized yeast] are not dependent upon the physiological condition of the reconstituted culture, since all that is necessary is that some viable cells retain sufficient genetic structure to permit their progeny to have characteristic fermentation capacities, Rogers ( 1914) applied lyophilization successfully to the preservation of bacteria and assessed the efficiency of his lyophilization on the basis of the biochemical activity as well as by the determination of the number of viable cells recovered. In fact he believed that measuring the acid produced by a given weight of dry Lactobacillus bulgaricus cells was a more reliable test of success than was the viable cell count. Volz and Gortner (1948) found that if lyophilized preparations of L. casei were used directly after reconstitution with saline or basal media, the acid production was lower than if the culture were transferred a few times before assaying for acid production. On the other hand, L. plantarum could be used either directly or as a transfer. They studied vitamin requirements for acid production but made no viable cell counts, Nei (1960) studied the effect of freezing and lyophilization on biochemical activity by measuring respiration rates. Although he did only a limited amount of work with lyophilized cultures he showed that the respiration rate of E . coli was not indicative of the number of viable cells after freezing and thawing. However, with Bacillus megaterium, a progressive decrease in respiration rate and viability on successive freezethaw cycles was noted. Januszewicz ( 1957) resorted to lyophilization because she observed a loss of dextran production by L. mesenteroides during storage on laboratory media. Although the growth requirements of the lyophilized cultures differed from the original, the yield of dextran of most strains was higher than before lyophilization. Januszewicz attributed this to the action of lyophilization as a selective factor, eliminating cells with low metabolic activity and low power of dextran production. Wasserman and Hopkins (1958)studied the oxidation of glucose and 2-ketogluconate by lyophilized and reconstituted preparations of S. marcescens. They found a general correlation between the number of viable cells, or percentage survival, and the rate of oxygen consumption, but under certain conditions of lyophilization and storage marked changes

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were produced in the enzyme systems of S. murcescens as indicated by the oxidative patterns. For instance, the action of arsenite, in concentrations which are not harmful to normal cells, sharply restricted the enzymatic activities of these cells. Verwey and Matt ( 1950) concluded that lyophilization produced no physiological damage of brucella vaccine since they showed that after reconstitution the culture could be stored at 2 O to 5OC. for 36 to 90 days with little or no loss of viability. This test is not necessarily critical because storage alone may permit restoration of the resistance of organisms to other adverse effects, as shown by Heckly et al. ( 1958). Serological specificity is another criterion that has been considered. Stillman (1941) reported that pneumococcus underwent no change in type specificity on lyophilization. Swift ( 1937), using relatively poor lyophilizing techniques, reported that bacteria maintained their original cultural, immunological, and biochemical characteristics for many years. Flosdorf and Kimball (1940) reported the maintenance of cultures of Bordetella pertusis in phase I after lyophilization and storage for 2 years at 5 O to 8OC.

3. Znf ectivity and Antigenicity It is well known that laboratory strains of pathogens which are repeatedly transferred without animal passage frequently show decreased virulence. Similarly, as will be discussed in Section VII, B, the virulence of lyophilized cultures also may change if the environment in lyophilized preparations is more favorable for one or another variant. There are, however, a number of reports in the literature, such as one by Swift (1937), in which a general statement is made that bacteria maintained their virulence as well as their original immunological and biochemical characteristics for many years in the dried state. Others, Stamp (1947), Stillman (1941), and Stockton and Newman (1950), state that virulence was unchanged or unaltered even though their techniques were possibly too crude to detect anything less than a 1,000-fold change. Velu et al. ( 1942) reported that the virulence of lyophilized Actinobacillus mallei was maintained for up to 8 months. Some cultures, however, were rendered avirulent after 10 to 25 months’ storage at lo to 5OC. Stein et al. (1949) showed in a crude way that cultures of Pasteurella bubaliseptica (author’s designation) obtained from lyophilized material were more virulent than the same cultures carried for 3 years in broth (one horse used for assay); 16 ml. of the broth culture failed to kill whereas 10 ml. from the lyophilized culture killed the horse. Recently Stein (1954) stated that cultures obtained from a preparation 13 years after lyophilization were still pathogenic. Similarly, Schoening et al.

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(1949) stated that the pathogenicity of Salmonella choleraesuis was preserved for at least 10 years. But in his text he listed 5 years as the longest period without subculture. However, neither Stein nor Schoening indicated the number of organisms administered but merely stated the volume of culture inoculated. It would perhaps have been more meaningful if they had included the dosage in terms of number of viable cells. Similarly Stamp (1947) studied a wide range of species preserved for 4 years and reported that virulence was maintained. Appleman and Sears (1946) lyophilized cultures of Rhizobium leguminosarum and showed that dried bacteria retained completely both viability and capacity to nodulate plant hosts and to fix nitrogen after 4 years’ storage. However, they reported no viable cell counts. The bacteria tested were isolated from a number of sources such as alfalfa, lespedeza, cowpea, pea, and clover. In all the above studies subcultures were tested rather than reconstituted cultures. That the use of reconstituted cultures, without allowing the organisms to metabolize, may be an important aspect in the use of virulence for assessing lyophilized cultures is born out by some work by Heckly et al. (1958). They found that immediately after reconstitution of P . pestis cultures, which had been lyophilized and stored for 9 months in rubber-stoppered bottles, as high as 8 x lo4 viable cells represented an LDSo,but in all instances, a single %-hour subculture in the ordinary culture medium restored the culture to its original virulence of 10 to 100 cells per LD,,. It is significant that those cultures which exhibited the marked loss in virulence suffered negligible loss of viability during the storage period. Apparently the lyophilized cells suffered some damage during storage and were unable to overcome the natural host defenses to produce an infection when inoculated into animals but yet were able to grow on nutrient medium. This may be another instance of metabolic injury comparable to that described by Straka and Stokes (1959). A change in virulence was characteristic of all three virulent strains studied and was demonstrated in guinea pigs as well as mice. The prevalence of this phenomenon is not easily estimated because most of those who studied the virulence of lyophilized cultures (Swift, 1937; Stein et al., 1949; Stein, 1954; Schoening et al., 1949) used subcultures obtained from the lyophilized preparation, rather than from the reconstituted cultures. Frobisher et al. (1949) lyophilized a number of acid-fast bacilli and tested these by inoculation of the reconstituted preparation directly into guinea pigs without subculture and concluded that all strains remained normal after 17 years, Unfortunately his results were presented only in terms of growth or no growth and he noted only whether cultures were virulent or not, without quantitation.

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Studies on lyophilized P s e u d o m m pseudomallei ( Heckly, 1957) indicate that cells remaining viable after drying and storage are fully virulent. Cultures were lyophilized and stored under conditions so adverse that the number of viable cells, when plated on a complete glycerin beef extract medium, was reduced from 1Olo to about lo6yet the number of viable cells constituting an LD,, was essentially the same as that before lyophilization. Organisms used for live vaccines, such as those for tuberculosis and brucellosis, are inoculated immediately after reconstitution without subculture and therefore the quality of the preparation can be assessed in terms of antigenicity, or protection, as well as the number of viable cells. Investigators in all parts of the world have worked on the problem of preserving BCG by lyophilization. Some, Kurylowicz et al. (1958) and Miller and Goodner ( 1953), evaluated their lyophilization and storage procedures primarily by measuring viability; apparently they assumed that if a cell is viable it has retained its immunogenical power. This may be a valid assumption but the majority of those concerned with the preparation of dry BCG have assessed their products in terms of ability to induce a tuberculin reaction and/or establish immunity. Ungar (1949) stated that his lyophilized cultures of BCG remained unaltered for at least 12 months and that they seemed to maintain their antigenic properties as indicated by tuberculin sensitivity and by inducing resistance in animals to subsequent infection with a virulent strain. More recently Birkhaug ( 1951), Rosenthal ( 1952b), and van Deinse (1951) showed that the rate of conversion to tuberculin reactivity was slower when inoculation was with lyophilized preparations than with fresh cultures. Birkhaug ( 1951) believed this delayed conversion was a result of a decreased number of viable cells since the lag in producing the conversion by dried bacilli could be eliminated by using 4 times the amount of cells on a weight basis. However, only 14%of the cells were viable. B@e and Evjen (1952) believed that full potency of a BCG vaccine could be retained even though only some of the cells in the dried preparation were viable. They reported that a certain number of viable cells were needed but beyond this minimal number, which they did not define, the numbers of viable cells could be greatly varied without influencing the effectiveness of the vaccine. If this is true then assaying for antigenic effect would be the most desirable method for determining the quality of the lyophilization and storage conditions. However, since B@eand Evjen failed to indicate the intervals between inoculation and subsequent conversion to tuberculin positive their conclusions should be re-evaluated, particularly in light of Birkhaug’s findings concerning

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the relationship between the rate of conversion and number of viable cells inoculated. The observations of Birkhaug and B@eand Evjen are not necessarily in disagreement since a large number of dead cells can interfere temporarily with the normal host defenses; thus even small numbers of viable cells can produce tuberculin sensitivity if given enough time. Reactions of guinea pigs inoculated with lyophilized preparations (van Deinse, 1951) lagged 10 days behind reactions noted in guinea pigs vaccinated with fresh BCG. The experiments showed that after 6.5 months the resistance to tuberculosis of those animals immunized with dried cells was equivalent to the reaction of animals immunized with fresh vaccine. Likewise the data presented by Leschinskaya (1946) showed that a group of guinea pigs inoculated with fresh BCG were more tuberculin reactive than those inoculated with dry culture that had been stored 18 months; yet on challenge both groups of animals were protected equally well. In view of the above observations it is obvious that although an assay based on production of tuberculin sensitivity is related to the practical objective it may not be sufficiently sensitive to detect a loss of activity until practically all cells are dead; in this way it is comparable to the "growth or no growth" method of assay, However, Geser and Azuma (1960), using allergenic potency as an index, showed a difference between preparations stored at 50° and 3OOC.; 3OOC. was best even though there was still considerable potency in the preparation stored at 5OOC. Infectivity is, of course, the only method available for demonstrating viability of viruses and rickettsia, whether it be by the inoculation of either susceptible hosts or tissue cultures of suitable cell lines. 4. Morphological Changes Although it is well known that repeated freezing and thawing disrupts many organisms the number of cells disrupted by a single freezing, drying, and reconstitution is so slight that observation of a lack of change in morphology is a poor index of how well lyophilization succeeded. For one thing bacteria are so small that details of structure can only be demonstrated by electron microscopy and the details may be distorted by the fact that organisms must be dry to be observed. Nei (1960) in his attempts to show morphological changes as a result of freezing (so that he could examine frozen bacterial cells without drying) mentions an unsuccessful attempt to utilize a refrigerated specimen holder in an electron microscope. The illustrations he presented of E. coli and B. megnterium showing changes as a result of lyophilization are not very convincing. However he offers convincing proof that the size of tobacco mosaic virus was reduced, particularly in a purified preparation, After

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the preparation was dried there was a large increase in the number of particles less than 120 mp and a reduction of the 240- to 360-mp particles but only a slight reduction in infectivity.

V. Comparison of Organisms Though many bacterial species have been preserved by lyophilization relatively few have been studied in sufficient detail to provide a basis for selecting conditions for optimal survival. Some of the suspending fluids that have been found to be satisfactory for lyophilizing a few organisms are listed in Table I. Survival values (as percentages of the original culture before lyophilization, wherever possible) are given as a guide to indicate what may be expected of lyophilized organisms in the group. Apparent discrepancies in the table are largely due to the effect of factors such as age of the culture, drying technique, moisture content, method of rehydration, etc., which are not indicated in the table. References were listed largely to provide a selected bibliography on the lyophilization and storage of a particular organism, or of groups of organisms. In addition to those listed, significant papers such as Stamp (1947), Proom and Hemmons (1949), Rhodes and Fisher (1950), Rhoades (1958), and Schmidt et al. ( 1958) should be consulted, since each of these was concerned with the effects of lyophilization and storage of most of the organisms given in Table I. Engley (1956) collected similar information on the persistence, under various conditions, of practically every known microorganism. He did not list the conditions of lyophilization but his extensive tables, 56 pages long, include data on viability and the references he cited might be helpful.

VI. Safety Aspects A. HAZARDS OF LYOPHILIZATION The fact that there is a hazard involved in the lyophilization of bacteria has been amply demonstrated. Cowan (1951), Swift ( 1937), Campbell and Pressman (1944), Stein and Rogers (1950), Reitman et al. (1945a, b), Busby (1959) and others have shown that without special precautions bacteria and viruses may be carried out of the lyophilizing ampule, presumably by the water vapor. If one considers the factors involved in lyophilization one can perhaps better appreciate the hazard of lyophilizing pathogens and how to minimize the risk involved.

I, Efect of Suspending Fluid The number of nonvolatile particles carried out of the ampule or bottle by the water vapor is dependent largely upon the nature of the suspend-

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ROBERT J. HECKLY

ing fluid. It seems obvious that if bacteria are suspended in distilled water before freezing there is little or nothing to hold them in place when the surrounding ice is removed, and the slightest “breeze” of water vapor would carry them out of the vessel. However, under conditions which favor the typical “biscuit” appearance of the dried culture, such as is obtained by drying skim milk or serum, a stable network is formed which effectively holds most of the particulate matter in position. Cowan ( 1951), using a fluorescent tracer (zinc oxine) demonstrated that there was more contamination of the drying apparatus when the tracer was suspended in water than when it was suspended in serum. In fact, he reported that occasionally no contamination could be detected after lyophilization of serum. 2. Efect of Rate of Lyophilization Apparently, the effect of drying rate on the dispersion of particles has not been studied, but it is apparent to anyone considering the problem that this is an important factor. Since drying begins at the exposed surface, a layer of dried material soon forms over the ice and all the water from the underlying ice must pass through the dried layer. Hence, at high velocity, the vapors would be more likely to dislodge particles and to carry them out of the ampule than at low velocities. The amount (weight) of water per square centimeter may not be large but at reduced pressure the volume of water vapor will be quite large. Because the temperature of the vapor varies little, the volume is largely a function of pressure. If one is drying a bacterial suspension at about -18OC. the pressure would be approximately 1 mm. Hg. At this pressure, about 470 liters of vapor would be evolved per square centimeter of surface assuming a thickness of 5 mm. Considering that such large volumes of vapor are moving through the uppermost layer of the dried preparation, often within a few hours, it is not surprising that some particles are carried along with the water vapor. Decreasing the drying rate would probably reduce the number of organisms carried out of the container, but this is not compatible with requirements for maximal preservation of the organisms. The rate of sublimation is governed by the heat input and the ice temperature is essentially dependent on the total pressure above the ice; thus, the rate can be effectively decreased by either reducing the rate of heat input, such as insulating or cooling the ampule, or by increasing the pressure (decreasing the vacuum) by means of a controlled leak into the system.

3. Effect of Container Size and shape of the container are attributes which ought to be considered, because the geometry of the vessel determines the thickness of

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the frozen layer. It is obvious that with thin ice layers less vapor need pass through overlying portions and consequently the chance of particles being dislodged and entrained is reduced. Cowan (1951), using the centrifugal freeze-drying technique of Greaves ( 1944) which freezes the sample as a wedge near the bottom of the tube, found that with a given container contamination of the lyophilization apparatus was a function of the volume of material in the ampule. Shell freezing, which should also reduce the loss of particulate material, has not been studied. No reports were noted which considered the effect of the container or method of freezing on the potential hazard of lyophilization. 4. Use of Filters At present the use of filters is the most effective method of preventing the escape of microorganisms. Campbell and Pressman (1944) stated that gauze placed over the opening of the flasks effectively prevented escape of material while dilute solutions were being dried. Others such as Cowan ( 1951, 1953), Annear ( 1956b), Scott ( 1958), the American Type Culture Collection (Catalog of Cultures, 1958), and the National Collection of Type Cultures (Rhodes and Fisher, 1950) employ a loose cotton plug inserted into each ampule or bottle; such filters seriousIy impede the flow of water vapor and viable cell counts of replicates may be extremely variable because of differences in the fit of the cotton plug. A filter inserted between the condenser and the vacuum pump is perhaps the best method for confining the organisms. Placed in this position the filter does not impede the flow of water vapor. The effective pumping speed of the pump is reduced but this can usually be tolerated, and, by measuring pressure in the condenser rather than on the pump side of the filter, comparable results from one experiment to the next are obtained. Furthermore, most of the organisms become trapped in the condenser which ensures a fair margin of safety. A particular disadvantage of a filter so placed is that additional precautions must be taken (Reitman et nl., 1954a) because manifolds, connectors, etc., are probably contaminated and the entire apparatus must be decontaminated before being disassembled. The use of individually plugged ampules is essential if one wishes to dry a variety of organisms simultaneously without a risk of cross contamination. Cowan (1951) reported that a considerable amount of cross contamination occurred when a number of cultures were processed simultaneously without cotton plugs.

B. HAZARDS OF OPENING AMPULES The opening of ampules containing lyophilized pathogens can be extremely hazardous, especially if they are sealed under vacuum. If an

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evacuated ampule is opened rapidly, there is a sudden inrush of air followed by an almost equally violent outrush carrying a large portion of the contents out of the ampule as an aerosol. Reitman et al. (1954b) showed that the amount of aerosol produced depended on the nature of the lyophilized product. Cultures suspended in serum before lyophilization produced the least aerosol when the ampule was opened. Although the numbers of organisms dispersed were reduced by adding serum to the culture before lyophilization, significant numbers were still released when the ampule was opened. The hazard encountered when ampules are opened has long been recognized and several different methods have been employed to reduce the hazard. Swift (1937) recognized the hazard and sealed his tubes by pouring wax onto the cotton plug after drying, rather than by sealing the tubes under vacuum. Proom and Hemmons (1949), Swift (1937) and others filled the tubes with nitrogen or other inert gases so that there would be no inrush of air when the seal was broken. Because some organisms survive prolonged storage best under vacuum, methods have been devised for reducing the hazard of opening evacuated ampules. Flosdorf and Kimbal (1940) wrapped a cloth soaked in antiseptic around the ampule. Recently, Reitman et al. (1954b) showed that such a procedure does indeed reduce the aerosol production. However, it is obvious that some disinfectant may be sucked into the culture by such a procedure. A procedure recommended by Cowan (1953) and Fry (1954), and one now commonly accepted, is to mark the neck of the ampule with a file and to then touch a piece of molten glass to the mark. If successful, a small crack forms which allows air to enter slowly until atmospheric pressure is reached; afterward the tip can be lifted off safely without producing an aerosol. Because Pyrex glass is rather difficult to crack by this procedure Cowan (1951) opposes its use. On the other hand, soft glass tubes have been known to develop cracks several days after sealing in cases where annealing was inadequate. A less commonly used procedure, but one which can be applied easily to Pyrex glass, is to make a small hole in the neck of the ampule with a pointed tungsten wire heated in oxygen-gas flame to a white heat. Obviously, care must be exercised to avoid heating the culture, Once mastered, this technique is effective and rapid. After the vacuum is released, the ampule is allowed to cool and it can then be opened by scoring with a file and breaking the tip in the usual manner. Another technique which is simpler than the above uses Scotch tape to prevent the inrush of air. After a file mark is made on the neck of the ampule, a wide piece of tape is wrapped around the file mark, When

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the neck is broken by the application of pressure opposite the mark, the tape prevents the rapid inrush of air and, furthermore, contamination of the culture was minimized because small bits of glass from the outer portion of the tube are held by the adhesive instead of falling into the ampule. Mechanical aids, such as heavy rubber balls over the bulb or rubber tubing on the smaller ampules, are recommended in all operations involving the application of pressure on glass to prevent injury from accidental breakage. All operations with pathogens should be performed in a bacteriological hood. An almost foolproof system employs an airtight metal container containing a steel ball and a measured volume of fluid used for reconstitution. After sterilizing the outside of the ampule, it is inserted into the sterilized container. Shaking the unit shatters the ampule and the culture is automatically reconstituted, After a suitable interval (to allow the aerosol within the container to settle) a small port is opened and the culture removed by pipetting. The double container system described by Weiss (1957) and employed by both the American Type Culture Collection and the National Collection of Type Cultures is worth considering for routine lyophilization because of its convenience and safety. A small inner tube containing the culture is plugged with cotton so that the outer tube can be opened by any convenient method. The culture can be safely stored under vacuum, and, by enclosing the label inside the outer container, the culture is permanently identified. Safety aspects of the ancillary operations, such as preparing the culture, making viable cell counts, and filling the ampules are considered by Chatigny (see Chapter 4) and will not be discussed here. It may be of interest that no recorded infections directly attributable to the process of lyophilization were found. Cowan (1951) described a case of brucellosis in one of two workers lyophilizing B . melitensis but the source of the infection was not determined.

VII. Discussion A. Loss

OF

VIABILITY

1 . Death Rate The fact that a plot of time of storage versus the number of viable cells remaining in a lyophilized preparation frequently approximates a straight line on semilog paper has led most workers to regard the death phenomenon as a rate mechanism strictly comparable to a monomolecular chemical reaction. However, in many instances the data points deviate

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significantly from a straight line. For example in Fig. 5, the points for the first 40 months of storage at room temperature exhibited a uniform logarithmic decay in number of viable cells, but after 40 months there was only a slight additional loss. If one examines the published data on viability of lyophilized preparations it seems that a straight-line logarithmic decay may be a coincidence or is obtained only during a certain period. For instance, in most of the experiments cited by Scott (1958) it seemed that only a curved line would fit all the data of each experiment. In view of these and similar observations the usual concept of rate, as applied to death of bacteria, may not be the only or even the most appropriate interpretation of the data. In an attempt to determine whether death was a matter of chance Fry and Greaves (1951) subjected a paracolon bacillus culture to repeated lyophilization and reconstitution “without allowing for further growth.” They concluded that the survivors were no more resistant than those that died, because approximately the same percentage of cells survived each cycle of lyophilization and reconstitution. However, it seems improbable that all cells in any given culture would have the same resistance because so many factors, including age of the culture, have been shown to influence the percentage of cells surviving lyophilization. Therefore the data of Fry and Greaves logically could be interpreted to show that the population was composed of cells having various degrees of resistance; most of the cells not being able to survive a single processing, others able to survive one but not two cycles, etc. If differences in resistance of a culture as a function of age is related to the physiological age of the individual cells, that is, the age after cell division, the use of synchronous cultures may be a profitable method of studying the problems involving “death rate” of lyophilized cultures. 2. Cause of Death

A variety of mutually exclusive theories have been provided to explain why bacteria die during lyophilization. Each of the procedures, freezing, drying, and storing in the dry state, have been shown to cause cell damage or death. Freezing is similar to drying in that in both cases the amount of water available to the cell is reduced. However, substances which protect cells against freezing do not necessarily protect them against desiccation. The answer may lie not in a basic, or underlying mechanism of protection but may be related to specific molecular properties of the agent. Glycerol, for instance, is a liquid and it follows that materials dried in the presence of such compounds may not become solids; other compounds incorporate water into a tightly bound crystalline structure.

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It seems fairly well established that colloids, as well as crystalline and some low-molecular-weight substances, appear to protect cells against the lethal or damaging effect of drying and they also protect these cells during storage in the dried state. Several theories have been proposed to account for these phenomena. For example, Fry and Greaves (1951) believe that glucose exerts its effect because it retains moisture at an “optimal” level. Scott (1960), on the other hand, feels that reactions between carbonyl compounds and amino groups may be involved. Evidence presented by Haines (1938) and Mazur et al. (1957a, b ) argues against mechanical injury, by the formation of ice crystals, as a cause of death. Studies by Herrera et a2. (1956) on yeast convinced them that loss of viability was not due to rupture since free nucleic acids were not found when the yeast was rehydrated with water at 4.5OC. despite the fact that a large number of cells had died and various other constituents, such as protein, phosphorus, carbohydrate, and ash, had leaked out of the cells. Furthermore they concluded that some of the extracellular nitrogen must have been contributed by cells which were subsequently shown to be viable. Biochemical or metabolic injury, as suggested by Straka and Stokes (1959), is an intriguing, and apparently valid, explanation of the observed facts but this concept raises another question. Not only are we faced with finding the cause, or causes, of death, but also with defining when the organism is truly dead, Straka and Stokes found that immediately after freezing, 63% of a culture of Pseudomonus ovalis grew on the minimal medium and 78% grew when plated on a richer or more complete medium; their interpretation being that 15%were injured and 22%killed. After 19 days’ storage of the same culture they showed that 18%were injured and 53%were killed. It is not improbable that 100%survival might be obtained consistently if a suitable medium were discovered. Perhaps none of the cells were killed by the procedure, but were merely unable to reproduce under the conditions of testing; certainly we do not consider higher plants and animals to be dead just because they are no longer able to reproduce. In terms of maintenance of a culture an organism may be dead, but if one measured other activities associated with living cells the picture might be considerably different. Many investigators have assumed that if one obtains a high survival of cells when they are assayed immediately after drying one is assured of good storage. Verwey (1945) did find a correlation between protection against drying and subsequent survival on storage, although this is contrary to some results on P . pestis reported by Heckly et al. (1958). They found essentially no difference between the percentage of P. pestis surviving lyophilization in a menstruum containing either glucose, lactose,

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or sucrose 1 month after lyophilization, but when tested 7 months after drying they found that less than 1000 organisms were present in both the control and in the preparation containing glucose, whereas lo7 cells remained in those preparations to which either sucrose or lactose had been added. Are dried bacteria truly dormant? Few facts are known, but some observations of Maister et al. (1958a) would indicate that at least partial metabolic functions are continuing in dried cultures, They described what may be considered to be a reactivation in the dry state since an increase in viable cell count was observed after an initial decrease in viability. After 12 months of storage at 5OC. the number of viable cells was restored to that observed immediately after drying. Of course other explanations are possible, For example, a change in assay efficiency, due to disaggregation of clumps of organisms or other uncontrolled variation in the assay procedures, might have raised the count. Some preliminary studies by Dimmick (1958) on the respiration of dry S. marcescens are also of interest. By using microrespirometers, with solid sodium hydroxide to absorb carbon dioxide and moisture, a definite and progressive gas uptake by dry S. murcescens was observed during a 6 weeks’ observation period at room temperature. Some of the slow rehydration studies particularly with yeast (Mitchell and Enright, 1957) also indicated that reactions may occur in the virtual absence of water. Results of Wasserman and Hopkins (1958) lend support to the idea that an imbalance in the cell’s enzyme system is a cause of death. They showed that lyophilization and storage affected the glucose oxidation enzyme in one manner and the 2-ketogluconate oxidation enzymes in another. Since differences were shown between these two enzymes it seems logical to assume that other, perhaps more vital, enzyme systems were affected. Sharp (1957)) Anglemier et al. ( 1960) and others have shown that chemical changes can occur in the absence of oxygen. Anglemier showed that lyophilized ham underwent a number of changes in addition to the development of off flavors, Therefore it is not surprising that changes in enzyme activity were demonstrated by Wasserman and Hopkins (1958). This does not answer the basic question, it only changes it to one of asking what causes inactivation of particular enzymes. Rahn and Schroeder (1941) discussed the death of bacteria at considerable length from the point of view of enzyme inactivation as the cause of death. Since there are many enzymes in bacteria, one would expect death only when all or most of a vital enzyme system were lost. As pointed ont by Rahn and Schroeder, if this were the case one would expect no deaths until a certain time and then a very rapid loss of viability. Their observations, however, were such that the decrease in

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number of viable cells appeared to follow monomolecular kinetics. Therefore, they concluded that it was far more probable that a very rare molecule or gene or other equally important entity of the cells was affected to produce death, rather than enzyme inactivation. OF KEEPINGLOSSES TO A MINIMUM B. IMPORTANCE

Most workers try to obtain the highest viability so that they are more certain of being able to recover the culture after prolonged storage periods. Because such large numbers are usually lyophilized and only a few viable cells are required to start a culture this is not an exacting requirement. Conversely, those who prepare live vaccines, such as BCG or B . abortus strain 19, are interested in keeping the loss of viability to an absolute minimum, since it is generally agreed that the efficacy is dependent on the live cells. A most important reason for keeping losses to a minimum which also has been mentioned by Fry (1954) is that if the majority of the bacteria die it is difficult to understand why there might not be some selection. Hence the recovered subculture may not be the same, genetically speaking, as the original culture. Because mutants are continually produced in every living culture most lyophilized preparations undoubtedly contain a few mutants, particularly if the mutants are more resistant than the parent culture, Few examples were found in which the recovered organisms were considered to be mutants of the original culture. Januszewicz ( 1957) observed that dextran production of Leuconostoc mesenteroides after lyophilization and storage for 51 months was higher than before lyophilization. The author concluded that the treatment was a selective factor, eliminating cells with a low metabolic activity which produced little or no dextran. Velu et al. (1942) noted marked niorphological changes after 21 months’ storage of lyophilized Actinobacillus mallei. Long filamentous forms became predominant; even after several passages on agar these filamentous forms persisted. Atkin et al. (1949) showed that some isolates obtained from lyophilized brewer’s yeast were distinctly different from the parent culture in their growth factor requirements; presumably as a result of selection since the percentage of organisms surviving was as low as 0.02%.In most instances growth of the parent culture exceeded that of the isolates in the various deficient media. Some observations on the effect of lyophilization on mixed cultures provide additional evidence that the properties of stock cultures could be changed by selection. Greaves (1960b) described an unsuccessful attempt to lyophilize a mixed culture in which he hoped the proportions of organisms would remain constant. He found, however, that after lyophilization the proportions had changed completely. Similarly, Leach

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and Scott (1959) lyophilized a mixture of S. marcexens and V. metschnikouii. They did not indicate the relative proportions before drying but reported the results in terms of change in population when rehydrated at 37OC. versus rehydration at 0%. Rehydration at 37OC. decreased the V . rnetschnikovii by 80 to 85% and increased the population of S. marcexens by about 301%.Appleman and Sears (1946) mentioned that they obtained cultures from each of their lyophilized Rhizobium legurninosarum preparations but one of the cultures failed to show nodulation. An examination of the culture showed that it was a sporeforming rod, not R. leguminosarum. Apparently all or most of the R. legurninosarum cells had died and only the sporeforming rod, which they subsequently found also to be in the original agar stock culture, survived the prolonged storage. A similar observation was made at the Naval Biological Laboratory. In one preparation of P . pestis a few mold contaminants were observed but since they represented less than 0.01%of the total number of viable cells they presented no problem immediately after lyophilization. However, after several years’ storage under rather unfavorable conditions the mold became the predominant organism. Therefore, although lyophilization may not be mutagenic the process could alter a stock culture by selecting a resistant population.

C. A SUGGESTED PROCEDURE It would be most helpful if it were possible to summarize or reconcile all the work on lyophilization and arrive at “the best lyophilization procedure” but, at present, there does not seem to be a single method which will consistently preserve all species ideally. Although it may seem presumptuous to make specific recommendations for general application a suggested procedure, which should yield acceptable results, will be outlined for the benefit of those who may not care to make lyophilization of bacteria a major project. Cultures may be grown in the usual liquid or solid medium. Mature, well-nourished, and well-aerated cultures should be used. Agar-grown cultures may be suspended in sterile skim milk or serum (if it is not bactericidal). A 3 to 6% sucrose solution may be substituted for the milk or serum if, for any reason, foreign proteins are to be avoided, Liquidgrown cultures may be added to an equal volume of sterile skim milk, serum, or 6% sucrose solution. The capacity of the ampule should be about 10 times the volume of the final suspension. In 2-ml. ampules 0.1 to 0.2 ml. per ampule generally can be processed satisfactorily. If contamination is to be prevented thc ampule should be stoppered by a loose cotton plug. Material may be snap-frozen after attachment to a manifold type of

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apparatus or it may be pre-frozen by slowly cooling the ampule to -lO°C. If snap-freezing is applied the pressure must be reduced slowly so that the cell suspension is degassed and slowly cooled to its freezing point. Apparatus design is not critical but the condenser should be adequately cooled to permit a final operating pressure of not less than 150 /A Hg, and the temperature of the culture as it is being dried should be between -loo and --20°C. Although the cultures may dry in less than 4 hours it is advisable to keep the ampules on the unit at room temperature for at least 6 hours; overnight drying is most satisfactory. The ampules should be sealed under vacuum and stored at Oo to 4OC., although room temperature storage may be satisfactory in many instances. The number of viable organisms at each step, before drying, after drying, and occasionally during storage, should be determined to assure that the procedures employed were appropriate.

D. FUTURE OF LYOPHILIZATION In many instances lyophilization may be the preferred method but other methods, such as preservation at liquid nitrogen temperatures, may prove to be superior to lyophilization for the preservation of stock cultures. However, as the reliability of preserving viability in lyophilized cultures is increased there will probably be an increased use of lyophilized organisms to be used directly as a reagent, as in microbiological assays. The empirical approach (trial and error method) has been successful in developing methods suited to the preservation of specific organisms but there are many basic questions which need to be answered, such as: Does death during drying relate to death during storage, and, do additives act only to regulate water content? These questions cannot be answered until sufEcient data become available to permit a resolution of the basic mechanisms involved. If sufficient effort is directed toward fundamental studies on the effects of various manipulations on a variety of organisms it may be possible to develop an understanding of those mechanisms directly responsible for death. Perhaps studies involving solid-state reactions will contribute to an understanding of those changes in dry preparations which result in death. It is generally assumed that without water metabolism is absent, but if the organisms are found to have metabolic activity in the dried state, as mentioned before, our concepts of how to preserve lyophilized cultures will have to be radically changed. ACKNOWLEDGMENT This work was sponsored by the Office of Naval Research under a contract with the Regents of the University of California. Reproduction in whole or in part is permitted for any purpose of the United States Government.

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Stamp, T. C. (1947). J . Gen. Microblol. 1, 251-265. Stein, C. D. (1954). Vet. Med. 49,469472. Stein, C . D., and Rogers, H. (1950). Am. J . Vet. Research 11,339-344. Stein, C. D., Mott, L. O., and Gates, D. W. (1949). Vet. Med. 44,338339. Stephenson, J. L. (1960a). Ann. N. Y. Acad. Sci. 85,535-540, Stephenson, J. L. (1960b). In “Recent Research in Freezing and Drying” (A. S. Parkes and A. U. Smith, eds.), pp. 121-145. C. C Thomas, Springfield, Illinois. Stephenson, J. L., Smith, G. W., and Trantham, H. V. (1957). Reu. Sci. Instr. 28, 381. Stillman, E. G. (1941). J. Bucteriol. 42, 689-693. Stillman, J. W. (1951). A w l . Chem. 23, 1058-1080. Stockton, J. J,, and Newman, J. P. ( 1950) Cornell Vet. 40,377-379. Straka, R. P., and Stokes, J. L. (1957). Appl. Microbiol. 5,21-25. Straka, R. P., and Stokes, J. L. (1959). J . Bucteriol. 78, 181-185. Strumia, M. M., McGraw, J. J., and Reichel, J. (1941). Am. 1. Clin. Pathol. 11, 480-496. Stull, J. W., and Herreid, E. (1949). J . Dairy Sci. 32,972-976. Swift, H. F. (1921). J . Erptl. Med. 33, 69-75. Swift, H. F. (1937). J. Bucteriol. 33,411-421. Tanguay, A. E. (1959). Appl. Microbiol. 7, 84-88. Thornton, H. G. (1954). “A Discussion on the Maintenance of Cultures by Freeze Drying.” Her Majesty’s Stationery Office, London. Thow, D. C. W. (1956). Can. J. Microbiol. 2,393-401. Topping, N. H. ( 1940). Public Health Repts. ( U.S.) 55, 545. Ungar, J. (1949). Tubercle 30, 2 4 . Ungar, J,, Farmer, P., and Muggleton, P. W. (19%). Brit. Med. J . 2, 568-571, van Deinse, F. (1951). Am. J . Public Health 41, 1209-1214, van Drimmelen, G. C. (1956). Onderstepoort J . Vet. Research 27, 215-225. van Drimmelen, G. C., and Steyn, H. S. (1959). J. Gen. Microbiol. 19, 324-329. van Rooyen, C. E., and Janes, C. (1954). J. Lab. Clin. Med. 43,489-494. Velu, H., Pigoury, L., and Courtade, R. (1942). Compt. rend. soc. biol. 136, 775-776. Venvey, W. F. (1945). Proc. 48th Ann. Meeting U. S . Livestock Sanit. Assoc. 1944, pp. 68-73. Venvey, W. F., and Matt, C. (1950). J . Am. Vet. Med. Assoc. 116, 296-297. Venvey, W. F., and Scheidy, S. F. (1946). I. Am. Vet. Med. Assoc. 59,362-365. Volz, F. E., and Gortner, W. A. (1948). Arch. Biochem. 17,141-148. Wachter, R. F., and Gilman, E. N. ( 1960). Bacteriol. Proc. ( SOC. Am. Bacteriologtsts) p. 113. Warren, J., Kugler, H., and Hall, H. M. ( 1951). J. Immunol. 67, 163-186. Wasserman, A. E., and Hopkins, W. J. (1957). Appl. Microbid. 5, 295-300. Wasserman, A. E., and Hopkins, W. J. (1958). Appl. Microbiol. 6,49-52. Webb, S. J. (1980). Can. J . Microbid. 6, 89-105. Weiser, R. S., and Hennum, L. A. (1947). J. Bactertol. 54,17-18. Weiss, F. A. ( 1957). In “Manual of Microbiological Methods” (H. J. Conn, ed.), PP. 101-104. McGraw-Hill, New York. Wickcrham, L. J., and Flickinger, M. H. (1946). Brewers Digest 21, 55-59, Willits, C. 0. ( 1951) . Anal. Chem. 23, 1058-1062. Wyckoff, W. G., and Lagsdin, J. B. ( 1944). Am. J. CUn. Pathol. Tech. Sec. 8, 10-16. I

Sphaerotilus, Its Nature and Economic Significance’ NORMAN C . DONDERO Department of Sanitation, Rutgers University, New Brunswick, New Jersey I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Technological Problems

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A. Orientation and Nomencl B. Infestation of Streams . . . . . . . . . . . . . . . . . . C. Pipe Clogging and Corro D. Bulking of Activated Sludge ..................... E. Miscellaneous Problems . . . . . . . . . 111. Identification and Taxonomy A. Stream Slimes, Gross and Microscopic Examination; Activated Sludge, Microscopic Identification ................................... B. Cultivation and Isolation .................... C. Cultural Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Morphological Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Taxonomic Considerations ................... IV. Causes of and Factors Affecting Stream Infestat A. Significance of Studies with Artificial Channels . .......... B. Factors Affecting Stream Infestations . . . . . . C. The Lower Columbia River . . . . . . . . . . . ....... V. Nutrition and Physiology of Sphaerotilus . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Organic Substances . . . . . . . . . . . . . . . . ............. B. Iron Transformations ............... .............. C. Composition and Formation of the She ............. D. Metabolic E5ciency . . . . . . . . . . . . . . . ............. E. Inhibition . . . . . . . . . . ................................. VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... References ........................... .....................

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1. Introduction The sheathed bacteria of the genus Sphaerotilus have been of interest and concern to industries, commercial and sport fisherman, limnologists, sanitary engineers, and others, but have received relatively little attention from bacteriologists. These bacteria proliferate abundantly in flowing waters with a production of masses of cells that absorb and entrap other materials, Adaptation of Sphaerotilus to the lotic environment rests mainly on the ability of these bacteria to form a sheath that confines cells Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers University, The State University of New Jersey, Department of Sanitation, New Brunswick, New Jersey.

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in a tubular filament and on their ability to become attached to a solid surface by adhesion or entanglement. The aggregations cause contamination of process waters, corrosion and obstruction of pipes, interference with commercial fishing, interference with waste disposal, and unsightly water courses. The correction of harm already done and the prevention of undesirable effects in the future has become an economic necessity. The literature on Sphaerotilus deals primarily with aspects of water and waste treatment, stream sanitation, and the effects of varying physical and chemical parameters on crude cultures of Sphaerotilus; the report of Lincoln and Foster (1943) on pollution in the lower Columbia River is an excellent exposition of the problems of stream pollution, Although information on the nature of Sphaerotilus itself is much less abundant, several papers cover the subject in broad scope (Harrison and Heukelekian, 1958; Pringsheim, 1949; Scheuring and Hohnl, 1956; Wurtz, 1956, 1957; Wolfe, 1958). Because the scope of the previous works is broad, it is the intent of the writer to be selective rather than exhaustive in covering the literature.

II. Technological Problems A. ORIENTATION AND NOMENCLATURE Although Sphaerotilus has been successfully cultivated in pure culture for some time (e.g., Schikora, 1899; Cataldi, 1939), microscopic identification is currently the main criterion for identification, and especially when considering natural or crude specimens and preserved material. Indeed, new species have been described from microscopic examination of plankton collected from lakes (Skuja, 1948). Microscopy is, however, sometimes subject to error, even though allowances are made for the recognized and reputed pleomorphism of Sphaerotilus. Specimens which, even with good modern microscopes, sometimes appear dubious require the added information which can be obtained from cultural methods for more certain identification. Bacillus cereus has some similar characteristics to Sphaerotilus and has sometimes been mistaken for it (Sack, 1925; Haag, 1927). Lackey (1941) reported that an organism isolated from the Columbia River possessed characteristics intermediate between those of Bacillus and Sphaerotilus. Contributing somewhat to the complexity of the literature prior to 1949 was the fact that Cladothrix and the iron bacterium, Leptothrix were not recognized as biotypes of Sphaerotilus (Pringsheim, 1949). Some of the difficulties due to “iron bacteria” can be charged against Sphaerotilus, but, since the identification of iron bacteria is more haphazard than that of Sphaerotilus, caution should be exercised. Little is known of the funda-

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mental relationships of the various iron bacteria and identification is based on rather dubious microscopic differences. The presence of rusty deposits, empty sheaths, or slimy growths has often sufficed for the implication of Leptothrix, Clonothrix, Cladothrix, Crenothrix, and others as the specific causative agents of the iron deposit in question. The term slime has often been used to signify accumulations of slippery materials. Biological slime, as distinguished from chemical slime, has been used to refer to films of microorganisms which form slippery layers on wetted surfaces of pipes and submerged objects in streams, lakes, and similar waters; the term has also been applied to the larger aggregates of Sphaerotilus, zoogleae, and fungi which occur in polluted streams. These terms will be used in the present paper, since they are convenient, are in accord with dictionary definition, and are based on the concept of the collective properties of the individual cellular capsules (which have been known as slime or slime layer), in spite of the possible criticism that such use perpetuates a nonspecific term for a specific condition and leads to a confusion of these with the capsular layer of bacteria.

B. INFESTATION OF STREAMS Upon introduction of nutritive materials, usually as wastes, into water courses, Sphaerotilus may develop to the extent that visible masses of slime appear as a woolly coating on submerged objects or as tufts and strands, sometimes 30 cm. or more long, streaming in the current from a point of attachment and varying in color from white, in fresh new growth, to dull grey-white, brown or rusty-red, depending on age, nutrition, and type and amount of solids entrapped from the passing water. At times, the tufts, or “slime blossoms” may contain a heavy gelatinous matrix. The gross appearance of Sphaerotilus infestation may be imitated to some degree by fungi, protozoa, inert fibers, or other substances. Although the productivity of polluted streams is difficult to estimate, some measurements on detached masses of floating Sphaerotilus have been made from which the amount of material passing the river cross section has been calculated: in the Danube and Main Rivers, about 64 tons, wet weight per day of drifting Sphaerotilus (Demo11 and Liebmann, 1952); in the Main 325 tons, wet weight per day (Liebmann, 1953); and in the Oker River, 12.5-100 gm. dry weight per cubic meter of water (Popp and Bahr, 1954). By using the factor 7%(Popp and Bahr, 1954; Liebmann, 1952, used S%),the wet weight values can be converted to 4.5 and 22.7 tons, dry weight, respectively. Such values indicate the potential for the deposition of large amounts of decomposing SphaerotiZus. Popp and Bahr (1954) measured deposition in layers 1 to 2 meters thick in areas of high oxygen deficiency. There was a high degree of secondary

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pollution resulting from the decomposition of the Sphaerotilus sludge for months after heavy pollution with sugar waste. The oxygen demand of dead Sphuerotilus sludge was about 11 times that of the same amount of living Sphaerotilus. Where commercial or game fishing exists drifting Sphuerotilus may foul salmon gill nets (Lackey, 1941; Lincoln and Foster, 1943; Wilson et al., 1960), interfere with fish hatching (Lincoln and Foster, 1943), and otherwise adversely affect streams by smothering eggs and aquatic fauna which serve as food for fish (Gaufin and Tarzwell, 1955). Although Sphaerotilus has been blamed for the decline of the Chinook salmon fishery in the Columbia River and for net rotting, it has been pointed out that the decline in the salmon landings may have been clue to other causes (McPherson and Gellman, 1958) since the area from which slime complaints were most prevalent accounted for only 1420% of the catch, There was no evidence that Sphaerotilus slime caused rotting of nylon gill nets, although the nets became fouled and were difficult to clean. A catch of 8.2 million pounds of Chinook salmon from the Columbia River was recorded for 1956. A similar problem of interference with gill netting operations existed in the Altamaha River in Georgia, where Sphaerotilus grew upon the waste from a cellulose manufacturing process ( Cawley, 1958). The deterioration of the aesthetic values of a stream is by no means a trivial effect, since, in addition to the offensive appearance of Sphaerotilus slime, it is an obvious sign of stream pollution which arouses the concern of the sport fisherman, the game warden, and the health officer, and often leads to the imposition of restrictive or punitive measures on the originators of the pollution along with the attendant expenses of corrective measures.

C. PIPE CLOGGING AND CORROSION Assuming, for the moment, that the iron bacteria are forms of Sphaerotilus, the source of the problems arising from the presence of these bacteria must also be considered. The slimes which develop from well waters, particularly those from deep wells which contain dissolved iron and carbon dioxide (Starkey, 1945) , may accumulate in pipes and tanks to the thickness of 2 cm. and contain Crenothrix and Leptothrix (Alexander, 1944). Nine of 19 wells in the supply system of a water company were affected by iron bacteria (Brown, 1934). The most severe trouble resulted from the precipitation and accumulation of Fe ( OH) The water was turbid and discolored and, in instances of severe infestations, was offensive in flavor and odors, probably due to the decomposition of the bacteria. Complaints of “red water” (Tenny, 1939) are a

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consequence of rust clogging which reduces the carrying capacity of pipes. Masses of rust and bacteria break off and are carried in the water to the consumer. Although most of the rust deposited in pipes is derived from iron originally dissolved in the water, iron bacteria have been implicated in the aerobic corrosion of iron pipes (Olsen and Szybalski, 1949a, b). By the reduction of oxygen tension over the restricted areas of pipe surface covered with the attached filaments of iron bacteria, electrochemical differential aeration cells are formed. These cells cause a difference of electrical potential between the more anaerobic area under the colony and the aerated free surface. The dissolution of the iron under the colony results in the formation of a tubercle composed of rust and bacterial filaments with underlying pitting of the pipe. Once corrosion has been established, the process continues practically independently of the metabolic activity of the iron bacteria. D. BULKINGOF ACTIVATED SLUDGE Separation of the biological sludge from the treated waste is an essential part of the effective biological treatment of sewage. In the activated sludge process, the separation is accomplished by settling the sludge and allowing the cleared supernatant to flow from the settling tank over a weir. Under certain conditions the sludge solids become “bulky,” develop poor settling characteristics, and pass over the weir in the effluent, thus defeating to some degree the purposes of the treatment. Sphaerotilus has been isolated repeatedly from bulky activated sludge (Ruchhoft and Watkins, 1928; Lackey and Wattie, 1940; Littman, 1940; Tkachenko and Droblyanets, 1959; Smit, 1934), but is not the sole cause of bulking. The conditions favoring Sphaerotilus bulking have been studied with small scale laboratory apparatus. Lackey and Wattie (1940) concluded that carbohydrate material caused bulking of activated sludge. A strain of Sphaerotilus was isolated and grown in the presence of a number of substances but no single one was identified as a cause of excessive growth; most of the substances tested, which included glucose and disaccharides, are not found in sewage. It was concluded that some factor in plant operation might be an important cause of bulking. In other studies (Ingols and Heukelekian, 1939; Heukelekian and Ingols, 1940) the principal causes of bulking of activated sludge were found to be ( a ) an excess of carbohydrate in relation to available nitrogen (carb0n:nitrogen ratio greater than 8:1), ( b ) an excess of nutrients in relation to activated sludge concentration, and ( c ) insufficient air. These conditions were felt to be more favorable to Sphaerotilus than to zoogleal forms, the predominant bacteria in normal activated sludge.

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Littman’s (1940) isoIate of Sphaerotilus did not grow well in sterile sewage unless carbohydrate was added, The pure Sphaerotilus sludge had a limited ability to remove turbidity and dissolved organic carbon compounds from sewage. The fact that the volume of Sphaerotilus was approximately three times that of an equal weight of zoogleal sludge provided a likely explanation of the inferior settling qualities of Sphaerotilus-bulked activated sludge in contrast to the normal zoogleal type. Ruchhoft and Kachmar (1941) concluded that Sphaerotilus was a delicate indicator of disturbances of the biological equilibrium of activated sludge but not a primary cause of bulking.

E. MJSCELLANEOUS PROBLEMS To complement the catalog of situations in which Sphaerotilus is found, the following may be mentioned: ( a ) Growths sometimes occur in sewer pipes, which break loose and descend on the treatment plant, clogging screens and choking filter nozzles and stone filter beds ( D e Martini, 1934; Skrinde, 1959). ( b ) Sphaerotilus has been found in paper machine wet felts, and clogging of the felts with Sphaerotilus was experimentally produced (Drescher, 1957). ( c ) There is little information regarding favorable properties of Sphaerotilus except for its capacity to utilize pollutants; it has been credited with lowering the ammonia concentrat’ion in polluted water to the level tolerated by fish (Schikora, 1899). ( d ) There may be several consequences of the iron bacteria in the water supply of a paper mill (Martin, 1955), i.e., objectionable discoloration of surfaces and products and interferences with water clarification and cooling of condensers.

111. Identification and Taxonomy A. STREAM SLIMES,GROSS AND MICROSCOPIC EXAMINATION; ACTIVATED SLUDGE,MICROSCOPICIDENTIFICATION The stream slime found in a Sphuerotilus infestation is composed, as would be expected, of a variety of microorganisms and has been likened to a natural community (Butcher, 1932). In streams inert material may be found in the form of silt, fibers, and chips. The slime may trail in the current as feathery, hairy masses from submerged objects, or it may become detached and float away. The filamentous masses offer shelter and support for other forms of life in polluted streams, such as other bacteria, protozoa, nematodes, rotifers, and insects. The fauna and flora may be expected to vary with conditions, especially those affecting nutrition and aeration.

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Often there is very little in the gross appearance of the Sphaerotilus slime to differentiate it from a slime formed from fungi, fibers, or other materials. It has in fact often been referred to in literature as “sewage fungus,’’ although the term also embraces some of the true fungi which may occur in polluted waters. Algal slimes are usually green unless the chlorophyll has disapperaed. The composition of the slime is readily established with the microscope. An abundance of sheaths is the striking feature of Sphaerotilus slime; many of the sheaths are often empty or show spaces between cells. The recorded dimensions of the cells which are rod shaped and vary in size, are 2 to 3 JA in diameter and 2.5 to 16 p in length (Harrison and Heukelekian, 1958). Cells retained within sheaths are aligned end to end as in a filament and may contain refractile granules or droplets. The sheaths, usually extremely thin when taken from polluted waters and so crumpled and entangled that it is impossible to estimate length, may often extend across the microscope field. Old or starved specimens may be entirely devoid of intact cells, whereas the sheaths are well preserved. Some stream slime specimens may be embedded in a rather firm matrix of mucous capsular substance. The associated bacteria are quite inconspicuous in a “predominantly” Sphaerotilus slime until an attempt is made to culture the Sphaerotilus, whereupon they appear in overwhelming numbers and variety. Since the sheaths are an essential characteristic of Sphaerotilus, they must be distinctly seen to make a diagnosis. For routine purposes, demonstration is most readily accomplished with the phase-contrast or dark-field microscope using thin wet mounts; lacking these, stains must be used with the bright field. A number of stains have been used with variable success, among them methylene blue, crystal violet, gram stain, and Ziehl-Neelsen. All stains seem to be ineffective for the sheath itself, but when there are many lysed cells or the preparation is quite dirty, stainable material is absorbed by the sheaths and they become distinctly revealed, Healthy cells fit tightly and obscure the sheath, which can be seen only where cells are missing. The sheaths of Sphaerotilus taken from streams are more visible than those from cultures, possibly because the sheaths seem to be thicker. When encapsulation is not dense, negative staining with nigrosin (Stokes, 1954) is an effective method to demonstrate sheaths for brightfield microscopy. The presence of sheaths alone in a stream slime is strongly presumptive, but not entirely conclusive, evidence of Sphaerotilus, since the sheaths may arise from other organisms such as algae, other bacteria, and protozoan residues. In massive growths there is probably not as much

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risk of error as in the examination of sparser growths obtained fromthin slimes, submerged slides, or sediments where many other organisms to some extent resemble Sphaerotilus. In activated sludge, some organisms resembling Sphaerotilus microscopically may be found regardless of the quality of the sludge. The filaments of Sphaerotilus become more numerous as the degree of bulking becomes worse. In a badly bulked sludge, the flocs are often composed mostly of Sphaerotilus; in better sludges a few filaments of Sphaerotilus project from flocs composed mostly of zoogleal bacteria and amorphous material. Because of the generally better nourished condition of activated sludge in comparison to some stream slimes, one may be on less certain ground in diagnosing Sphaerotilus in sludge unless sheaths are distinctly visible. Sporeformers, which may closely resemble Sphaerotilus, are usually present in abundance, Another common pitfall for the inexperienced and unwary is the resemblance of the sheath to the headless, empty stalks and holdfasts of the smaller stalked, ciliate protozoa, Where feasible, cultivation and isolation give more certainty to the diagnosis.

B. CULTIVATION AND ISOLATION

1. Continuous Flow Methods The effective adaptation of Sphaerotilus to the stream habitat makes possible a method of cultivation in crude cultures which are really massive enrichment cultures produced by simulating stream conditions in troughs or channels. The basic procedure is to furnish nutrients to a chamber or trough through which water flows. With sufficient velocity of flow and with proper nutrient concentration, Sphaerotilus proliferates attached to surfaces in the trough or channel. The liquid may be subject to some detention time and then passes from the chamber, carrying with it all the organisms which cannot attach to or entangle themselves with the Sphaerotilus, or maintain themselves by rapid multiplication. The last possibility can theoretically be minimized by low nutrient loading and short detention times. In such an apparatus concentration of nutrient, nutrient loading, velocity of flow, and detention time can be controlled, as well as variables which can be studied by conventional batch methods. The results can be measured in terms of weight or volume of Sphaerotilus produced or in terms of utilization of the nutrient. In experiments to determine which wastes or waste fractions from pulp mills promote the appearance of Sphaerotilus in the Columbia River, Lincoln and Foster (1943) added known amounts of wastes continuously to the Columbia River water passing through troughs. The water was taken from the river above the sources of pollution. Flow through the

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troughs and additions of wastes were maintained at constant rates. Although the troughs were not artificially seeded, Sphaerotilus appeared spontaneously in two days in troughs to which enough waste sulfite liquor was supplied to give concentrations of 100 p.p.m. or above. At the highest concentrations, flocs typical of those seen in the Columbia River appeared. In these experiments the water was obtained from the stream, the nutrients were fed from the barrels, and dependence was placed on the selective nature of the physical and chemical conditions to bring forth and maintain the desired culture. Trough experiments were carried out more extensively by Amberg and Cormack (1960) using 12 indoor channels to simulate stream conditions. Temperature was controlled by heaters, flow velocity by power-driven paddle wheels in the troughs, and waste feed by multiple-feed metering pumps. The total weight and volume of the growth produced in each of the channels was measured. Scheuring and Hohnl ( 1956) described somewhat different apparatus designed to permit recirculation as well as flowthrough. The results obtained by these methods will be discussed in subsequent sections. Many of the continuous culture vessels or continuous fermenters which have been described elsewhere could be conveniently used with little or no modification for the study of Sphaerotilm or other sessile microorganisms. In the previously cited methods, most of the Sphaerotilus remain attached in the trough or culture vessel while the liquid flows past, In principle, these are perfusion methods, similar to that of Audus (1946) except that the liquid is not completely recirculated. The ability to absorb the nutrients, albeit extremely dilute, from the passing water allows Sphaerotilm to flourish where the low concentration would not permit comparable growth in stagnant water. Continuous culture or continuous flow laboratory apparatus might very possibly be useful for studying Sphuerotilus. A comparison of some of the pertinent characteristics of the types of apparatus may be useful. In chemostats, or continuous culture apparatus ( Bryson, 1952; DeHaan and Winkler, 1955; Finn and Wilson, 1954; Moser, 1958; Novick and Szilard, 1950; Owen and Johnson, 1955; Zubrzycki and Spaulding, 1958), the attempt is made to maintain bacterial solids (cells) at a constant ratio to nutrients by adjusting the concentration of a limiting nutrient and by regulating the washout or detention time. The bacteria are carried out of the culture vessel with the overflow of medium. They appear to be more dependent on concentration and diffusion gradients (unless motile) for contact with their food than would be attached forms growing in an environment where the food is constantly renewed in the passage of liquid past the cells.

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The activated sludge process, in laboratory apparatus, as well as in full scale plants combines some of the features of the chemostat and perfusion methods. The flocs of activated sludge are carried through the tank with the sewage. The tendency to settle out of suspension is counteracted by the turbulence of aeration and mixing. The opposing forces keep water passing the floc. The sludge leaves the tank in the effluent as fresh sewage flows into it. As in the chemostat, sludge solids are maintained at a constant ratio to nutrients, not by restricting the multiplication rate of the bacteria in the tank but by returning a part of the activated sludge separated from the tank effluent. Most troughs, continuous flow, or perfusion arrangements inherently have a detention period unless they are constructed as linear channels without recirculation, The detention period is a departure from their similarity to a stream. An advantage of perfusion or channel apparatus is found in the latitude of nutrient loading permitted by varying the hydraulic load or the amount of liquid passed through. By utilizing this relationship, materials can be fed at low concentrations as nutrients which may easily be toxic at higher concentrations, Phenolic wastes, lower fatty acids, or cyanides have been biologically treated in this fashion on trickling filters. 2. Batch Enrichment Methods Preliminary enrichments before isolation have been used successfully with Sphaerotilus and Leptothrix by several investigators. The methods employed have been influenced by the biotypes desired and the observations that Sphaerotilus thrives in a natural environment low in nutrients or one which has received pollution from wastes containing carbohydrates or allied oxidizable materials. Iron or manganese seemed to be important to the economy of the organisms, and associated organisms did not thrive on the low level of organic matter. In streams and filter beds many of the associated organisms were washed away. A favorite method of enrichment has been Winogradsky’s (1888) technique, which consisted of placing some thoroughly boiled and extracted hay and ferric hydroxide in a 50-ml. cylinder and filling it with water from which enrichment was expected. After 10 days or so the filaments of Sphaerotilus or iron bacteria appeared, The method was used by Cataldi ( 1939) for Sphaerotilus, who observed both Leptothrix and Sphaerotilus in enrichment cultures and distinguished them by the absence of visible iron deposition in the sheaths of Sphaerotilus. On subculture, the Sphaerotilus developed readily, but subcultures of Leptothrix were obtained only with great difficulty. Better results with Leptothrix were obtained by the substitution of manganous carbonate for ferric hydroxide, Stokes ( 1954) obtained Sphaerotilus, omitting the ferric hydroxide.

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On several occasions the present writer tried the boiled hay method without success. The cultures were prepared with ferric hydroxide, with manganous carbonate, with both, and with neither. Preliminary enrichment was abandoned when it became apparent that Sphaerotilus could be isolated directly from unpolluted stream sediments, sewage, and activated sludge by direct plating on a suitable medium after overnight incubation, whereas the hay enrichment cultures produced neither Sphaerotilus nor Leptothrix after weeks of incubation. Collins (1952) used spring water containing a bit of iron wire in a petri dish. From this, rusty floc was transferred to two media made up of tap water and inorganic salts which supported Leptothrir. Since the media were liquid, it may be assumed that they served as secondary enrichment cultures. Lackey and Wattie (1940) produced heavy growth of Sphaerotilus by placing strands of Sphaerotilus taken from bulky activated sludge in carboys containing 0.1%sucrose which were aerated for a few days. The bottles were emptied and refilled twice daily. 3. Isolation Methods and Culture Media

After some growth has become visible, either from pollution or enrichment, it has been the practice to select bits of the slime or floc and to rinse them in broth or water to reduce the numbers of adherent organisms. The cleaned filaments may then be streaked on solid media directly or macerated mechanically before plating. After incubation, filaments of Sphaerotilus extend from the areas inoculated and are conveniently picked up by cutting out a small piece of agar with the needle. Repeated purification is advisable. The Biisgen-Hoflich medium, or some modification, has been used by several workers. This originally contained 0.05% beef extract solidified with 1%agar, but beef extract up to 0.5%has been used for isolation of pure cultures (Bahr, 1953; Hohnl, 1955; Pringsheim, 1949; Skerman et al., 1957a). Other modifications have been reported effective: addition of 0.25% manganous carbonate (Cataldi, 1937); yeast extract or proteose peptone instead of beef extract; and addition of 0.002 to 0.005% manganous sulfate for the isolation of S. discophom (Pringsheim, 1949). Glucose, peptone, and salts agar were used by Lackey and Wattie ( 1940), Ruchhoft and Watkins ( 1928), and Stokes (1954). A medium which was used by Cataldi and tried a few times by the writer gave excellent results with Sphaerotilus consisted of peptone, 0.5 gm.; manganous acetate, 0.1 gm.; tap water, 1000 ml.; and agar 15 gm. Lackey (1941) isolated Sphaerotilus on agar made from Columbia River water polluted with spent sulfite liquor; and Lackey and Wattie (1940) isolated Sphuerotilus on filtered sewage plus 1.5%agar.

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Standard nutrient agar is unsuitable for Sphaerotilus, but when supplemented with carbohydrates, e.g., 0.1%dextrose plus O , l % starch (Littman, 1940), it has been used for isolation. Incubation temperatures used for the enrichment and isolation procedures ranged from 20° to 3 O O C . When isolating Sphaerotilus by the media cited, either excessive numbers of extraneous microbial colonies or some degree of suppression of all organisms, including Sphaerotilus, must be contended with; because Sphaerotilus is usually outnumbered, the media are not efficient for isolation purposes. To be reasonably confident that typical strains were being isolated from the material under investigation, the medium would be expected to support the growth of numerous colonies of Sphaerotilus. This is not the case, however, when seemingly healthy stream slimes are inoculated on beef extract media; the few colonies which developed could be regarded as atypical of the bulk of the slime. An attempt was made to remedy the difficulty with new media (Dondero et al., 1961). The isolation medium consisted of pancreatic digest of casein 0.13 to 0.258, agar 1.5!%,and water. This medium depressed the number of associated organisms. By addition of either yeast autolysate ( u p to 0.2%)or glycerol (up to l % ) ,growth of Sphaerotilus was improved, but the numbers of extraneous colonies also increased. Incubation at 2OOC. promoted the appearance of extraneous colonies and slowed the growth of Sphaerotilus whereas the opposite was the case at 28OC. 4. Preservation of Cultures The isolation of Sphaerotilus is not difficult when the appearance of the colonies has been learned. Typical colonies appear on 0.05 to 0.5% beef extract agar and on many other media; it was the experience of this writer, however, that the media described were not altogether satisfactory for preservation of cultures. Many cultures did not survive when transferred on several of the media already described. Furthermore, the available formulations seemed unsatisfactory in that few colonies appeared after massive inoculation of agar, abundant growth was not often obtainable in broths, and the survival of stock cultures was irregular even over short periods. Media for isolation and conservation which seemed more suitable were developed and are the subject of a separate report (Dondero et al., 1961). A medium found to be suitable for both isolation and conservation of Sphaerotilus is as follows: pancreatic digest of casein, 5.0 gm.; glycerol, 10 gm.; yeast autolysate, 1.0 gm.; water, tap or distilled, 1000 ml.; and, for solid medium, agar, 15 gm. The formulation was not an ultimate refinement, but it gave profuse growth with most isolates

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when adequatedaeration was provided for the broth. A few isolates grew less profusely in the medium. The casitone glycerol yeast autolysate medium ( CGY) encouraged excessive colony formation of the bacteria and fungi associated with Sphaerotilus, necessitating some modification for suppressing the growth of contaminants. Except for actidione, p-aminosalicylic acid, and isonicotinic acid hydrazide, all the inhibitors tested suppressed Sphaerotilus more than the accompanying bacteria when incorporated into the agar. The three substances were of no practical use for primary isolation of Sphaerotilus. An improved isolation medium was obtained by eliminating glycerol and yeast autolysate from CGY and reducing the casein digest to 0.13 to 0.25%.In this way a maximum number of Sphaerotilus colonies grew in the presence of contaminant colonies. Stock cultures were preserved at room temperatures in broth tubes for 6- to 12-month intervals between transfers by sealing the tubes after good growth had developed. Stocks were more conveniently preserved by lyophilization in 20% skim milk after abundant growth had been obtained and concentrated from CGY cultures aerated on the shaker.

C.CULTURAL CHARACTERISTICS Sphaerotilus colonies present a distinctive flat, arborescent aspect from their first appearance, which is maintained persistently on media that do not contain excessive carbohydrate. When growth is abundant some strains form smooth or mucoid colonies and lose the characteristic appearance. Stokes’ (1954) paper contains excellent illustrations of both types of colony, Sporeformers of the cereus-mycoides type may resemble Sphaerotilus closely when very young (18-24 hours, 28OC.) but the resemblance disappears as the sporeformer colony matures. Numerous short branches which appear on the rhizoid projections from the colony produce a bristle-like appearance which changes later to the typical smooth colony of a Bacillus. Cultural characteristics of Sphaerotilus were adequately presented by Stokes and given in detail by Pringsheim, who demonstrated the relationship of the Leptothrix and Cladothrix forms of Sphaerotilus. In broth Sphaerotilus tends to grow as a thick, coherent pellicle at the surface of the medium or in the depths of the tube as a cottony meshwork or web (arachnoid form). In weak beef extract broth turbidity is absent or slight. When carbohydrates or other growth-promoting substances are included in the medium, growth of some strains is more vigorous and cells released from the sheaths swim freely in the medium, producing marked turbidity. Some strains grow in broth as small pellets similar to those formed by fungi growing submerged. The Leptothrix form was

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described (Cataldi, 1937) as forming beneath the surface of agar containing 0.01%manganous acetate small, filamentous, dark-chestnut-colored colonies which are almost black when pure or reddish when mixed. The Cldothrix form, according to Pringsheim, grows in solutions of low nutritive value and would not be expected to have a distinctive colony form.

D. MORPHOLOGICAL CHARACTERISTICS Morphology of artificially cultivated Sphaerotilus resembles closely that of the wild forms in nature. It was by changing the conditions of cultivation that Pringsheim showed that cultures of microorganisms which conformed in microscopic morphology to the descriptions of the genera Sphaerotilus, Cladothrix, and Leptothrix could be converted interchangeably to the other forms. Tubular sheaths enclosing the cells are a characteristic of the order Chlamydobacteriales [and are found in the marine organisms, Leucothrix (Pringsheim, 1957)] but are not confined to bacteria alone. Sheaths are also found among the algae and may be thin or very thick. Some sporeforming Bacillus from pseuclosheaths i.e. the remnants of autolyzed cells remain together in a filament and may be mistaken for the authentic structure. The presence of chlorophyl or endospores in an apparently ensheathed microorganism therefore exclude it from the genus Sphacrotilus. Endospores were described in Sphaerotilus (Sack, 1925), but the organism was thought actually to be Bacillus megaterium (Haag, 1927). The sheath may be absent from Sphaerotilus when growth is very abundant in smooth colonies or in very nutritive broths but it must be demonstrable under some condition of cultivation for the organism to be classified as Sphamotilus, since a set of distinctive biochemical or other data sufficient for identification and differentiation does not yet exist. In the absence of the sheath it is difficult to differentiate Sphaerotilus from a Bacillus vegetative cell. The individual cells vary in length and may contain refractile droplets. The free cells are usually motile and have monotrichous flagellation. Although they have been called conidia, spores, or swarmers, there is no indication that they are resistant cells or possess special reproductive properties. The terminology probably was derived from the similarity to zoospores of algae and fungi. The typical Sphaerotilus growth habit is said to exhibit dichotomous false branching rarely or not at all, whereas it is characteristic of CZadothrix. The branching is sometimes difficult to see in preparations from cultures, but is often seen in specimens from natural waters and activated sludge. The sheath of the Leptothrix form is quite characteristic and was

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aptly described by Pringsheim as resembling smooth lengths of glass capillary tubing. These tubular sheaths are often curved and usually appear empty of cells. The thickening of the sheaths results from the deposition of iron or manganese. Cladothrix and Sphaerotilus are commonly surrounded by capsular material outside the thin sheath. The capsular substance is quite inconspicuous microscopically unless present in copious amounts. It is most readily noticed, when present, by the thick mucous quality of the growth from broth cultures and in stream slimes it may become conspicuously gelatinous. Regarding the nomenclature of the extracellular structure Pringsheim is, at times, somewhat indefinite. The term "primary sheath" was used to designate the thin, tubular membranous structure immediately outside of and enclosing the cells. When the primary sheath becomes quite distinct and somewhat thickened, it is referred to simply as the sheath; however, although mucilage is mentioned, the mucilaginous material is sometimes intended to represent a very much thickened sheath or sometimes a structure analagous to the slime layer or capsule of bacteria. Dense mucilaginous deposits of this nature are called sheaths in the descriptions of some algae. In this chapter, the sheath is regarded as the thin, flexible tube, the outer slimy layer is referred to as capsule or slime layer. Disk-shaped holdfasts have been repeatedly described as the means by which the filaments attach to the solid substrate. The writer has searched carefully for these structures but has never been able to distinguish such an organelle from the protoplast-like cells frequently found terminating free-floating filaments. Holdfasts were not seen on slides submerged in artificial channels following seeding with Sphaerotilus, although adherent cells of Sphaerotilus covered the slides.

E. TAXONOMIC CONSIDERATIONS The interrelationships of the Chlamydobacteriaceae had been speculated upon for some time previous to the work of Cataldi (1939) and Pringsheim (1949). Although pure cultures had been obtained, much of the comparative work had been done on naturally occurring organisms and much importance was attached to microscopic morphology. It was not until pure cultures were studied intensively that the relationships of the genera began to appear. Cataldi isolated 255 strains of chlamydobacteria from waters, muds, and soils using hay enrichment cultures supplemented with ferric hydroxide or manganous carbonate. The organisms were separated into two

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NORMAN C. DONDERO

genera, Sphaerotilus and Leptothrix, the distinction arising from the deposition of iron in the sheaths of the latter, but not in those of the former. Since all the cultures required organic matter for development, this work did not substantiate Winogradsky’s assertion that the organisms could utilize inorganic iron as the sole source of energy. Pringsheim’s (1949) dissertation on the three genera of filamentous ensheathed bacteria did much to show the biological relationship of the genera and to remove the confusion of taxonomic nomenclature which surrounded the organisms, Pure cultures of the bacteria were obtained by washing threads of starting material with capillary pipettes under the microscope, transferring to agar plates, streaking, and isolating. The starting material was obtained from aquatic growths identified as Sphaerotilus, Cludothrix, and Leptothrix. By inoculating the isolates into several culture media which resembled approximately the different ecological conditions in which the respective natural growths were found, each growth form could be reversibly interconverted. The Sphnerotilus nntans form was obtained in beef extract broth; in soil-barley medium or dilute hay decoction, the dichotomous false branching habit of CZadothrix appeared. The Leptothrix ochracea form appeared after prolonged incubation in dilute beef extract solution, or hay decoctions, to which had been added 0.01 to 0.021 ferrous ammonium citrate. All strains could be induced to incorporate iron in the sheath or capsule by addition of ferrous ammonium citrate to the medium: the iron was detected by the Prussian blue reaction, and with high concentrations of iron in the culture medium the sheaths became visibly yellow or rusty. The appearance and, presumably, the composition of the sheath and capsular material varied according to whether iron and maganese were available to the organisms. All the strains required some organic matter for growth. From these observations it was concluded that the names Leptothrix ochracea, Cladothrix dichotoma, and Sphaerotilus natans were synonyms and that the differences in growth habit resulted from modification by environmental influences. The name SphuerotiZus was retained by reason of priority. The names Chlamydothrix, Megalolthrix, and Clonothrix were considered to be synonyms of Leptothrix and no longer valid. Beger and Bringmann ( 1953) characterized and differentiated Sphaerotilus and Leptothrix into many species, which are included in Bergey’s Manual (Breed et al., 1957). Most of the characteristics considered by Beger and Bringmann were based on microscopic morphology of naturally occurring forms. Biochemical and cultural characteristics obtained under controlled conditions were not considered, not available, or did not provide sufficient information for differentiation. In view of the pleomorphic responsiveness to changes in the environment previously shown by Pring-

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sheim for these organisms, extensive subdivision into species seems premature. The status of Crenothm'x, as Pringsheim noted, appears dubious but, lacking cultures for study, there is no firm basis for rejecting the genus. The possession of nonmotile, spherical conidia and tapering expansion of the sheath toward the unattached end of the filament are the characteristics which differentiate Crenothrix from Sphaerotilus. One may entertain the suspicion that these differences are not so extreme as to be excluded from the category of phenotypical variations, Published photographs of Crenothrix growing in water treatment plants (Wolfe, 1960a, b ) bear a strong resemblance to Sphaerotilus forma eutrophica and forma ochracea. The cultural and biochemical characteristics of the sheathed bacteria have been of minor significance from a taxonomic viewpoint. The chief recorded characteristics may be summarized as follows: gram negative; nitrates utilized; indol, hydrogen sulfide, and acetyl methyl, carbinol not produced. Gelatin is not liquefied, or is liquefied very slowly and slightly. The chlamydobacteria should not be confused with the obsolete designation for actinomycete, anaerobic, animal parasites, Leptotrichia ( Lepto-

thrix buccalis)

.

IV. Causes of and Factors Affecting Stream Infestations A. SIGNIFICANCE OF STUDIES WITH ARTIFICIAL CHANNELS The flourishing blooms of Sphaerotilus which appear in water courses following the introduction of organic pollution have been the principal focus of chemical, hydrographic, and microbiological research into the nature of Sphaerotilus. Fortunately, the slimes which have been induced to grow in experimental channels were similar to those of streams and much of the circumstantial information obtained from the streams could be tested experimentally in channels. In the experimental channels, artificial seeding may be effected with river slimes, or the water or wastes entering the channel may carry the latent inoculum naturally (Lincoln and Foster, 1943). In any event, the population establishes itself in an equilibrium which is determined by environmental factors, since the apparatus is unshielded from contamination and is subjected to the constant passage of unsterilized water. Nutrients or wastes to be tested are fed separately to the channels at rates predetermined to give the required dilutions. The water-nutrient mixture may be allowed to flow directly through or may be circulated by some device. In the latter instance, there is provision for overflow, thereby permitting a continuous flow of water through the apparatus which carries off metabolic products and unattached particles that remain sus-

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pended at the flow velocities employed, The effects of varying the several parameters can be measured quantitatively from the weight or volume of the slime in the channel or the weight of growth attached to slides, laths, wooden blocks, or other solid substrates. Data from experimental channels with mixed populations are of practical significance for stream sanitation work in that the interactions of the mixed biota are taken into account. The channel flora, by its adjustments to changes in the environment engendered by the addition or withdrawal of nutrients or inhibitors, adjustment of pH, and other influences, can be expected to give some idea of the consequences to the stream, for which it serves as a model, beyond stimulation or inhibition of Sphaerotilus alone; for example, the replacement of Sphaerotilus by fungi or zoogleal bacteria or the effect on waste stabilization, Although the intrinsic properties of Spliaerotilus in pure culture are of practical as well as academic interest, the use of mixed channel cultures is a measure of insurance against the possible idiosyncrasies and limited response of pure strains. Pure cultures are more easily handled by conventional methods, but the high concentration of nutrients required by batch lot cultures produces not only an unnaturally enriched environment, but may also produce unnatural bacteria. Pure cultures have been used in connection with the utilization of specific compounds (Hohnl, 1956; Lackey and Wattie, 1940; Stokes, 1954) of which more will appear later in this chapter.

B. FACTORS AFFECTINGSTREAMINFESTATIONS Much of the work on Sphaerotilus in streams is reviewed by Harrison and Heukelekian ( 1958). The material for the immediately subsequent paragraphs will be drawn largely from that review without further bibliographical reference, since most of the references in that review are also given in this one. References to specific authors will be made in connection with pertinent points of information drawn from other sources. Sphaerotilus appears as the dominant organism in zones of heavy pollution in streams which have received domestic sewage and certain industrial wastes that contain mono- and disaccharides, lower organic acids, alcohols, and amino acids. Wastes of such content originate from the manufacture of beet sugar, wood pulp, rayon viscose, flour, glue, dairy products, coke, and textiles. The disposal of spent sulfite liquor from pulp manufacture creates a special problem because of the large volume of production and the high nutritive composition. The volume of sulfite waste liquor (101%solids) varies from 2500 to 3000 gallons per ton of pulp produced (Eldridge, 1960). The liquor is mixed with wash water before discharge from the mill, the average output being 60,000 gallons per ton of pulp. The composition of sulfite waste liquors, as compiled by Eldridge is given in Table I.

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Approximately 3 million tons of spent sulfite liquor solids are produced annually in the United States alone (Lueck, 1960). Although the lignosulfonate ( lignin ) and inorganic components are not used by Sphuerutilus most of the remainder of the liquor is especially effective in promoting infestations of streams. Dilution of the spent liquor (as low as 1.4 mg. per liter biochemical oxygen demand) does not prevent Sphaerotilus from growing in streams provided that the required nutrients are continuously present; neither does treatment of the spent TABLE I SULFITEWASTELIQUORSOLIDS Component

Per cent of total solids

Formic acid Acetic Methyl alcohol Ethyl alcohol Acetone Furfural Pentose Hexose Tignin Miscellaneous Calcium Sulfur

0.5-0.8 3.8-4.0 0.5-1.0 0.14-0.17 0.07-0.11 0.18-0.24 2.16-2.43 12.3-14.8 50.2-52.2 24.8-29.5 4.5 9.15

liquor by the activated sludge process effectively remove slime-promoting substances (Cawley, 1958). The substances which promote Splzaerotilus are present in untreated sewage, which is inhibitory until diluted, but they are removed by biological treatment ( Wuhrmann, 1954). The nitrogen requirements of Sphaerotilus can be satisfied by ammonia, nitrates, or amino acids, and sewage may contribute sufficient nitrogen and phosphates to make up these deficiencies in spent sulfite liquor (Amberg and Cormack, 1960). Sphaerotilus has not been reported in salt water but is found in streams of neutral or mildly alkaline p H (pure culture range is from p H 5.5 to 10, optimum is about pH 7 ) . Cooler water temperatures permit large amounts of slime formation (Cawley, 1958). In the Altamaha River, with a constant food supply and with dissolved oxygen present in the water, beds of Sphaerotilus slime extended 180 meters downstream from the waste outfall in the summer ( 3O0c.), but at the 10°C. winter temperature the growth extended downstream 24 kilometers. This effect seems to be an expression of the phenomenon ( Jordan and Jacobs, 1947, 1948), that lower temperatures

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promote a greater efficiencyof conversion of food into cells. The temperature range permitting Sphuerotilus development in streams corresponds with that reported for cultures, i.e., 5 O to about 4OoC., abundant growth occurring at 23OC. The velocity of the stream has a profound influence on Sphaerotilus. Where polluted water is stagnant Sphaerotilus does not develop; at least 19 cm. per second (0.6 ft. per second) was thought to be required, even though oxygen is available (Liebmann, 1937, 1939) although pure cultures grow in quiet broth. Excessive velocity tears loose quantities of slime from points of attachment and scours sedimented masses from the stream bed where decomposition has begun. Sphaerotilus is an obligate aerobe but is sometimes found growing in waters so heavily polluted that dissolved oxygen is undetectable. The streamers are then found close to the surface and below riffles. It is supposed that the cells constantly deplete the oxygen as it diffuses to them. The flux of the stream carries away the unattached organisms that would otherwise remain in position to compete for nutrients, and similarly carries away metabolic products. There are low flow velocities in which the microbial character shifts away from predominance by Sphaerotilus (Amberg and Cormack, 1960; Phillips, 1960). The chief significance of stream flow is that the microorganisms which grow attached to the surfaces over which the water passes are exposed to a continuously renewed supply of food. C. THELOWER COLUMBIA RIVER From a series of investigations of factors affecting slime development in the Columbia River, utilizing river data and the results of experimental channel studies (Amberg and Cormack, 1960) it was reported that maximum concentrations of slime occurring in the fall and spring were probably promoted by land surface runoff of nitrogen and phosphate from agricultural fertilization in the watershed area. The runoff supplemented the nitrogen and phosphate-poor sulfite waste liquor, the principal waste contributing to slime formation, to make a balanced nutrient solution in the river. There appeared at times to be competition for phosphate between Sphaerotilus and algae. It was experimentally established that growth in the channels was a function of the amount of nutrient passing the slime surface and not of the concentration. Between limits set by low and high flow velocities, increases in the flow, at constant nutrient concentration, caused a proportional increase in the total amount of food passing over the slime surface, resulting in the increase in the amount of dime. By varying the flow rate and nutrient concentration it was found to be almost impossible to eliminate slime growth by reduction of waste concentrations,

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The established slime growth exhibited a high capacity for assimilating nutrients, removing as much as 8741 of the sulfite waste during 33 minutes of contact time. It had been noticed that intermittent discharge of pulp waste caused the disappearance and prevented the reappearance of slime in streams ( Engineer Grevemeyer, in discussion, Liebmann, 1952). This observation was confirmed experimentally (Amberg and Elder, 1956) and the intervals necessary to prevent slime growth were determined. The most favorable method for obtaining maximum growth of Sphaerotilus was by continuously discharging spent sulfite liquor, whereas discharging the same quantity over an interval of 6 hours produced no slime growth. In a “field trial” on logs moored in the Columbia River, which were exposed to sulfite liquor fed through diffusers, the slime formed by discharging liquor for 24 hours once in 6 days was less than that obtained from onefifth of the amount of liquor continuously discharged. The method of slime control by intermittent release of wastes may not be generally applicable since, on at least one stream, it has been found ineffective, as practiced ( Cawley, 1958). Circumstances affecting intermittent discharge are volume of stream flow, location of the outfall, retention period required, storage facilities for the retention of the required volume of waste, sensitivity of the stream biota to slugs of waste, and other limitations.

V. Nutrition and Physiology of Sphaerotilus A. ORGANIC SUBSTANCES

Investigators have disagreed somewhat on the utilization of various nutrient compounds. Stokes (1954) and Hohnl (1955) disagree on the utilization of the carbon compounds arabinose, xylose, and ethanol. There was agreement that propionate, methanol, and lactose were not utilized. Both workers found that the following were utilized: dextrose, galactose, sucrose, maltose, succinate, fumarate, lactate, and glycerol. Results differed concerning availability as organic nitrogen sources of leucine, cystine, tryptophan, tyrosine, methionine, threonine, urea, and acetamide. Considered to be available were glutamic acid, asparagine, alanine, and arginine. Stokes’ strains could utilize ammonia N when sucrose or glycerol were present in the medium, but not dextrose; Hohnl’s strains did not tolerate ammonium salts but could use nitrate when dextrose was the carbon source. Possible explanations for the discrepancies obtained by different investigators, of which the above serve as examples, may be sought in the use of different basal media and different substrate concentrations. Strain

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variation may also account for differences in requirements. There is some indication that isolates from the same source may have unlike nutritional demands ( Dondero et al., 1961) . Six morphologically typical cultures isolated from Altamaha River slime were all different in terms of their ability to grow in a synthetic medium supplemented with casein digest or yeast autolysate. On considering the vast numbers of SplzaerotiEus cells which must exist in massive stream infestations or bulking sludge, it seems reasonable to expect large numbers of genetic variants to be present, particularly when the varying characteristics are those which may have no direct bearing on the survival of the organism in the natural state. Microenvironmental differences, which must certainly exist, would probably contribute further to variability. One might also speculate, although there is no evidence, that, if such variability exists, a given population may be able to adapt to a specific environment conditioned by climate, water composition, or prolonged pollution by a particular type of waste, and that the population could differ, statistically at least, from that of another stream, or geographical area. Synthetic, defined media, lacking vitamins, have been used for the cultivation of Sphaerotilus (Lackey and Wattie, 1940; Stokes, 1954; Cataldi, 1939; Hohnl, 1955) but the amount of growth so obtained was less than that resulting from the inclusion of complex organic substances. These growth-promoting substances include casein hydrolyzate, beef extract, peptone, sewage, soil extract, or spent sulfite liquor. The stimulating effect of each substance may vary, depending upon the other components of the culture media. Gelatin may not support growth (Lackey and Wattie, 1940) or it may be liquefied slowly (Cataldi, 1939; Pringsheim, 1949). Starch was not hydrolyzed by Lackey’s strains. Waitz and Lackey (1959) observed improved growth of Sphaerotilus in the presence of sulfur-containing amino acids and -SH groups. Elemental sulfur was seen to appear in cells of Sphaerotilus after exposure to H2S, confirming previous observations by others (Skerman et nl., 1957a) who observed the same effect in Sphaerotilus and Begginton. There seems, however, little justification for supposing a close relationship to the sulfur bacteria on this account, since the ability is also possessed by some yeasts and a species of Alternuria (Skerman et nl., 195%).

B. IRON TRANSFORMATIONS The question of the importance of iron in the metabolism of Sphaerotilus biotypes deserves more than passing comment because of Winogradsky’s (1888) interpretation that the iron deposited in the sheaths of

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Leptothrix was the metabolic end product of the oxidation of ferrous iron by the bacteria. Winogradsky’s strains were obtained from enrichment cultures containing extracted hay and ferric hydroxide. He believed that ferrous bicarbonate was formed during decomposition of the organic matter. The ferrous bicarbonate was thought to be oxidized then by Leptothrix, the resulting ferric hydroxide being deposited outside the cells. Bits of the Leptothrix were observed microscopically in slide cultures in which water bearing ferrous iron was renewed several times daily. Winogradsky saw growth of the cells and deposition of ferric hydroxide. Filaments decolorized with carbonated water did not absorb ferric hydroxide from suspension in typical fashion, but filaments placed in a solution of ferrous bicabonate formed typical, rusty, homogeneous sheaths. The fact that sheaths deposited oxidized iron in areas where iron was not precipitated by spontaneous atmospheric oxidation was interpreted as necessarily resulting from bacterial oxidation. There was no visible oxidation where the sheaths did not contain cells. When the waterbearing reduced iron which supported growth of Leptothrix was allowed to become oxidized, growth of Leptothrix ceased. It was recorded, however, that organic compounds, such as calcium butyrate, would promote good growth. Cataldi (1937, 1939) reported that she could not isolate Lepfothrix in completely mineral media nor grow her isolates from organic media in completely mineral media. Omission of iron or manganese from the medium was not noticeably deleterious to the development of most cultures. The organisms grew best with complex organic matter. Manganous acetate and ferric ammonium citrate were salts from which manganese and iron were deposited in the sheaths. Cataldi believed that ( a ) manganese did not participate in the metabolism of the bacteria because manganese was deposited by dead cells, or ( b ) an exoenzyme was responsible for the deposition, since the activity of the oxidizing factor was destroyed by the action of weak or strong acids. Oxidized iron did not appear in the sheaths until the reaction of the medium exceeded p H 6.8. The demonstration that cultures of the heterotrophic bacteria, Sphnerotilus and Cladothrix, became transformed into Leptothrix the iron bacterium, and that Leptothrix could be made to revert (Pringsheim, 1949) , removed the requirements of obligate dependence upon oxidation of ferrous iron for Leptothrix. In a series of papers dealing with microbial transformations of iron ( Halvorson and Starkey, 1927; Starkey and Halvorson, 1927; Halvorson, 1931; Starkey, 1945) the theoretical principles and biological conditions concerned with the solubility and precipitation of iron compounds and

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their availability of microorganisms were examined. It was shown that iron could be oxidized, reduced, precipitated, and dissolved both by biological and nonbiological reactions in the presence of microorganisms without necessarily entering the metabolic plexus. In a simple aqueous system in which there is no complexing of ions, at atmospheric pressure and at a reaction pH above 4, the solubility of ferrous iron is very low (0.027 mg. per liter or less) and excess iron comes out of solution as ferric hydroxide. With a reduction of oxygen concentration or a decrease in pH more ferrous ions are formed at the expense of ferric iron according to the equilibrium 4 Fe++

+ 02 + 2 H+ F! 4 Fe++++ 2 OH-

and the derived expression showing the relationship between reduced iron, hydrogen ion concentration, and oxygen concentratinn :

in which A is activity and [AOZlp is the difference between the activity of oxygen at pressure p and activity at atmospheric pressure. From the equation it may be seen how the alteration of pH or oxygen concentration will shift the equilibrium to favor deposition or solution of iron. Utilization of oxygen, formation of acids, and production of carbon dioxide favor the solution of iron, whereas aeration, loss or utilization of carbon dioxide, or the formation of ammonia would tend to cause precipitation of iron. The utilization of the anion of sodium acetate or sodium nitrate would likewise favor iron precipitation by raising the pH of the medium. The production of insoluble iron sulfides under anaerobic conditions removes iron from solution. Complexing agents keep iron in solution at pH levels which would normally cause precipitation. Iron-binding compounds, such as ferrichromes and related substances are produced by microorganisms (Neilands, 1957). Sequestering substances are produced by Bacillus megaterium and Aspergillus niger, which hold ferric iron in solution at pH values of 10 to 11 (Garibaldi and Neilands, 1956). On the other hand, decomposition of the organic portion of the complex, as in ferric citrate or ferric ammonium citrate, results in precipitation of iron. Halvorson (1931) found that the deposition of iron in mineral springs was caused by contact of the water with oxygen and that the remaining iron concentration was that of the equilibrium value rather than a lower value which might have resulted from depletion of ferrous iron by biological oxidation to insoluble ferric iron. The carbon dioxide tension of waters, soils, and culture media are of

Sphaerotilus

I01

importance in the solution and deposition of iron. Large concentrations of carbon dioxide, such as those found in anaerobic decomposition, may cause the precipitation of iron as the carbonate. Lesser amounts, such as those from aerobic decomposition, favor solution of iron by depressing the pH. With the escape of carbon dioxide or its utilization by algae, iron may be precipitated. In view of the foregoing discussion, taking into consideration that the lower limit for growth of Sphaerotilus is about pH 5 and that the organism is an obligate heterotroph, it seems that the available evidence does not support Winogradsky’s interpretation that the oxidation of iron is vital for growth of the organism and deposition of iron. As an alternative to Winogradsky’s indirect proof that iron deposition was due to biological oxidation, it is suggested that ammonia in the cells and sheaths would cause precipitation of iron from ferrous solutions where air did not penetrate beneath the cover glass. The failure of the organisms to grow when the iron is already oxidized is not as easily explained. Although the weight of circumstantial evidence bears strongly away from autotrophy, the lack of a body of consistent information about the physiology of the genus Sphaerotilus, the known pleomorphism, and the lack of well-studied pure cultures has a counterbalancing influence which recommends that judgement be held in abeyance for lack of strictly conclusive evidence. It is quite possible that the identity of the organisms in question is uncertain. The biological transformations of manganese are not as well known as those of iron. They appear to be somewhat similar but not identical. In the bottoms of reservoirs and slow-moving waters, manganese is dissolved from soil, rocks, and organic matter by biological activity. In streams and water treatment plants, much as with iron, the manganese is removed from solution by oxidation, elevation of pH, and extraction by microorganisms (Griffin, 1960).

c. COMPOSITION AND FORMATION OF THE SHEATH There is little information about the composition of the sheath and capsule or the factors which influence their formation. The primary sheath of Sphaerotilus has been reported to consist of a hemicellulose membrane containing dextrose, galactose, arabinose, xylose, and ribose units (Hohnl, 1956). As previously noted (Winogradsky, 1888; Pringsheim, 1949), the glassy sheaths of Leptothrix, which are formed in ironand managanese-containing media, are composed, at least in part, of ferric hydroxide and probably manganese oxide. When sufficient iron is present in the medium the sheaths give a positive Prussian blue reaction. Winogradsky (1888) observed that the deposited mineral became more

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difficult to dissolve with hydrochloric acid as the age of the culture increased. The difference in solubility was attributed to changes in the composition of the sheath with age. The sheathed bacteria growing in water mains and galleries of water treatment plants have the remarkable ability to remove iron and manganese from water in which the concentration of these elements is too small to be chemically detectable (Wilson, 1945), and to incorporate it, presumably, primarily in the sheath. An organism identified microscopically as Clonothrix ( Wolfe, 1960b) incorporated manganese and iron from water which contained chemically undetectable manganese and iron up to 0.02 mg. per liter. Twenty-eight per cent of the total dry weight of the bacteria consisted of iron and manganese in approximately equal quantities. The conditions which promote the formation of the sheath are not known. There are more free-swimming cells and fewer ensheathed filaments when the medium is concentrated (Pringsheim, 1949). In smooth colonies on agar, most of the cells are free (Stokes, 1954). It is not unusual to see sheaths which contain several rows of cells. Such an occurrence is suggestive of the possibility that the cells are synthesized at a proportionately greater rate than the sheath in rich media.

D. METABOLICEFFICIENCY The voluminous development of Sphaerotilus in streams with low nutrient concentrations is suggestive of a high efficiency of conversion of substrate to bacterial substance. Stokes (1954) reported a high degree of oxidative assimilation, especially with sugars and sugar acohols (80%) which was only slightly inhibited by sodium azide and 2,4-dinitrophenol. The productive potential of substrates for Sphaeratilus has been investigated with the objective of estimating the extent of sliming to be expected in rivers from the disposal of pulp mill wastes (Hohnl, 1955; Scheuring and Hohnl, 1956). In streams, part of the mass of the Sphnerotilus slime is derived from extraneous substances. The composition varies according to age and conditions of growth. The ash content of stream slimes reported by several investigators varied from 24 to 64%dry weight (Scheuring and Hohnl, 1956); trough cultures contained 13 to 28% ash from the precipitation of sulfates and phosphates from neutralized spentliquor-water mixtures. The ash of pure cultures in dextrose-nitratc medium with basal salts (0.03%)was 6 rt 0.5%. The nitrogen content of flask cultures declined with age because of the higher proportion of hemicellulose sheath substance to cells. Growth from beef extract (0.2%) medium contained 10.5%nitrogen, about twice that of cultures grown in 1%dextrose-nitrate-salts medium.

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The efficiency of synthesis, in terms of bacterial substance formed per unit of substrate oxidatively utilized, was determined experimentally. The values obtained were as follows: dextrose, 19%;xylose, 12%;pine spent sulfite liquor, 14%; and meat extract, 36%.From these data it was calculated tnat 5.1 to 5.9 kg. of Sphaerotilus (dry weight, 6%ash) can be formed for each cubic meter of undiluted pine waste liquor (Hohnl, 1955) E. INHIBITION The substances which cause the inhibition of Sphaerotilus have an important bearing on the cultivation as well as the control of the organism since some of them are present in culture medium ingredients and others may be required for slime control in the plant and in the field. Wuhrmann (1949) believed that the inhibition of Sphaerotilus by undiluted sewage, raw or sterile, resulted from the presence of trytophan, cystine, and methionine. It was estimated that these amino acids were each present in concentration of about 500 pg. per liter and that they were strongly inhibitory in excess of 200 to 500 pg. per liter. At least a twentyfold dilution of raw sewage was necessary for good growth in rivers, channels, or pure cultures, In river disposal, other microorganisms were believed to reduce the level of inhibitory substances. Stokes (1954) obtained good growth, at 1000 mg. per liter, with cystine and tryptophan, and Waitz and Lackey (1959) found no injurious effects from methionine using concentrations greatly in excess of 200 mg. per liter. Ruchhoft and Watkins (1928) found beef extract in culture media to be inhibitory to Sphnerotilus, whereas Pringsheim (1949) found it to be satisfactory as the sole nutrient for Sphaerotilus. Manganous sulfate was considered by Pringsheim to be inhibitory at 50 mg. per liter, but Stokes observed no inhibition at 100 mg. per liter. Lackey and Wattie (1940) tested substances for to,xicity for Sphaerotilus in activated sludge or in heavy cultures and found that the following were toxic at the doses indicated (milligrams per liter): chlorine (0.5 residual), silver nitrate (0.5-2.0), phenol (5.0), acetic acid (50), brilliant green (5.0), malachite green (5.0), Janus green ( 20), methylene blue (20), and gentian violet (10). Chlorine was considered to be the most feasible substance for large scale use, as in water and sewage treatment plants. In water treatment plants, high initial doses of chlorine are required to remove established slimes. Repeated dosing to 50 to 100 mg. per liter residual chlorine followed by high-pressure air and flushing has been used (Alexander, 1944). After dislodgment of the slimes, 0.75 to 1.0 mg. per liter residual chlorine was maintained in the effluent water.

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Opinions on the efficacy of copper sulfate differed. Waitz and Lackey (1959) found 1 mg. per liter toxic in cultures, and De Martini (1934) recommended 2 mg. per liter in wastes for Sphaerotilus control in sewers. Brown (1934) considered copper sulfate unsuitable for control in water works. Nakamura and Dunstan (1958) found that 5 mg. per liter of copper sulfate in cannery waste used for vegetable irrigation inhibited Sphaerotilus and other slime completely without apparent harm to the vegetables. For removal of preformed slime 50 mg. per liter were required. The latter investigators tested other compounds and mixtures in the field and in uitro. Thirty-seven compounds which inhibited Sphnerotilus and 35 which did not were listed. Ammonium chloride at 10 mg. per liter was reported by Hohnl (1955) to inhibit oxidation of carbohydrates and growth of Sphaerotilus. Hohnl detected ;I depression of respiratory activity with 0.067 M (0.93%) phosphate buffer, but 0.01 and 0.02 M buffer did not interfere. Sodium chloride added to 0.5%meat extract depressed growth at 0.2%NaCl and prevented growth completely at 0.3%and above.

VI. Conclusion It would be difficult to estimate the total cost of the difficulties caused by Sphaerotilus. To do so, it would be necessary to have more information than is readily available. It would be necessary to know the number of sulfite process mills and the volume of their wastes discharged into rivers, the value of fisheries involved, the number of nets fouled, the cost of waste treatment systems, and waste impoundments. The extra costs of water treatment resulting from fouling and corrosion by “iron bacteria” in numerous water works would have to be included. Also to be considered is the loss of efficiency in sewage and industrial waste treatment when Sphaerotilus bulking takes place. Not the least of the problems is the degradation of the aesthetic qualities of streams which have become fouled with the unsightly growths. The problems of Sphaerotilus slimes in streams are not confined to the United States but are also found in Europe and are probably widespread throughout the temperate zones, where most of the wood pulp is produced. Because of its connection with sewage and industrial wastes, Sphaerotilus is perhaps better known among sanitary engineers and sanitary chemists than among bacteriologists; at least, the bulk of pertinent information is found in the scientific journals of the sanitation field. Taking into account some few notable exceptions, most of the work in recent years may be said to have been motivated by the ultimate objective of suppression of the nuisances caused by Sphaerotilus and the iron bacteria. Although this work represents most of the progress achieved in obtaining

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information on this group of bacteria, there yet remains much to be done of both practical and scientific interest. As has been obvious in this chapter, there are many contradictory and confusing results to be reconciled and information to be confirmed. A few of the points yet to be investigated are the morphogenesis, composition, and destruction of the sheath; the possible utility of the organism; the kinetics of growth; the existence of a bacteriophage and its relation to the biology of ensheathed bacteria. In the future, no doubt, many more pertinent points will come to light.

REFERENCES Alexander, L. (1944). J. Am. Water Works Assoc. 36, 1349-1355. Amberg, H., and Elder, R. (1956). J. Sanitary Eng. Diu. ( A m . SOC. Civil Engrs.) 82, 929-1 to 929-11. Amberg, H., and Cormack, J. (1960). Pulp Paper Mag. Can. 61, 1960, T-70 to T-80. Audus, L. (1946). Nature 158, 419. Bahr, H. (1953). Schweiz. Z. Hydrol. 15, 286301. Beger, H., and Bringmann, G. (1953). Zentr. Bakteriol. Purasitenk. Abt. I I 107, 318-334. Breed, R. S., Murray, E. G. D., and Smith, N. R. (1957). “Bergey’s Manual of Determinative Bacteriology,” 7th ed. Williams & Wilkins, Baltimore, Maryland. Brown, K. ( 1934). J . Am. Water Works Assoc. 26, 1884-1700. Bryson, V. (1952). Science 116,48-51. Butcher, R. (1932). Trans. Brit. Mycol. SOC. 17, 112-125. Cataldi, M. ( 1937). Folia Biol. (Buenos A i m ) Nos. 79-82, 337-344. Cataldi, M. ( 1939). Rev. inst. bacteriol. dept. nacl. hig. (Buenos Aires) 9, 1-96. Cawley, W. (1958). Sewage and Ind. Wastes 30, 1174-1182. Collins, F. (1952). Australian J . Exptl. Biol. Med. Sci. 30, 279-286. DeHaan, P., and Winkler, K. (1955). Antonie van Leeuwenhoek J . Microbiol. Serol. 21, 33-44. De Martini, F. (1934). Sewage Works J. 6,950-955. Demoll, R., and Liebmann, H. (1952). Schweiz. Z. Hydrol. 14, 289-297. Dondero, N., Phillips, R., and Heukelekian, H. (1961). Appl. Alicrobiol. 9, 219-227. Drescher, R. (1957). Tuppi 40, 904-910. Eldridge, E. (1960). Robert A. Tuft Sanituy Eng. Center Tech. Rept. W60-3, 255256. Finn, R., and Wilson, R. (1954). J . Agr. Food Chem. 2,6t?-69. Garibaldi, J., and Neilands, J. ( 1956). Nature 177, 526-527. Gaufin, A., and Tarzwell, C. ( 1955). Am. Midland Naturalist 54, 78-88, Griffin, A. ( 1980). J. Am. Water Works Assoc. 52, 1326-1334. Haag, F. (1927). Zentr. Bakteriol. Parasitenk. Abt. I I 69, 4-14. Halvorson, H. ( 1931). Soil Sci. 32, 141-165. Halvorson, H., and Starkey, R. (1927). J. Phys. Chem. 31, 626-631. Harrison, M., and Heukelekian, H. ( 1958). Sewage and Ind. Wastes 30,1278-1302. Heukelekian, H., and Ingols, R. (1940). Sewage Works J. 12, 694-714. Hohnl, G. ( 1955). Arch. Mikrobwl. 23, 207-250. Hohnl, W. (1956). Wochbl. Papierfabrik. 84,564-565. Ingols, R., and Heukelekian, H. (1939). Sewage Works 1. 11. 927-945.

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Jordan, R., and Jacobs, S. (1947). J . Gen. Microbiol. 1, 121-136. Jordan, R., and Jacobs, S. (1948). 1. Gen Microbiol. 2, 15-24. Lackey, J. (1941). On the Biological Aspects of Paper Mill Pollution in Certain Parts of the Columbia River Watershed with Especial Reference to Sphaerotilus natans, pp. 1-11. Prelim. Rept. Stream Pollution Invest. Lab., U. S. Public Health Service (mimeo). Lackey, J., and Wattie, E. (1940). Public Health Rept. ( U . S . ) 55,975-987. Liebmann, H. ( 1937). Gesundh. Ing. 60,517-518. Liebmann, H. (1939). Gesundh. Ing. 62, 515-521. Liebmann, H. (1952). Ber. Abwassertechn. Ver. H3, 118-133. Liebmann, H. (1953). Vom Wasser 2 0 , 2 4 4 3 . Lincoln, J., and Foster, R. (1943). Report on Investigation of Pollution in the Lower Columbia River. pp. 1-143. Wash. State Pollution Comm. and Oregon State Sanitary Survey. Littman, M. (1940). Sewage Works J . 12, 685-693. Lueck, B. ( 1960). Robert A . Tuft Sanitary Eng. Center Tech. Hept. W60-3, 257-260. McPherson, W., and Gellman, I. (1958). Cooperative Study of Slime Occurrence in the Columbia River in the Vicinity of Vancouver, Washington During 1957-58, pp. 1-85. Natl. Council Stream Improvement, Inc. and Eng. Expt. Sta., Oregon State College, Corvallis, Oregon. Martin. R. (1955). Tech. Assoc. Pulp Paper Ind. Tappi Monograph Ser. N o . 15, 55-74. Moser, H. ( 1958). Camegie Inst. Wash. Publ. 614, 1-136. Nakamura, M., and Dunstan, G. ( 1958). Studies on the Control of Slime in Cannery Waste Water in the Walla Walla Area. Div. Ind. Research, State College of Washington, Pullman, Washington. Neilands, J. (1957). Racteriol. Revs. 21, 101-111. Novick, A., and Szilard, L. (1950). Science 112, 715-716. Olsen, E., and Szybalski, W. (1949a). Acta Chem. Scand. 3, 1094-1105. Olsen, E., and Szybalski, W. (1949b). Acta Chern. Scand. 3,1108-1116. Owen, S., and Johnson, M. ( 1955). J . Agr. Food Chem. 3,606-608. Phillips, R. ( 1960). The study of Sphaerotilus under simulated stream conditions. Thesis, Rutgers University, New Bnmswick, New Jersey. Popp, L., and Bahr, H. (1954). Wasserwirtschaft 45, 29-33. Pringsheim, E. ( 1949). Phil. Trans. Roy. SOC. London Ser. R 233,453-482. Pringsheim, E. ( 1957). Bacteriol. Reos. 21, 69-76. Ruchhoft, C., and Kachmar, J. (1941). Sewage Works J . 13,3-32. Ruchhoft, C., and Watkins, J. (1928). Sewage Works J . 1, 52-58. Sack, J. (1925). Zentr. Rakteriol. Parasitenk. Abt. 11 65, 116-118. Scheuring, L., and Hiihnl, G. (1956). Schriften Ver. Zellstoft Papier-Chern. Ing. 26, 1-151. Schikora, F. (1899). Z . FZscherei 7, 1-29. Skerman, V., Dementjeva, G., and Carey, G . (1957a). J . Bacteriol. 73, 504-512. Skerman, V., Dementjeva, G., and Skyring, G. (1957b). Nature 179,742. Skrinde, R. (1959). Experimental Treatment of Food Processing Waste Waters at Walla Walla, Washington in 1959. Sect. Rept. No. 19, pp. 1-75. Div. Ind. Research Inst. Technol., Wash. Sta. Univ., Pullman, Washington. Skuja, H. (1948). Syrnbokze Botan. Upsalienses D, 1-391. Smit, J. (1934). Sewage Works J . 6, 1041-1053. Starkey, R. ( 1945). J . Am. Water Works Assoc. 37, 963-984.

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Starkey, R., and Halvorson, H. ( 1927). Soil Sci. 24, 381-402. Stokes, J. L. (1954). I. Bacteriol. 67,278-291. Tenny, M . (1939). J. Am. Water Works Assoc. 31, 9&104. Tkachenko, N., and Droblyanets, B. ( 1959). Mikrobiologiya 28, 708-712. Waitz, S., and Lackey, J. (1959). Quart. J . Florida Acad. Sci. 21, 335-340. Wilson, C. ( 1945). 1. Am. Water Works Assoc. 37, 52-58. Wilson, J., Wagner, R., Toombs, G., and Becher, A. (1960). J. Water Pollution Control Federution 32, 83-89. Winogradsky, S. (1888). Botan. 2. 46, 261-270; (1949). In “Microbiologie du Sol” pp. 52-55. Masson, Paris. Wolfe, R. ( 1958). 1. Am. Water Works Assoc. 50, 1241-1249. Wolfe, R. ( 1960a). J. Am. Water Works Assoc. 52, 915-918. Wolfe, R. (1960b). 1. Am. Water Works Assoc. 52, 1335-1337. Wuhrmann, K. (1949). Verhandl. Intern. Ver. theoret. angew. Limnol. 10, 580-586. Wuhrmann, K. (1954). Sewage and Ind. Wastes 26,212-220. Wurtz, A. (1956). Bull. fraw. piscicult. No. 182, 5-25. Wurtz, A. (1957). Bull. franC. piscicult. No. 184, 89-116. Zubrzycki, L., and Spaulding, E. ( 1958). 1. Bucteriol. 75, 278-282.

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Large-Scale Use of Animal Cell Cultures DONALD J. MERCHANT AND C.RICHARDEDAM Department of Bacteriology, The University of Michigan, Ann Arhor, Michigan I. Introduction

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11. Current Areas of Large-Scale Use of Tissue Cultures A. Virus Isolation and Identification . . . . . . . . . . . . . .

B. Virus Vaccine Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 C. Chemotherapy Screening ................. TJI. Some Biochemical Activities of Animal Cells Gro . . . . . . . 113 A. Synthetic Activities . . . . . ....... B. Chemical Conversions ................. IV. Some Trends in Animal Cell and Organ Cultur A. Definition of Nutritional and Physical Requirements for Animal Cells in . Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .119 B. Characterization of Metabolic Pathways . C. Large-Scale Cell Production ....... D. Continuous Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . 123 V. Speculation on Applications of Cell and References ...........

1. Introduction It has been just a half century since Harrison (1907) first demonstrated the cultivation of animal cells in vitro, about thirty-five years since the introduction of the organ culture method by Strangeways and Fell (19251926), and approximately thirty years since the cultivation of plant tissue by Gautheret ( 1932) and White ( 1932). Subsequently these techniques have been utilized in almost every phase of research in biology. Prior to the past decade, however, cell, tissue, and organ culture were almost exclusively research tools, considered to be too exacting and uncertain for use on a large scale or on a routine basis. Indeed the cumbersome technology, the poor definition of physical and chemical requirements, and a general lack of quantitative methods discouraged all but the most painstaking and determined workers. Beginning in the mid 1940's, however, the efforts of these workers began to bear fruit in the form of fairly rapid technological advances. As is often the case, technological advances in allied fields played a major role in these developments. The application of cell culture methods to the study of animal viruses is a notable example of the progress made during the past decade. Improvement of cell culture procedures made possible a rapid adaptation 109

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of methods used with great success in the bacterial virus field. Such developments have opened entire new areas of investigation and of application of fundamental knowledge, not only in virology but in many other fields as well. Nevertheless, an application of potentially even greater significance would be the utilization of animal cells for the production, in vitro, of chemicals by fermentation methods. Compounds unique to the animal cell, such as hormones, would be of particular concern. The possibilities of such an application were considered early by several investigators ( Carrel and Burrows, 1910; Ebeling, 1925; Haymaker, 1935; Lewis and Geiling, 1935) and much effort has been directed toward the in vitro cultivation of normal, functioning cells either as established strains of cells or as organized tissues, While the majority of the studies with cell strains or serially cultured tissues were not notably successful many important and encouraging observations were recorded. In almost all instances the lack of technological development and of supporting information prevented the studies from being pursued further. Heavy demands for the application of cell culture techniques in research, particularly in the burgeoning fields of virology and cancer research, have tended to focus attention on these areas in recent years. Recently, however, a general interest again has been mainfest in the possible applications of large-scale cell culture for other uses. While there is little in the literature, as yet, specifically related to this subject, an analysis of available information and of current trends in cell culture may provide a basis for useful speculation.

II. Current Areas of Large-Scale Use of Tissue Cultures While it is intended to limit this discussion primarily to applic at'ions other than those directly related to virology and cancer research it will be useful to consider briefly the areas in which large-scale use of cell cultures has been most successful to date. A. VIRUSISOLATIONAND IDENTIFICATION Within a relatively short time monolayer cultures of animal cells have become an indispensible tool for the isolation and identification of animal viruses. The ability to distinguish variations in types of cytopathogenic action, plus the ability to neutralize these effects with specific antisera, has made possible rapid identification of animal viruses. Quantitative techniques similar to those which have characterized the bacterial virus field have added precision to animal virus research, The first real test of cell culture on a large scale came with the application of the monolayer culture method to the evaluation of the polio-

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illyelitis vaccine during the 1954 field trials (Francis, 1957). To titer the many thousands of serum specimens, cultures of susceptible cells were required in vast numbers with each participating laboratory using thousands of cultures per week. To meet this demand it was necessary to remove cell culture from the realm of specialized research tools and put it on a mass production basis. A technique previously considered practicable only for highly skilled technicians with long experience thus almost overnight was handed to laboratory workers with little or no previous experience in cell culture. They were required to prepare and handle cultures in numbers far in excess of those handled in most laboratories and at the same time it was necessary to achieve a much higher degree of uniformity than had been common previously. Success in this undertaking was the result, to a marked degree, of several advances in technique made concurrently or in the years just prior to the field trials. Among the more important were ( a ) development of the monolayer culture technique (Evans and Earle, 1947) and its subsequent refinement and application to a variety of cell types, ( b ) perfection of nuclei and cell enumeration procedures (Sanford et al., 1951; Scherer et al., 1953), ( c ) development of replicate culture methods (Evans et al., 1951), and ( d ) refinement and simplification of growth and maintenance media for cell cultures. It was shown conclusively in this instance that cell strains could be grown and handled on a large scale in several different laboratories and yet give comparable results. The widespread adoption of these simplified procedures for routine isolation and identification of viruses has in turn led to advancements in technique at a continuously accelerating rate. The role of antibiotics in the development of tissue culture techniques for virus isolation should not be minimized. As recently as fifteen years ago it would have been considered foolhardy to attempt routine isolation of viruses from most clinical specimens. In the relatively few instances where such direct isolations were made filtration was required to render the specimens free of bacteria, yeasts, and molds. Such treatment often resulted in a significant loss of virus titer. The control of contamination by the simple addition of antibiotics has been an exceptionally useful procedure.

B. VIRUSVACCINEPRODUCTION A second large-scale application of cell culture techniques was in the production of the poliomyelitis vaccine itself. Though other virus vaccines had been prepared from cell culture materials this had not been done on a comparable scale.

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While the field trials required replication of cultures in large numbers, the individual cultures were grown in tubes, small bottles, or vinyl cup panels. To produce virus in the quantities required for mass immunization it not only was necessary to have many cultures, but also masses of cells per culture. Methodology already established (Evans and Earle, 1947; Evans et al., 1951; Scherer ct al., 1953) made this transition feasible in a short time. In addition the experience of the pharmaceutical and fermentation industries in scaling up production of molds, yeast, and bacteria proved to be very helpful. The standard cell culture method used for the production of virus vaccines has been growth of cells as monolayers in large bottles (32-oz. prescription bottles, Blake bottles, etc. ) . This has been dictated largely by the requirement for use of primary monkey kidney cells for vaccine production. Several laboratories have experimented with growth of animal cells in fermentors (Nickel1 and Tulecke, 1960; Rightsel et al., 1960; Ziegler et al., 1958) or in continuous culture apparatus (Cherry and Hull, 1960; Cohen and Eagle, 1960; Cooper et al., 1959; Graff and McCarty, 1957; Lindbergh, 1931; Merchant et al., 1960). The feasibility of these methods using serially cultured cell lines is well established but little success has been attained with primary isolates. The unsolved question of the dangers of tumor viruses is thus blocking, for the present, the use of a potentially efficient method. The success of large-scale tissue culture in the production of the poliomyelitis vaccine has stimulated similar development of vaccines from adenoviruses, influenza, and numerous other viral agents. In the case of some of the important animal virus diseases the threat of latent tumor viruses does not prohibit use of established cell strains for vaccine production and fermentation methods may prove to be quite useful. C. CHEMOTHERAPY SCREENING Currently the tissue culture method is the most widely used primary screening procedure for anticancer agents and for antivirus compounds. Such studies were a natural outgrowth of the large-scale use of cells for virus isolation and titration. Certain differences exist, however, in the requirements for cell systems to evaluate anticancer and antiviral agents. To study antiviral compounds a single cell line with a wide spectrum of virus susceptibility may suffice to evaluate compounds for their inhibitory effects against a number of different viruses. At most, parallel tests in 3 to 4 cell lines of varying susceptibility would be required. In contrast, any potential antitumor agent must be tested against a variety of tumor lines as well as being checked for its effect on “normal” cell lines. Thus, from the standpoint

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of the handIing of cell cuItures, the cancer chemotherapy program is considerably more complex. Since serious questions have been raised as to whether any long-term tissue culture cell line can be considered normal, it becomes necessary to include many primary cell cultures in a cancer chemotherapy screening program. Such cultures are more tedious to prepare and handle than established cell lines. Moreover, many of the highly refined quantitative methods which have been applied with certain cell lines cannot be readily applied to most primary cultures. Media for the latter are generally more complex in composition or are ill defined. In spite of these shortcomings, cell culture has proven to be the method of choice for primary screening of potential antitumor agents (Eagle and Foley, 1958; Siminoff and Hursky, 1960).

Ill. Some Biochemical Activities of Animal Cells Grown in Vitro A comprehensive review of the biochemical activities of animal cells in vitro is not attempted here. Rather it is intended to present some of the evidence available concerning synthesis and conversion of compounds of unique biological interest such as hormones. A. SYNTHETICACTIVITIES At a very early date Carrel and Burrows (1911b) succeeded in growing thyroid glands of dogs, cats, and guinea pigs in plasma clot cultures. While both epithelial and fibroblastic elements emerged, epithelial outgrowth predominated. The tissue was maintained through three serial subcultures but no attempt was made to assay for hormone. Fifteen years later Ebeling (1925) isolated a strain of epithelial cells from chick embryo thyroid. This culture was maintained by serial subculture in plasma clots where the cells grew at the surface of the clot as “pavement epithelium” and within the clot as “glandular structures.” After four months in culture the lumen of the acini which formed in the clot were filled with “colloid” as demonstrated by staining. No attempt was made to measure activity levels. Interesting observations were subsequently recorded by Demuth ( 1932-1933) who maintained chick embryo thyroid in chick plasma, without embryonic extract, for periods up to 90 days. There was evidence of some epithelial differentiation in his cultures though no acini or colloid were noted. After 4 weeks in culture a substance was produced which caused metamorphosis in tadpoles. Thyrotropic extract of the pituitary failed to stimulate growth of thyroid epithelium though this was accomplished when the tissue was grown in plasma from a cockera1 which had been fed potassium iodide.

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Numerous workers have studied thyroid function in vitro using the tissue slice method and several have employed organ culture techniques to study uptake of iodine and synthesis of thyroxine (Baker, 1938; Carpenter, et al., 1954; Gonzales, 1956; Levenstein et al., 1940; Roche et al., 1957; Scamen and Stahl, 1956). However, little work has been done with cell culture methods since these early observations by Ebeling and Demuth. A few short-term studies utilizing Il3I have been reported. Thus Gonzales (1956) added I 1 ? I to the culture medium and demonstrated synthesis of thyroxine and of mono- and diiodotyrosine by explants from 7-day-old chick embryos. Identification was made with autoradiographic techniques after the compounds had been separated from the growth medium by chromatography. Peak production occurred at 6 days. After 14 days mono- and diiodotyrosinc were still being synthesized but at a reduced rate. Colloid was readily observable in histological sections prepared from the explants but was present to a lesser degree in the outgrowth, Similar results were reported by Oppenheimer et al. (1956) with rabbit thyroids grown as explants in roller tubes. Though mono- and diiodotyrosine and thyroxine were produced no follicle formation or histological evidence of colloid production was observed in the outgrowth. Thyroid-stimulating hormone (TSH ) did not exert an observable effect. Pulvertaft et nl. (1959) have employed the technique of trypsin dispersion to develop stable lines of cells from human pathological thyroids. Thcy utilized both toxic and nontoxic goiters as well as carcinoma of the thyroid. In the majority of instances the cell lines underwent transformation, in vitro, as described by Westwood et al. ( 1957). Kadioiodine was incorporated into organic compounds by untransformed cells while the transformed cultures lost this ability. Striking differences in viriis susceptibility were also noted between transformed and untransformed cells. A t least three different groups of workers have reported production of gonadotropic hormone by human placental tissue maintained for extended periods in vitro. An early study by Gey el al. (1938) demonstrated production of hormone by cultures derived from a 3-month-old placenta which had been maintained in culture for 2 months and by cultures from a liydatidiform mole after 1 month. The tissues were cultivated in a medium composed of human cord serum, beef embryo extract, chicken plasma, and balanced salt solution (BSS ). Subsequently Gey et al. (1940) reported hormone production by such cultures after 6 months in vitro. No gonodotropic substance was produced by control cultures of cells from ectopic chorion tissue. Cells which produced hormone were identified as being of the “I, type.”

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Jones et al. (1943) later reported that although hormone was produced in some cultures of placental tissue and hydatidiform mole for as long as 6 months in vitro, there was a steady decrease in the quantity of hormone produced. These authors also noted that no clear-cut evidence of estrogen synthesis was obtained. Quite similar results were reported by Stewart et nl. (1948) who maintained gonadotropic hormone production by normal placental cultures for 75 days. There was progressively diminished production of gonadotropin over this period, however, and there was no evidence of estrogen synthesis. Waltz et nl. (1954) used strip-type explants of hydatidiform mole which they imbedded in plasma under perforated cellophane. The fluid medium was composed of horse serum, chick embryo extract, and BSS. Little or no outgrowth occurred, but one culture produced gonadotropic hormone for 413 days and two other cultures produced hormone for 377 days each. During these intervals the increase in content of hormone over that present in similar fragments of tissue at the time of explanting ranged from ten to one hundred and fiftyfold. Again, no estrogen activity was noted. Information concerning cultures of other endocrine tissues or organs is considerably more fragmentary. While a number of endocrine glands have been maintained as organ cultures for brief periods and studies have been made of hormone production by them there have been relatively few attempts to maintain cells from endocrine glands in continuous culture. However, several of these short-term studies using explants are worthy of mention. Maintenance of adrenal explants in hanging drop cultures with continued production of epinephrine was reported by Lewis and Geiling (1935). Over a period of 3 days there was a fivefold increase in epinephrine which was assayed by blood pressure changes in the cat and dilation of the pupil in the frog. LUXet al. (1937-1938) reported studies of homografts of adrenal glands in rats. The glands were maintained in vitro for 10 days and were then tested by transplantation into adrenalectomized rats. Ten of 20 such grafts were successful and 7 were still functional after 5 months. Anterior and posterior pituitary glands of 8-day-old rats were grown in culture by Anderson and Haymaker (1935-1936). After 6 days in vitro the cultures were sacrificed and assayed for hormone production. Equal weights of freshly excised glands were used as controls. The only hormone produced was melanophore-expanding factor which increased tenfold in concentration. Subcultures were prepared using only the outgrowth of the original colonies. The melanophore-expanding factor continued to increase in amount during the first subculture. Experiments were not carried further. However, Engel and Werber (1937) and Cut-

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ting and Lewis (1938) failed to demonstrate hormone production by the pituitary in vitro. Of particular interest is the report by Thompson et al. (1959) on production of hormones by serially passaged human anterior pituitary cells. In this study several lines of epithelial-like cells were derived from anterior pituitary glands and maintained as monolayer and as suspension cultures. Somatotropin, corticotropin, and gonadotropin (both FSH and ICSH) were produced in suspension cultures as well as in the monolayers cultures though yields were not high. Of great practical as well as theoretical interest are the experiments of Gaillard ( 1953) with the parathyroid gland. After maintenance for varying periods in vitro glands were grafted into the axillary region of persons suffering from parathyroid deficiency. Glands from newborn humans were maintained in organ culture for periods up to 1 month. During the last 10 to 15 days in culture the organs were fed with plasma and serum from the prospective recipients. Functional glands were maintained in 7 of 18 patients under 36 years of age. No successful grafts were obtained in persons over 36 years old. The glands remained functional for periods of 4 to 12 years as shown by freedom from symptoms of parathyroid deficiency. In a study of the pancreas Murray and Bradley (1935) grew two island-cell adenomas in culture and later grafted them into the axilla of a diabetic. No significant drop in the requirement for therapeutic insulin was noted. Ovaries and testes have frequently been the object of study in vitro ( Champy, 1920-1922; Ingram, 1956; Long, 1940; Martinovitch, 1937; Mendelsohn, 1937; Payne and Meyer, 1942). Martinovitch ( 1937) reported maintenance of rat ovaries, with maturation division of ova and differentiation of Graafian follicles, with some follicles remaining ‘%ealthy” for 22, days. After 11 days in culture testes of newborn rats showed partial development of spermatocytes from spermatogonia. Long (1940) reported that in addition to ovum development in rat ovaries he could detect follicular hormone production after 4 weeks in vitro. The presence of hormone in the supernatant fluid was tested by the ability to induce estrus in immature mice. Payne and Meyer (1942) detected estrogen in cultures of ovaries maintained in vitro for 5 days. Presence of the hormone was detected by transplantation of the explants into the anterior eye chamber of castrate female rats. While the foregoing selected examples of endocrine function in vitro are very intriguing and, perhaps, are most pertinent to this presentation it will be worthwhile briefly to mention a few other examples of noteworthy biosynthetic activities by animal cells in culture. Ebner and associates ( 1959) have reported significant synthesis of

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lactose and P-lactoglobulin by bovine mammary cells both during primary explantation and after extended periods of culture. Synthetic activity was intimately related to the physiological condition of the cultures. Production of collagen by connective tissue cells in culture was recognized early by Lewis (1917) and has been reported by many workers under a variety of conditions (Bloom 1929-1930; Kuwabara, 1959; Maximov, 1927-1928; Porter and Pappis, 1959; Pumper, 1958; Yardley et al., 1960). While many of these studies utilized freshly transplanted tissue Merchant and Kahn (1958) noted a collagen-like protein in cultures of a mouse fibroblast after 17 years in culture. Ebeling and Fischer ( 1922) described a homogeneous colloidal secretion in the lumen of tubules which formed in mixed cultures of Carrel’s chick heart fibroblast (Carrel and Burrows, 1911a) and Fischer’s chick lens epithelium (Fischer, 1922). The mixed cultures were maintained through seven transplantations. In recent years several workers have reported the synthesis of acid or neutral mucopolysaccharides in cell cultures. Particular attention has been paid to cultures of synovial cells and the production of hyaluronic acid (Castor, 1959; Gaines, 1959; Grossfeld, 1957; Hedberg and Moritz, 1958, Kling et al., 1955). Heparin also has been shown to be synthesized by mast cells in culture (Paff and Bloom, 1949) . The possibility of antibody synthesis in vitro intrigued investigators early in the history of cell culture techniques. Carrel and Ingebrigsten (1912) reported production of hemolysin against goat red blood cells by guinea pig bone marrow and lymph nodes. In recent years Stevens and McKenna (1958) and Fishman (1959) also have reported primary response to antigen by cells in vitro. While most workers have not been able to confirm the above findings, it has been well established that tissues from previously immunized animals will demonstrate a secondary antibody response in culture (Askonas and Humphrey, 1958; Fagraeus, 1948; Kuening and Van Der Slikke, 1950; Sterzl, 1959). B. CHEMICAL CONVERSIONS While the literature on fermentation by animal cells in culture is limited, a few reports have appeared which demonstrate significant conversions of steroids by animal cells or cell fractions in vitro. Several of these studies have utilized established strains of animal cells. Sweat and co-workers ( 1958) reported metabolic conversion of cortisol and progesterone by uterine fibroblasts, strain U12-750. This strain of human uterine cells was isolated by Swim and Parker (1957) and is an established cell line, Stock cultures of the cells were maintained in roller

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tubes and C"-labcled steroid was added to the medium. In this system cortisol was converted to 4-pregnene-llp,l7,20~,21-tetrol-3-one, 4-pregnene-17,20p,21-triol-3,20-dione, corticosterone, and llp-hydroxyandrostenedione-3,17. Progesterone was converted to 4-pregnene-20/3-01-3-one, 4-pregnene-20-01-3-one, and allopregnene-3,20-dione. Berliner et al. (1960) likewise have studied the metabolism of C"labeled corticosterone by fibroblasts, strain U12-79, while Perlman eE nl. ( 1960) have examined the metabolism of progesterone and testostcrone by mammalian cells growing in suspension culture. The latter workers grew L strain mouse fibroblasts in l-liter stirred bottles. Both 4-pregnene20-01-3-one and 4-pregnene-20p-ol-3-one were produced from progesterone with an approximately 2058 yield. Testosterone was converted to androstene-3,17-dione. Velle and Erichsen (1960) have reported the conversion of estrogens by bovine kidney cells in culture. The kidney cortex was trypsinized and planted in Carrel flasks in a lactalbumin hydrolyzate medium. The cells were allowed to grow until a monolayer of epithelial cells was formed. Hormone was added in maintenance medium and after 24 to 96 hours the medium was removed, fractionated, and analyzed. Estradiol-17P was converted to estrone with a 22.9% of yield. The reverse process also occurred and yielded 26.8% estracliol-17p from estrone. Estradiol-17a was also converted to estrone, but the reverse reaction did not take place. The same rate and amount of conversion occurred with the cell cultures irrespective of the sex of the donor animal. In a system composed of human liver homogcnate, nicotinamide, citrate, and reduced TPN, Atherden (1959) observed rapid conversion of progesterone to 5- and 5a-pregnene-3,20-dione, 3- and 3p-hydroxy-5and 5p-pregnene-3,20pregnene-2O-one, 3-hydroxy-5p-pregnene-20-one, diol. Similarly Ryan (1959) noted conversion of androgens to estrogens in high yield by a system consisting of human placental microsomes, reduced TPN, and oxygen. The ability of stallion testis, human term placenta, and a human feminizing adrenal cortical carcinoma to convert Cl4-testosterone to G4labeled estrogenic steroids was demonstrated by Baggett et al. ( 1959). Isolation of 5 ( 4 5 ) -dihydroaldosterone and 3p-hydroxy-5( 4,s) -tetrahydroaldosterone from incubation mixtures of d-aldosterone and rat liver homogenates has been reported by Pechet and co-workers ( 1960).

IV. Some Trends in Animal Cell and Organ Culture Research The practical application of cell or organ culture on a large scale must depend, significantly, upon the ability to obtain and define adequate in vitro systems. It therefore is pertinent, as background for a discussion of

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possible uses of cell cultures, to consider briefly certain areas in which rapid advances are being made toward a clear understanding of the in vitro system and perhaps of its relation to the in vitro situation. A. DEFINITION OF NUTRITIONAL AND PHYSICAL REQUIREMENTS FOR ANIMAL CELLSIN CULTURE Rapid progress has been made in the past decade in defining nutritional requirements of animal cells in culture both on a qualitative and on a quantitative basis. While the subject has been discussed in several excellent reviews (Eagle, 1958; Morgan, 1958; Paul, 1960) it is important to focus attention here on several points which relate rather specifically to the subject of large-scale cell culture and its applications. Definition of nutritional requirements of cells, along with other developments, has made it possible to simplify greatly the medium used for cultivation of animal cells and tissues, This has been characterized by the use of nutrient solutions in which all the components are defined (Eagle, 1955; Evans et al., 1956; Waymouth, 1959) or in which the undefined portion has been greatly refined and reduced to a small fraction of the total medium. Such a situation makes possible the sensitive biochemical determinations and control required to evaluate the system in terms of synthetic or fermentative activity. An additional, and perhaps equally important, benefit which has accrued with the simplification of nutrient solutions has been the elimination of many inhibitors and toxic factors which are common to complex natural supplements such as serum, embryonic extract, ascitic fluid, etc. While this has been documented most frequently with respect to virus inhibitors (Burnet and Lind, 1954; Ginsberg and Horsfall, 1949; Hilleman and Werner, 1953; Sampaio and Isaacs, 1953) and cytotoxic factors (Bjorklund, 1960; Bolande and Todd, 1958; Federoff and Cook, 1959; Rose, 1958) it is reasonable to suppose that specific or nonspecific inhibitors of hormones or other physiologically active compounds produced by cells may be present also in many natural products. The progress to date in the area of animal cell nutrition in uitro, impressive as it is, must be assumed to be only fragmentary. In particular we know little concerning the nutritional requirements for specific cell synthetic activities or specialized function. The criteria used in determining the qualitative and quantitative nutritional requirements to date have been: ( a ) ability to support growth or multiplication; ( b ) maintenance of viability; ( c ) plating efficiency. The latter measures a combination of growth requirements and attachment factors. The notable exception with regard to requirements for specific function are the studies of nutritional needs for virus synthesis (Ackermann, 1951; Bader and Morgan, 1958;

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Daniels et al., 1952; Eagle and Habel, 1956; Tyndall and Ludwig, 1960). It is not known whether this is a unique situation. While much effort has been directed toward definition of nutritional needs and the development of defined media, a significant amount of attention also has been directed toward finding simple and inexpensive supplements. Included among the materials thus evaluated are many components such as peptones, protein hydrolyzates, and bacteriological media (Ginsberg et al., 1955; Mayyasi and Schuurmans, 1956; Melnick, 1955; Waymouth, 1956), some of which were recognized as potential animal cell nutrients by early investigators (Carrel and Baker, 1926; Lewis and Lewis, 1911). Other materials which have been used widely or have shown promise are yeast extract (Melnick, 1955) and skim milk (Rabson et al., 1958). Parallel to the developments in cell nutrition there have been a number of studies in recent years which emphasize the role of various components of the medium in affecting the physical environment of cells apart from a direct nutritional action, These effects have consisted in part of direct action of the materials on the cells and in part with interactions of the substances with the surface on which the cells were growing or with other components of the medium. A few examples will serve to point up the significance of such observations. Fisher et al. (1958) have described a factor in fetal calf serum which they designate fetuin and which, in addition to serving as a nutrient, acts in promoting attachment and spreading of cells on glass. It has been characterized as a glycoprotein and accounts for as high as 80% of the total protein in fetal calf serum. Lieberman and Ove (1958b) likewise have reported a glycoprotein from beef serum which has the property of promoting attachment of cells to glass, They further state that certain basic polypeptides could be substituted for the protcin factor. Additional studies by Lieberman et al. (1959) suggest that the “attachment factor” is not identical with fetuin. On the basis of their studies with the “attachment factor,” and with polypeptides, Lieberman and Ove (195813) suggested a mechanism of cell attachment to glass which involves a protcin on the cell surface and polyvalent cations. Results of studies by Weiss (1959) of the effect of various serum fractions on attachment of cells to glass surfaces are in general agreement with such an hypothesis. The differences between trypsin-treated and non-trypsin-treated cells in their attachment are particularly convincing. The findings of Kuchler et 01. (1960) extend the mechanism of cell binding ascribed to protein in the above studies to include methylcellulose. Earle et al. (1954) and McLimans et al. (1957a, b ) added methyl-

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cellulose to suspension cultures, primarily to increase viscosity. In both instances the medium also contained serum. Merchant et al. (1960) were able to replace the serum of the medium with peptone and methylcellulose. In this case (Kuchler et al., 1960) methylcellulose coats the cell surface and functions in prevention of cross binding. Merchant et al. (1960) also reported the use of a different type of compound to prevent aggregation of cells in suspension and to prevent binding to glass surfaces. These compounds are known as “dispersing agents” and are polysulfonic acids. They are effective primarily in increasing the net negative charge on the cell surface. A very interesting and significant series of papers by Rappaport and others (Rappaport, 1960a, b; Rappaport and Bishop, 1960) further elucidate the nature of the glass surface required for attachment of cells, particularly in defined medium. Thus it would seem likely that many factors interplay to affect the binding of cells to solid surfaces or to other cells. A clearer knowledge of this subject is essential to the expanded application of cell culture techniques. B. CHARACTERIZATION OF METABOLIC PATHWAYS Much of the early work on metabolism of cell cultures was concerned with the question of utilization of amino acids and other small molecules as opposed to incorporation of large molecules such as serum proteins and components of embryonic extract ( Davidson and Leslie, 1950; Fischer, 1953; Winnick and Winnick, 1953). Recent developments in medium simplification and the application of isotope-labeling methods largely have clarified the situation. A question which has been troublesome from the inception of the cell culture method, is the possibility that all cells grown in culture are abnormal and indeed may become malignant. This has arisen because of the many similarities between cultured and malignant cells such as abnormal chromosome patterns, growth rates, etc. Warburg ( 1956) has focused attention on the question of carbohydrate metabolism and has emphasized the high “glycolysis” to “respiration” ratio in most cultured cells, As a result much attention has been given to study of carbohydrate metabolism and a fairly clear picture is beginning to emerge (AbdelTawab and Broda, 1959; Barban and Schulze, 1956; Eagle et al., 1958; Jones and Bonting, 1956; Leslie et al., 1957; Lieberman and Ove, 1958a; Munyon and Merchant, 1959; Paul, 1959). Likewise amino-acid metabolism (Eagle, 1959; Manson and Thomas, 1960; Pasieka et’al., 1958), protein synthesis and turnover (Eagle et al., 1959; Francis and Winnick, 1953; Jordan et al., 1959; Morgan and McCrone, 1957; Salzman, 1959; Sinclair and Leslie, 1959), and nucleic

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acid metabolism (Feindendegen et al., 1960; Gerarde et al., 1952; Lu and Winnick, 1954; McFall and Magasanik, 1960; Paul, 1959; Salzman, 1959) have received increasing attention. While only a very few points are clearly defined in these areas studies are now underway in many laboratories and rapid advances are to be expected. The development of rapid cloning techniques by Puck et al. (1956) and the advances in quantitative technology and in development of simplified nutrients, which we have already discussed make possible definition of metabolism which is essential for the practical application of cell culture. Of equal significance, however, is a realization of the interdependence of metabolism and the general physiological status of the cell culture. Recently there have been several studies, such as those of Merchant et nl. (1960), which have related metabolic activity to the physiological growth cycle of cell populations. Further use of this method would seem to be in order. C. LARGE-SCALE CELLPRODUCTION

The possibility of massive cultures of animal cells was recognized when the development of the monolayer technique (Evans and Earle, 1947) freed cell cultures from the plasma clot. Shortly thereafter larger and larger flasks were employed (Earle et al., 1950; Shannon et al., 1952). The maximum practical use of the monolayer method was achieved in its application to the production of virus vaccines as described earlier. Earle et nl. (1951) attempted an improvement of the monolayer system by providing a three-dimensional surface. This did not prove to be practical though a different three-dimensional surface has been used by Leighton ( 1954) to encourage cell organization and differentiation. The recognition by Owens et al. (1953, 1954) that animal cells could proliferate while in free suspension and the practical development of this method by Earle et al. (1954, 1956) opened an important new area of approach. Earle et al. (1956) recognized the similarity of this system to bacterial and fungal cultures and suggested the possible use of fermentation methods. The growth of cells in suspension on a rotary shaker, however, limited the expansion of the method and further development came largely as a result of adaptation of stirred cultures. While numerous workers have contributed to the development of this methodology the initial credit perhaps goes to Cherry and Hull (1956) who adapted the magnetic stirrer to give a closed system which, in many ways, is ideally suited to the cell culture system. Scaling up of cell cultures has moved ahead rapidly in the past 4 to 5 years. McLimans and associates ( McLimans d al., 1957a, b; Ziegler et al., 1958) have been particularly active in this regard, though a number of

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other laboratories have grown cells on a large scale. Thus Nickell and Tulecke (1960) obtained 7-lb. wet weight of plant cells from a culture maintained in a stainless steel fermentor using 135 liters of medium. Rightsel et al. (1960) similarly have grown monkey kidney cells in a 30-liter stainless steel fermentor. The chief problem with additional scale up would appear to be the cost of the medium aside from the difficulties normally encountered in any fermentor scale up.

D. CONTINUOUS CELLCULTURE The application of continuous culture methods to the growth of animal cells has been a comparatively recent development though the Lindbergh apparatus (1931) perhaps was an abortive attempt. Two distinct methods of continuous culture have been developed to date. Graff and McCarty (1957) reported an apparatus for continuous exchange of the fluid medium thus providing a “constant” chemical environment. The population was allowed to increase so that a truly constant condition was never achieved and the length of time over which the system could be operated was limited. This system is well suited for many chemical analyses and has achieved wide application. Continuous culture methods analagous to the “chemostat” of Novick and Szilard (1950) have been devised by Cooper et al. (1959), by Merchant et al. ( 1960), and by Cohen and Eagle (1960). In these systems the population is maintained at a constant level and cells have been maintained in a “steady state” for extended intervals.

V. Speculation on Applications of Cell and Organ Culture The information currently available would seem to warrant the following general conclusions: ( a ) Animal cell cultures can be scaled LIP to almost any desired level; ( b ) a wide variety of cell strains can be grown on a mass scale; ( c ) many cells can synthesize physiologically active substances in vitm; ( d ) biochemical conversions can be achieved with cell culture systems. The acceptance of the above statements does not imply that biological synthesis or conversion of any compound can be performed in vitro on a practical basis. It does indicate that avenues of approach are open and that the feasibility of the method can be put to experimental test. Several approaches suggest themselves. Pursuit of these might prove to be quite fruitful. A continued simplification of the nutrient medium for large-scale growth of cells would serve to eliminate inhibitors or antagonistic components and in addition would aid in the detection and isolation of any

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active compounds which might be produced, The elimination of such ill-defined natural components as serum would seem to be particularly important. This does not imply, necessarily, that a completely defined medium is required or desirable, While large quantities of cells would be necessary for the commercial production of any compound it is extremely important to recognize that rapidly growing cells may not function maximally in the synthesis of specialized compounds. It long has been recognized that growth and differentiation, to a degree, may be antagonistic (Fawcett, 1955; Fischer and Parker, 1928). Cells in a rapidly growing population are likely to be preoccupied with the synthetic activities associated with replication. The answer to these problems would seem to be growth of large populations and then maintenance under conditions more favorable for specialized function (Merchant et al., 1960). Actually, the fermentor, operating on a continuous basis, might be the answer. Agitation and flow rate could be so adjusted in a fermentor that once the cell population was grown to a desired maximum, controlled additives of nutrient or substrate would permit the desired product to be bled off in constant yield over long periods of time. Additional points which have not been adequately studied are the possible need to supply appropriate precursors and the need to add physiologically active substances to the culture system. The significant interactions of the endocrine glands in vivo suggest that we might expect one hormone to affect production or conversion of another hormone. Using organ culture methods Schaberg ( 1957) has demonstrated, in vitro, the interdependence of endocrine glands. Adrenals were cultured and demonstrated to synthesize steroid hormones, Gradually the synthesis of steroids ceased but could be reactivated either by co-culture of the anterior pituitary or by addition of ACTH. It is not clear that this was a direct action on steroid synthesis rather than a release of preformed hormone or of activation of an inactive precursor. Nevertheless, an important, and possibly a very rewarding area of investigation is suggested. A very intriguing possibility is the utilization of mixed cell cultures or of “organ” or tissue cultures rather than cell strains. Mixed culture fermentations by bacteria long have been utilized and the subject has been reviewed by Gerhardt and Bartlett (1959). The possibility of a chain reaction requiring more than one cell type is not at all unlikely in animal systems where cells often reach a high degree of specialization. Likewise a certain degree of organization of cell types might be required for the efficient production or conversion of some compounds. Finally, the success of large-scale use of cell cultures for production of biochemicals will depend in large measure on the application of well-

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established microbiological procedures. Cell strains must be assayed and selected. These must be cloned and characterized, and stock strains must be rigidly controlled, Storage of prototype cultures under conditions which minimize change is imperative. If these conditions are met and the problems which have been outlined above are solved we confidently expect that large-scale use of cell cultures will be commonplace.

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Stewart, H. L., Jr., Sano, M. E., and Montgomery, T. L. (1948). J. Clin. E n d o c r i d 8, 175-181. Strangeways, T. S. P., and Fell, H. B. (1925-1926). Proc. Roy. S O C . B99, 34@-366. Sweat, M. L., Grosser, B. I., Berliner, D. L., Swim, H. E., Nabors, C. J., Jr., and Dougherty, T. F. ( 1958). Biochim. et Biophys. Acta 28, 591-596. Swim, H. E., and Parker, R. F. (1957). Am. J. H y g . 66,235-243. Thompson, K . W., Vincent, M. M., Jensen, F. C., Price, R. T., and Schapiro, E. (1959). Proc. SOC. Erptl. B i d . Med. 102, 403-408. Tyndall, R. L., and Ludwig, E. H. (1960). J. Bacteriol. 80, 96-103. Velle, W., and Erichsen, S . (1960). Acta Endocrinol. 33, 277-286. Waltz, H. K., Tullner, W. W., Evans, V. J., Hertz, R., and Earle, W. R. (1954). J. Natl. Cancer Inst. 14, 1173-1185. Warburg, 0. ( 1956). Science 123,309414. Waymouth, C. ( 1956). 1. Natl. Cancer Inst. 17,315-325. Waymouth, C. (1959). j . Natl. Cancer Inst. 22, 1003-1017. Weiss, L. (1959). Exptl. Cell Research 17, 499-507. Westwood, J. C. N., Macpherson, I. A., and Titmuss, D. H. J. (1957). Brit. J. Exptl. Pathol. 38, 138-154. White, P. R. (1932). Arch. exptl. ZcUfoTsch. 12, 602-620. Winnick, R. E., and Winnick, T. ( 1953). J. Natl. Cancer Inst. 14, 519-525. Yardley, J. H., Heaton, M. W., Gaines, M. L., and Shulman, L. (1960). Bull. ]ohm Hopkins H o s p . 106,381-393. Ziegler, D. W., Davis, E. V., Thomas, W. J., and McLimans, W. F. (1958). Appl. Microbiol. 6, 305-310.

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Protection against infection in the Microbiological Laboratory: Devices and Procedures MARKA. CHATIGNY Naval Biological Laboratory, School of Public Health, University of California, Berkeley, California I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 11. Recent History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 111. Routes and Sources of Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Inoculation through the Skin . . . . . . . . . . . . . . . . . . . . . . . B. Ingestion ....................... C. Respiratory Tract Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 IV. Safety Devices and Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 A. The Safety Hood . . . . . . . . . . . . . . . . . . . B. The Culture-Shaking Machine . . . . . . . . C. The Centrifuge . . . . . . . . . . . . . . . . . . . . . . . . 157 D. The Blendor and Similar E. The Pipette ....................... F. The Syringe and Needle and the Inoculat G. The Incubator .............................. . . . . . . . . . . . . 162 H. The Steam Pressure Sterilizer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 I. Animal Exposure Equipment ( Respiratory) . . . . . . . . . . . . . . . . . . . 163 J. Animal-Holding Devices . . . . . . . . . . . . . K. Protective Clothing . . . . . . . . . . . . . . . . . L. The Use of Plastics in Laboratory Safety Devices . . . . . . . . . . . . . . . . . . 171 V. Decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 A. Thermal Decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 B. Liquid Decontaminants . . . . . . . . . . C. Gas or Vapor Phase Decontaminant D. Ultraviolet Irradiation ..................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 VI. Laboratory Design . . . . . . . . A. Location and Traffic P . . . . . . . . 179 B. Room Finishes ......... C. Services . . . . . . . . . . . . ........................ 181 D. Ventilation Systems . . . . . . . . . . . VII. Safety Programs and Adm'nist-.Ition VIII. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 IX. Commercially Available Safety Devi X. Motion Pictures and Fil Procedures . . . . . . . . . . . . . ................................. 186 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

I. Introduction In the past two decades there has been a vast growth in research, teaching, and clinical laboratory work in microbiology. In spite of many 131

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advances in protective measures made during this period, laboratoryacquired infections appear to have increased at a rate nearly paralleling this growth, and the risk of acquiring infection is still a severe problem to the laboratory worker. The existence of the problem has long been recognized and research scientists, clinicians, engineers, and many other workers have all contributed corrective measures from their own areas of special competence in a beginning of a scientific evaluation of the problem of laboratory safety. While a comprehensive set of standards has not yet been devised for safe microbiological work, sufficient information has been collected to permit development of reasonable precautions and safeguards which should be employed whenever applicable. Frequently, the use of such precautions is required by law, For example, the California Labor Code requires that an employer “shall furnish and use safety devices and safeguards . . . reasonably adequate to render such employment and place of employment safe” (Kleps, 1957). Even without such regulations common sense alone dictates the necessity of protecting workers from injury and death. It is time, therefore, for the laboratory infection problem to be included in the portfolio of the industrial hygienist, to have accident data more faithfully recorded, and to have people whose primary concern is safety blend their experience and knowledge with those of the laboratory worker in a continuing and coordinated effort to evaluate and to control laboratory-acquired diseases. This article will attempt to review the recent background of the laboratory infection problem; it will discuss some sources of such infections and will examine some of the more significant protective measures that have been employed for their control. It will be concerned only with protection against laboratory-acquired infectious diseases rather than with the general problems of industrial safety.

II. Recent History The early recognition accorded the problem of laboratory-acquired infection was the result of work on highly infectious diseases such as brucellosis, tularemia, tuberculosis, and Q fever. Prior to World War I1 microbiology laboratory safety was primarily the concern of the individual worker. However, by the 1940’s the problem of laboratory infections was of such magnitude as to attract attention, and several reports described the hazards of employment in infectious disease hospitals and laboratories and noted that such workers were subjected to greater occupational risks than those in comparable pursuits. Morris ( 1946), Gruber

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(1949), and Mikol et al. (1952) reported the incidence of occupational infection in tuberculosis hospitals and laboratories. Brucellosis was seen as a hazard by Meyer and Eddie (1941) who recorded 74 cases of infections acquired in the, laboratory. Forty-seven cases of Q fever infections in a laboratory group were reported by Huebner (1947). Sulkin and Pike ( 1949, 1951a, b), in extensive surveys employing literature search and the analysis of questionnaires returned by' cooperating laboratories, collected extensive data for the period 1930-1950 on laboratory infections caused by a wide variety of bacteria, viruses, rickettsia, fungi, and other parasites in addition to those agents noted above. While this survey is not current, many of the facts presented in it are still pertinent and useful. A total of 1342 laboratory infections was reported. Of these, approximately 16%(215) were attributed to proven laboratory accidents and the remainder to laboratory operations in which no overt accident was known to have occurred: The total number of deaths (39) was 3%of the total cases, Brucellosis, tuberculosis, tularemia, typhoid, and streptococcal infections accounted for 31%of all infections. Viral hepatitis, psittacosis, Q fever, typhus, and coccidioidomycosis accounted for another 26.7%. In all, 69 different agents were implicated. About onethird of the infections noted were recorded in the literature, the remainder being ascertained from questionnaires. Most infections ( 75.31) occurred in trained laboratory workers and animal caretakers; janitors and dishwashers accounted for the next largest grouping with 135 (10%) incidents, with students, clerical workers, and others accounting for the remainder. With a few notable exceptions there appears to have been little change in the pattern of laboratory-acquired infection in the decade since the Sulkin and Pike survey. Tuberculosis is still the cause of a great number of cases due, in large part, to the great number of workers involved. Reid (1957), in reporting the incidence of pulmonary tuberculosis in medical laboratory workers, found a tuberculosis rate 2 to 4 times higher than that expected from natural causes in each age group examined. Merger (1957) stated that the incidence of the disease in tuberculosis laboratory workers in Ontario, Canada was approximately 28 times that of the general population for that province and 8.4 times that for all of Canada (less Quebec province), Considering other infections, Merger (1957) reported the occurrence of 5 infections with Brucella abortus and 1 with Pnsteurella tularensis in a small staff engaged in preparing antigens. Van Metre and Kadull (1959) described 62 cases of laboratoryacquired tularemia. A newly discovered hazard, Monkey B virus, has been reported by Davidson and Hummeler (1960) as responsible for 12 deaths in 15 cases, 9 of which occurred in 1957 and 1958. This virus

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disease is considered statistically rare, but the tremendous increase in the importation and use of the Macncn mulnttn (Rhesus) and M . phillippinensis (cynomolguS) monkey for research and production of biologicals has caused the infection incidence to rise to a level attracting notice. Although the risk must be low, if one considers the vast number of monkeys handled in the past few years, it presents a serious problem since there is no known method of effective therapy or of immunological protection. Coccidioides imniitis appears still to be a prolific generator of laboratory infections. Fiese (1958) cites several cases and notes that “the only occupations more hazardous ( in respect to coccidioidal infection) than agricultural and allied pursuits are those which involve handling CoccidioWes in the laboratory.” In this disease it is very likely that the cases reported represent only a fraction of actual infections, since it has been estimated that substantially less than 40%of natural infections reach the attention of the clinician. As a case in point, asymptomatic infections occurred in 5 workers and a clinical case in 1 worker out of a group of 9 studying this organism at the Naval Biological Laboratory in the period 1953-1959 ( Levine, 1960). These workers were all utilizing “reasonable” precautions and protective equipment. Similarly, Dickie and Murphy ( 1955), in describing laboratory infection with Histoplasma cnpsulntum, noted that 18 students in medical technology developed strong positive skin tests but only 2 showed overt symptoms. Accidents with other bacterial, viral, and rickettsia1 agents reported in the literature include infections such as viral hepatitis, Russian spring summer viral encephalitis, shigellosis, poliomyelitis, leptospirosis, psittacosis, ancl many others. The statistics of laboratory infection are of little interest to the laboratory worker until he becomes a participating statistic. However, it is helpful to the industrial hygienist and to management to know the relative risks of laboratory work. It would be valuable to develop accident rates and injury severity rates from the data available for comparison with rates in other occupations. However, up to this time the reporting of laboratory-acquired infections in the open literature has not been sufficiently reliable to permit anything more than intelligent approximations. On this basis, Sulkin and Pike (1951a) estimated the incidence of laboratory-acquired infections in various types of laboratories over a 20-year period. They calculated an average infection-producing accident rate of 0.5 infections per year per 1000 workers (0.25 infections per million man hours) for all the reporting laboratories. Research institutes had the high rate of 4.1, whereas a low of 0.2 was recorded for clinical laboratories. To use the now standard terminology of “accidents per

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MICROBIOLOGY LABORATORY SAFETY

million man hours," these rates change to 0.25, 2.05, and 0.1 accidents per million man hours, respectively, These accident rates were based on estimates of the number of personnel in the laboratories and the total number of hours worked. Since most of the data were not current at the time of the surveys and since many of the accidents were listed from memory by personnel involved, these rates -may be conservative. Wedum (1957) proposed that the infectious disease accident rate of 2.05 accidents per million man hours given by Sulkin and Pike be combined with the 1953 hospital laboratory rate of 4.2 given by the National Safety Council for all injuries other than occupational disease, to develop a rate of 6.25 accidents per million man hours for a large infectious disease institution. He reported average annual rates ranging from 4.0 to LOSTTIMEACCIDENTS

PER ~~

TABLE I MILLIONMAN HOURS EMPLOYMENT ~

Clinical laboratories (TB hospital) (1953) All industry (1955) Hospitals (general) (1953) Infectious disease institutes (1957) Clinical laboratories (general hospital) (1953) Chemical industry (1954)

11.8" 6 . 96b 6.5" 6 . 25c*d 4.5a 4.12d

U. S. Department of Labor ( 1958). Safety Council Statistics ( 1955). ' A theoretical combination of mechanical, chemical, and infectious hazards. Wedum ( 1957).

* National

7.9 over a period of several years for work at the U. S. Army Chemical Corps Biological Laboratories, Fort Detrick, Maryland. A recent study of work injuries and work injury rates in hospitals made by the U. S. Bureau of Labor Statistics (U. S. Department of Labor, 1958), indicated an average rate of 4.5 accidents per million man hours (all types of accidents) for clinical laboratories in general hospitals, but a rate of 11.8 for tuberculosis hospital laboratories. It was observed that infections constituted one-third of all the laboratory accidents. These rates and others are compared in Table I. In this study there was also an indication that the nature of injuries in hospital clinical laboratories is similar to that reported by Sulkin and Pike ( 1951b). Cuts and lacerations and occupational diseases accounted for the majority of accidents. Additional data acquired from the California State Public Health Laboratories are similar and tend to confirm this distribution (see Fig. 1). Data on work time lost in each accident causing disabling injury, given in Table 11, further emphasizes the se-

136

MARK A. CHATIGNY INJURY 0 I

STRAINS AND SPRAINS

FREQUENCY: PERCENT OF A L L DISABLING INJURIES 10 20 30 40 50 I

I

*

+

t--]

BRUISES AND CONTUSIONS CUTS AND LACERATIONS FRACTURES

A

OCCUPATIONAL DISEASES BURNS AND SCALDS HERNIAS I R R I T A T I O N S DUE TO FOREIGN AMPUTATIONS

0 DATA

( ' ) H O S P I T A L , ALL D E P T S . 1953 ( 1 )CLINICAL

LABORATORIES I N H O S P I T A L S 1953

(

2 ) S T A T~E PUBLIC ~ HEALTH LABORATORIES 1956

~

DATA FROM CALIF. S T A T E P U B L I C H E A L T H L A B O R A T O R I E S INCLUDES NON D I S A B L I N G I N J U R I E S S O U R C E : ( I ) U.S. DEPT. OF L A B O R B U R E A U OF L A B O R S T A T I S T I C S , B U L L E T I N NO. 1219 (2) C A L I F . S T A T E D E P T . O F P U B L I C H E A L T H L A B O R A T O R Y ACCIDENT REPORTS.

FIG.1. Work injuries in hospital laboratories (by nature of injury),

riousness of work-acquired infections. For e,xample, the average time lost for all accidents in hospital clinical laboratories (214 days per disabling injury) is more than three times greater than that observed for all manufacturing industries (85 days per disabling injury). When only workacquired infections are considered, the comparison becomes even more disparate with an average of 441 days lost for each hospital occupational disease incident and 912 days lost for the infective disease incidents acquired in hospitals and hospital laboratories.

~

~

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It is seen from Table I that the accident rate for laboratories varies widely and, in the case of the tuberculosis hospitals, appears to be affected by the infectiousness of the disease agent involved, as is the time loss from the accident as seen ip Table 11. The theoretical rate proposed by Wedum for a large research institute appears to be of comparable magnitude to those for hospitals and industry. As one might expect, it is higher than that for the chemical industry wherein somewhat similar occupational hazards prevail. It is of further interest to note that the observed death rate of 3%for laboratory-acquired infections ( Sulkin and Pike, 1951b) is high compared with the average of 0.3%for accidents in several major manufacturing groups (U. S. Department of Labor, 1954). Recent information TABLE I1 AVERAGE NUMBEROF DAYSLOSTOR CHARGED rE6 DISABLING INJURY All manufacturing industriesa (1952) All hospitals,* all injuries (1953) All hospitals,* occupational diseases Infective and parasitic diseases in hospita1s:b (1953) total Virua diseases Tuberculosis Other diseases All hospital clinical laboratoriesb (average)

85 62 441 912 128 1284 32 214

" Based on reports which furnished details regarding duration of the resulting disabilities during the year 1952 (consisting of 80% of the sample); U. S. Department of Labor ( 1954). * Includes all data for year 1953; U.S. Department of Labor ( 1958).

( Sulkin, 1960) indicates that the laboratory infection death rate appears to have risen to approximately 5%in recent years. The history of laboratory infection showing disease and sometimes death caused by every infectious agent studied or employed (Anonymous, 1951), the magnitude of the accident rate, and the lack of substantial improvement in accident prevention during the past decade all show that improved corrective action is necessary.

111. Routes a n d Sources o f Infection It is noteworthy that in all the recent surveys of laboratory-acquired infections, the sources of a large majority of these infections have not been precisely identified. As a result, correction of the circumstances creating the exposures has been difficult or impossi6le. Knowledge of the exact source of infection would be the single most useful tool available for solution of this problem. If this information is

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not available, one is limited to examining what is known concerning the most probable routes and sources of infection. A. INOCULATION THROUGH

THE

SKIN

Most of the proved cases of accidental infection reported have resulted from very obvious incidents. As Long (1951) has suggested, a majority of such cases depended on the skin or the alimentary tract as the portal of entry. Sulkin and Pike (1951a) noted that some 26.5%of accidental infections involved the use of the needle and syringe. Similarly, of 33 laboratory infections reported elsewhere in the literature, 16 involved this device. Skin contamination by spilling or spattering of organisms was the second most frequent type of accident, accounting for 21.48 of the total (Sulkin and Pike, 1951b). Cuts and puncture wounds caused by surgical instruments are no small part of the total, and it is thought that many of the tuberculosis laboratory infections reported by Smith (1953) were caused by this means. Laceration with broken glass or with autopsy instruments is of very frequent occurrence in hospital clinical laboratories (U. S . Department of Labor, 1958). If glassware is contaminated, the possibility of accidental inoculation is obvious. Abrasion of the skin with contaminated grinding compounds or other materials (usually small breaks in the skin are implicated) is less obvious but nonetheless a real hazard. More dramatic is the problem of animal bites and scratches. Most of the 12 fatal Monkey B virus cases cited earlier were attributed to monkey bites or to penetration through broken skin when infected tissues were handled, Dolan et nl. (1958) noted the accidental transmission of ringworm infection to laboratory workers from mice during a mouse colony epizootic. The combination of animals biting or scratching, coupled with the inherent danger of the syringe and needle, points up the existence of a real hazard to those workers engaged in inoculating large mammals, particularly primates.

B. INGESTION Mouth operation of pipettes in the transfer of liquid cultures or specimens appears to be the most frequent cause of overt ingestion. Thirtythree out of a group of 216 laboratory accidents reported by Sulkin and Pike (1951b) occurred in pipetting operations. In spite of the widely used precaution of plugging the proximal ends of the tubes with cotton, mouth pipetting presents the hazard not only of direct ingestion but also that of hand and finger contamination with subsequent manual dissemination of infectious material throughout the laboratory.

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C. RESPIRATORY TRACT EXPOSURE A large majority of the cases noted in the surveys listed above are of undefined origins. The area and the type of work involved in the exposure were usually known, but the specifics of exposure were not. In most instances, airborne infection was presumed and the incident was reported as such or as from unknown causes. Respiratory tract exposure requires the greatest attention in any consideration of laboratory safety, not only because of its frequency but also because it affords the greatest difficulty in establishing and subsequently controlling the sources of contamination. A number of investigators, most notably those of the Biological Laboratories, U. S. Army Chemical Corps, who have made extensive studies of laboratory hazards, have attempted to evaluate the aerosol hazards associated with various laboratory operations. In some instances, typical laboratory procedures of pipetting, inoculating cultures, and many more were performed employing innocuous microorganisms as simulants for the pathogens. Samples of air and surface contaminants were taken at strategic locations on the work surface and in the air around the workspace. In other studies, workers sampled the air and surfaces around actual laboratory 0perations.l Morris ( 1960) has reviewed some of these tests and has discussed the mode by which many of these operations produce aerosols, The following data are drawn from these simulant trials, tests of actual procedures, and from some personal experiences. 1. Aerosols Produced by Common Laboratory Techniques Many common bacteriological and viral laboratory techniques produce aerosols. Johansson and Ferris ( 1946) photographed aerosols generated in the process of pipetting samples onto agar petri plates. For example, when the last drop of fluid in the tip of a pipette was forcibly expelled, a spray of more than 10,000 droplets was produced. While they did not examine the aerosol particle size distribution in detail, it is apparent from their photographs that this aerosolization produced many small particles (less than 5 p in diameter) in addition to some very large droplets which fell immediately to the work surface. Other common laboratory techniques were examined by Anderson et al. (1952) who used indicator organisms (Serratia indica) in conjunction with sieve-type air samplers to detect aerosols. They studied aerosols produced by pipetting, by operating the Waring blendor, by causing small droplets of liquid culture Many of these tests, along with corrective safety measures, have been described and recorded in filmstrips and motion pictures. A listing of these films.and others describing various microbiology laboratory safety procedures is given in Section X.

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to fall from varying distances onto several types of materials, by removing stoppers from bottles, by removing an inoculum from a vaccine bottle, by flaming a loop, by inoculating cultures, and by other typical laboratory operations, Their tests showed aerosol colony counts ranging from an average of 0.3 from a simple slide agglutination technique to “too numerous to count” (greater than 2000) from opening a Waring blendor immediately after use. Similar studies on various techniques employed with viruses were made by Reitman et al. (1954b). Using coliphage-T-3 as a simulant for a pathogenic virus, they demonstrated area contamination and aerosol generation from such operations as intranasal instillation of culture suspensions into mice, intracerebral inoculation of mice, egg inoculation, egg harvesting, serial diluting, and the grinding of brain tissue in a TenBroeck grinder. Wide variation in results was observed, but the presence of aerosols from each operation was undeniable and was accompanied by heavy contamination of the workers’ hands, the work surfaces, and of the animals, eggs, or culture vessels. More detailed studies on each of several common operations have been made by Reitman and Phillips (1955) who examined pipetting, by Hanel and Alg (1955) who studied syringe and needle manipulations, and by Phillips and Reitman (1956) who made tests on operations with an inoculating loop. These tests again indicated the generation of aerosols in varying concentrations from every one of the operations. Reitman and Phillips ( 1956) also demonstrated that aerosols were produced during operations concerned with centrifugation of culture samples, as did Salvador et al. (1956) who described the escape of a “mist” from a centrifuge containing tuberculin. In the latter case severe allergic reactions occurred in 4 people who were in the room for a short time. Wedum (1953) and Reitman and Wedum (1958) have collected and tabulated much of this information and, in addition, have provided further data on aerosol generation from simulated trials of various manipulations connected with the processes of lyophilization, centrifugation, animal inoculation, and autopsy, Quantitatively, the results showed wide variations, ranging from no bacterial counts for many operations to recovery of more than 2000 colonies from breaking an ampule containing 2 ml. of lyophilized S. indica culture. Tomlinson (1957), in studying the generation of an aerosol from the act of opening a screw-capped bottle of culture material, recovered up to 48 colonies from the air. The aerosol came from the contamination occasioned when the inoculating loop touched the sides of the bottle or when vapors of the contents condensed inside the cap and permitted bacterial growth on the rims. It was estimated that many of the particles aerosolized were less than 5 in

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diameter. Hambleton (1957) noted that the trisodium phosphate frequently used in the diagnostic culture of the tubercle bacilli tends to dry to a friable crust around the screw threads and is discharged as a contaminated fine powder when the tube or bottle is opened after incubation. Dekking (1954) took flash photographs of the operation of making a sputum smear between glass slides and observed aerosol formation. Thus, it is apparent that even the simplest operations can produce aerosols. TABLE I11 EXAMPLES OF AEROSOLSPRODUCED FROM SOME COMMON BACTERIOLOGICAL TECHNIQUES" Average number of colonies on air sampler plate Laboratory operation

Minimum

Maximum

Agglutination, slide drop technique Animal injection (guinea pig) undisinfected inoculation site Centrifuge, tube broken, culture stayed in cup Centrifuge, tube broken, culture splashed outside of centrifuge One drop S . indicu falling 3 in. onto: Stainless steel Dry hand towel Towel wet with 5% phenol Insert hot loop in culture Insert cold loop in culture Break ampule of lyophilized S. indicu Streaking, rough agar plate with loop Pipetting, inoculate culture Using blendor with poor fitting parts Opening screw-cap bottle

0 15 0

0.66 16 20 1800

80

0.2 0.0 0.0 0.68 0.0 1939 7 0 77 0

4.7 0.35 0.05 25 0.22 2040 73 2 1246 45

Condensed from Reitman and Wedum ( 1956) and Tomlinson ( 1957).

Autopsy of infected animals presents not only the obvious hazard of direct inoculation but also that of aerosolization. Sloan (1942) demonstrated recovery of airborne tubercle bacilli disseminated from freshly excised lungs. Admittedly, the lung tissue is a spongy, air-perfused mat that lends itself to aerosolization of fluid contents, but also at any autopsy, blood smears or splashes on the hands and arms of the worker are commonplace. Aerosol generation must be expected, in addition to massive hand and work area contamination. Table I11 includes some of the data from these reports cited above in an abbreviated form. It must be noted here that most of the tests referred to above provided results that are subject to considerable interpretation before such data

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can be used for development of protective procedures. Typically, the work reported by Wedum, Reitman, and others was done using mechanical sampling devices with direct impingement of the sampled air onto agar covered petri plates. The results tend to be conservative because of the efficiencies of 47 to 73%reported by Dubuy and Crisp (1944; cited by Reitman et al., 1954b, c, d ) for the sieve-type sampler most frequently employed. Further, Dubuy et al. (1945; cited by Anderson et al., 1952) indicated that most of the colonies found on the receptpr agar of this type sampler are from clumps of bacteria, representing up to 10 single organisms per colony, indicating that an additional upward adjustment of the data may be necessary. Aerosol particle size is one of several factors which may tend to reduce the potential hazard. Davies (1949) and Sonkin (1951) showed that large particles may not penetrate the respiratory tree to a favorable site for infection and that very small particles may not be retained. Since the sampling device employed collected all particles, large and small, it may be desirable to use an “effective particle count” using more refined sampling methods which permit some analysis of particle size distribution; viz., the cascade-type sampler described by Andersen (1958) or other suitable devices with particle size discrimination characteristics (Wolf et al., 1959). The presence of some particles in the size range for optimal penetration of the respiratory tree does not always lead to infection. As Wedum (1953) has reiterated, development of illness from laboratory infections depends on four major interrelated factors: ( 1 ) dosage, ( 2 ) virulence of the organism, ( 3 ) route of infection, e.g., skin, eyes, mouth, and lungs, and ( 4 ) resistance of the individual, i.e., state of health, response to vaccines, natural resistance, and previous infection. The practice of using simulnnt organisms and air samplers in test trials of laboratory operations is one of the best ways to evaluate the potential hazard. However, the problems peculiar to the survival of the airborne microorganisms and the sampling of the viable organisms must be considered. Mechanical sampling devices are many, and each has its own particular use and limitation. Wolf et ul. (1959) discussed thoroughly the problems of sampling bacterial aerosols and also the results obtainable with currently available devices; more recently, Ratchelor ( 1960 ) has reviewed the literature on aerosol samplers. In certain circumstances it may be of value to monitor and sample actual operations in the laboratory, provided the sample can be evaluated in a reasonable time. Work involving the agents of tuberculosis and coccidioidomycosis are examples. The onset of the disease symptoms may be very slow and early therapy may be helpful. Mechanical samplers, as suggested above, can be used, but for some microorganisms a susceptible animal is a better sampler. For example, guinea pigs have frequently been

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used as sensitive detectors for tuberculosis (Solotorovsky et al., 1953; Bogen, 1959; Riley et al., 1959). This technique would appear to be particularly useful in monitoring viral and rickettsia1 contaminants which are difficult to detect by in vitro methods. In the event of a laboratory accident in which the presence of aerosols is suspected, the most definitive sample may be provided by the worker himself through the use of the mouthwash sampling technique. This method, described by Rogers et al. (1955), involves a simple mouthwash or gargle with a sampling medium which must be taken within about 30 minutes of the incident and subsequently examined for the potential pathogen. It can provide confirmation of an exposure within hours. The method has been successfully employed in detection of Mycobacterium tuberculosis (Rogers et al., 1955) and of Bacillus anthracis (Carr and Rew, 1957). 2. Aerosols from Laboratory Apparatus Lyophilization operations, usually done with concentrated suspensions of viable organisms, can create a severe hazard, and the paucity of reported cases of infection incurred in this operation is a better indication of the difficulty of determining sources of infection than of the inherent hazard. Filling ampules with concentrated culture in media usually selected to provide optimal survival of the microorganisms produces obvious aerosol hazards, as previously noted. Breakage of an ampule of dried material can have even worse consequences, when one considers the inherent characteristics of the dried material. It is light, friable, and viable, in a protective menstruum, and almost an ideal aerosol material. Reitman et al. (1954c, d ) discussed the hazard of both operations and showed that widespread contamination of a room occurred in a very short time when an ampule containing viable organisms in a milk and broth menstruum was dropped. Heckly? considers the problem of equipment contamination and ampule opening in further detail and suggests means for minimizing the hazards associated with these processes. Many other devices also contribute hazards which are difficult to evaluate but are nonetheless real. Steam-operated sterilizers are usually connected to discharge the chamber condensate into open cup drains near the machines, The initial entry steam frequently condenses over the materials in the machine, is mixed with cool air, expelled from the chamber, and is forced out into the open cup with sufficient pressure to generate a fine aerosol. At least one incident of recovery of Coccidioides immitis from this condensate has been observed (Chevrefils, 1960). Petri plate and culture bottle incubators are another unexpected source of hazard. There is, first, the problem of direct surface contamination 2 See

page 63 in this volume.

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from handling contaminated materials and, secondly, the aerosol hazard from necessarily loosely sealed petri plates or culture bottles which may become overgrown with the subject agent. Incubators with forced convection would, of course, offer the greatest hazard. The danger generated by the various flask-shaking machines is almost immediately obvious, even if only minor leakage from contamination of screw caps, bottle sides, or cotton plugs is to be expected from each culture vessel. Shaking a culture disturbs the surface violently and creates a fine mist in the flask. Reitman and Wedum (1956) demonstrated the release of such aerosols when caps were removed from shake flasks immediately after removing them from the machine, Even higher colony counts were noted from this operation when the cotton plug was wetted as a result of the violent shaking of the culture in the vessel. The breakage of a culture container on such a machine may constitute a major laboratory accident. Even assuming no immediate exposure of personnel, there is the possibility of unobserved widespread area contamination and the need then to resolve the possibility of exposure of all the people who seem to traverse the area at such inopportune .times. More specialized items of laboratory apparatus also should be considered. Devices for exposure of animals to aerosols of pathogenic organisms, similar to those described by Leif and Krueger (1950), by Henderson (1952), and by others offer a serious source of danger due to the deliberate generation of massive aerosols. Bulk culture fermentation equipment, continuous flow centrifugation, spray drying, and other operations involving large volumes of infectious agents offer comparable hazards. Because these are usually fairly complex installations, the designer usually has considered the mode of operation very carefully and has taken steps to obviate as many potential hazards as possible. It is not altogether surprising to note the infrequency of reports of infection originating in such specialized equipment.

3. Aerosols from Animals Although there are relatively few animal-caused laboratory infections recorded, the infection potential of transmission from animal to man should not be overlooked, and the long history of infections acquired from animals has been well recorded. The work of Lurie (1930) gave early quantitative indication that tuberculosis was transmitted throughout the air in an animal room housing infected and normal guinea pigs. More recently, Phillips et al. (1956a, b ) described intracage cross infections among BruceZZa and anthrax-infected guinea pigs and demonstrated the presence in the room air of other test organisms ( B . subtilis var. niger) aerosolized from coat contamination.

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Hagan ( 1955) relates many instances of reported laboratory animal-toman transmission such as infections of salmonellosis, Erysipelothrix, tuberculosis, leptospirosis, viral ( lymphocytic ) choriomeningitis, and psittacosis. Additionally, he notes the transmission of parasites and other pathogens such as those causing echinococcosis, acariasis, toxoplasmosis, and other diseases. Sinha et al. (1954) reported the transmission of Newcastle’s disease between chickens by airborne virus. Considering humananimal transmission, Pagano et ai. (1960) isolated human strains of staphylococci from animals during an epidemic in a veterinary school, showing that the reverse is a possibility. While aerogenic dissemination is not exclusive in all these examples, its possibility cannot be excluded.

4. Other Aerosol Hazards Many less obvious hazards exist in the microbiology laboratory. Laboratory ventilating systems may collect relatively harmless, dilute aerosols, then concentrate them by lodging the microorganisms in an environment permitting multiplication, and subsequently disseminate them as a continuous low-level airborne contamination. The possibility of such contamination is noted in the “Heating Ventilating Air Conditioning Guide” ( 1958). Cases of ventilation unit contamination have been reported by Anderson ( 1959) who recovered Pseudomonas pyocyanea disseminated from an air cooling unit into a hospital operating theater, and Solberg et al. (1956) who observed contamination of the air from a cooling coil in a ventilating system. Ventilation systems can also cause drafts across work benches into the laboratory worker’s face or circulate dusts which may act as carriers for transmission of airborne organisms from animal rooms or janitorial sweepings. Bentzen et al. (1947), using a slit sampling device to sample air in a laboratory working on staphylococci and p-streptococci, observed that a background count of 50-75 viable particles per cubic meter of air rose four- to fivefold immediately after the floors were swept. The organisms recovered were identified as those under study. Knowledge of such air contamination is not new, and one has only to examine the many reports on hospital contamination to verify that its control presents a problem of considerable magnitude. Constant, low-level contamination can and should be controlled by good housekeeping (Adams et al., 1959) and by the use of the various devices described here, Accidental, heavy contamination in one area which may rapidly be disseminated to another by the ventilating system presents a different and most difficult hazard, and methods for correction are given in Sections V and VI below. Contaminated waste and laundry disposal are frequent sources of aerosol exposure. In a large institution, such as a hospital building or a

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research center, there may be considerable distances between the laboratory and the trash incinerator or laundry collection center. Hurst et nl. (1958) have described a laundry chute as a source of staphylococcus infections in a hospital and demonstrated rapid dissemination of material throughout several floors of a multistory building when material was dumped from only one floor. Infected dressings, wastes, and linens all produced aerosols which were rapidly spread through the building by the ventilating system. Packaging the wastes and linens gave little improvement, since too often a package broke open or the cloth bags became wetted with the infectious material. Other laboratory wastes can also be a hazard to the laboratory worker and to others outside the laboratory. Contaminated glassware, trash, animal wastes, or other solid debris present obvious materials-handling probIems. Liquid wastes are discarded into the sink or drain, contaminate the drain lines, and permit viable pathogens to be retained and even to multiply in traps prior to discharge into public waste pipes, Recovery of viable tuberculosis from sewer lines has been demonstrated many times, as has recovery of poliomyelitis and Coxsackie viruses (Kelly et al., 1955, 1957; Bloom et al., 1959). Stoker (1957) reported 2 cases of laboratory infections of Q fever which were traced to an overflow of water from a blocked drain. The water had been contaminated in another part of the building.

IV. Safety Devices and Techniques The most frequent source of infection in the laboratory is, as already indicated, one or more of the routine operations which permit accidental ingestion, inoculation, or inhalation of a disease agent. It is of interest to examine a few of the methods used to control infection from these operations. First, an effort must be made to reduce the hazard by the use of safe techniques developed through operator education. Secondly, devices and systems must be designed to contain the hazards and to dispose of dangerous wastes in a safe manner. In any practical system of laboratory safety these methods must be considered interdependent, and the dichotomy indicated here is used only for convenience in treating the subject, If equipment is to be of value it must be employed with the application of much common sense. There is, after all, no safe substitute for a careful worker, but even such a worker requires certain equipment if he is to perform his assigned tasks with minimal risk to himself and his co-workers. A. THESAFETYHOOD The safety hood or enclosure is perhaps the most useful protective device in the laboratory. A properly designed and properly used hood

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provides a device to contain most of the routine laboratory procedures which produce aerosols or offer the possibility of squirting infectious material directly at the worker.

1. Development Van den Ende (1943) used a very simple ventilated fume hood in which to perform intranasal inoculations of animals. The air was exhausted from this hood by induction into a gas flame. Shepard et al. (1945) described a hood after the same pattern for housing a Waring blendor and other aerosol-producing devices. Their wooden cabinet, similar in form to a chemical fume hood, depended on a draft created by a gas burner in a stack through which the cabinet ventilating air was exhausted at approximately 500OF. This cabinet could be used with the front closed or with the addition of a two-hole armport board. Electrical power to devices inside the hood was disconnected automatically when the hood door was open. Simple unventilated hoods have frequently been utilized far animal autopsy, and a typical unit was described by Krueger (1949). This hood was approximately 6 ft. long with sloped viewing glass sides, below which were horizontal openings through which operations were conducted. Ultraviolet (UV) lamps were installed in the hood which was open to the room through a vent at the top. Kantorowicz and Rees (1950) described a somewhat similar device utilizing two sheets of plate glass as simple spatter shields. Krueger (1949) further described a hood for use with agents such as plague bacillus or psittacosis virus. The hood was single-sided, 80 in. long, 26 in. deep, and 30 in. high. The front of the hood had sloped safety-glass view windows below which were mounted 2 removable front panels bearing arm-length rubber gloves. The chamber was equipped with fluorescent tubes and UV lamps, and the ventilating air was exhausted through a gas-fired air incinerator. Materials and equipment were put into the hood through a double-door air lock at one end. Wedum (1953) described a hood unit which was similar in configuration except for the addition of a blower and a spun glass protective filter on the hood exhaust. Jensen ( 1954), working towards a standardization of laboratory methods for a tuberculous clinical laboratory, described a very simple hood unit fabricated of transparent rigid plastic in the familiar form of a chemical fume hood with a fixed glazed front and a large access opening. Others have used hoods more closely adapted to the operations in the microbiology laboratory. Williams and Lidwell ( 1957) designed a sheet metal cabinet with exhaust ports strategically placed to direct the inflowing air current across the work area. The hood was exhausted at the rate

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of 100 cubic feet per minute (c.f.m.) through a filter pad. When tested with B. subtilis and Serratia marcescens aerosols, samples from immediately in front of the hood showed concentrations approximately 2%of those noted in the exhaust. Couling and Rees (1959) described a unit in use since 1951 for post-mortem examination of small animals at the Tuberculosis Isolation Unit, National Institute for Medical Research, London. This hood was provided with a sloping glass front hinged at the top for access to the unit, and with two 18-in. turntables to which the animals could be affixed for dissection. These freely turning tables could be lowered by a lever and immersed in a disinfectant solution. The hood was ventilated through an opening along the face at the rate of 300 lineal feet per minute (1.f.p.m.) and exhausted to the laboratory-air-sterilizing incinerator. The simple hoods, either the modified chemical fume hood type or the bacteriology hood, have not always been successful when a highly infectious microorganism was involved. For example, while Keeney (1946) was quite successful in containing Coccidioides immitis in a stainless steel hood patterned after that described by Shepard et al. (1945), another group of workers in California was unable to use similar equipment successfully and was forced to discontinue growth of the fungus on solid media in order to avoid the aerial mycelial growth phase and thereby reduce the hazard of infection ( Sulkin and Pike, 1951a). Other workers in this field recognized the need for tighter enclosures for extremely hazardous operations; willing to sacrifice some loss of access for gain in safety, they looked to the use of airtight hoods or safety cabinets. Wedum ( 1953) published photographs and descriptions of sloped-front cabinets with fixed view glass and arm-length rubber gloves attached to fixed glove ports on the front of the hood. The cabinets of varying sizes and shapes were made of stainless steel and were patterned after the low-level radioactivity “dry box” (Fitzpatrick et al., 1949). These units, in use at Army Chemical Corps Biological Laboratories, were described in further detail by Gremillion (1959), who discussed some of the more recent designs of this type of bacteria-tight cabinets, He described a system of gastight stainless steel cabinets made in various sections or modules, and bolted or cemented together to form extensive housings for all procedures. Centrifuges, incubators, sterilizers, animal cages, and other equipment were all housed in, or made part of, such assemblies. Exhaust air from the cabinets was drawn through a bacterial filter and an air incinerator to provide a sterile exhaust. All materials removed were withdrawn from the cabinets through dip tanks of liquid disinfectant or through a double-ended autoclave attached to the cabinet assembly. All manipulations were carried out through arm-length rubber

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gloves tightly attached to the fixed glove ports on the hood. Similar, though less complex, assemblies have been in use for some time at the Naval Biological Laboratory, where they are employed to house aerosol generation apparatus for respiratory inoculation of small animals ( Goldberg et al., 1958).They differ from those described by Gremillion ( 1959)

FIG. 2. Open-front bacteriological safety hood as used at U. S. Naval Biological Laboratory ( U . S. Naval Biological Laboratory photograph).

only in details of construction and in the ventilation system which provides a greater ventilating air change rate to provide rapid dilution of aerosols in the cabinet.

2.Current Designs Currently, two distinct types of hoods appear to meet most needs in the microbiology laboratory: (1) the large single hood usually seen with sloped view glass, open front, and a filtered exhaust providing a specified flow rate of air inward and across the work surface (Fig. 2 ) , and ( 2 ) the smaller, fully closed type safety cabinet with air lock entry port, fixed

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rubber gloves, sloped view glass, and a filtered exhaust system (Fig. 3 ) . The latter are designed usually to maintain a specified negative pressure in the hood rather than an air flow rate. Selection of the type of hood depends on several factors which must be appraised by the user. At the Naval Biological Laboratories, the open-front, high-flow-rate hood is employed where the hazards are accidental and are of low order, the agent employed has low infectivity, and ready access to the process is desired. Tasks carried out in this hood include pipetting, inoculating animals with a needle and syringe, inoculating cultures, removing samples from vaccine bottles, and small animal intranasal instillations. For work in which aerosols are generated deliberately or where a highly infectious agent is employed, the closed-cabinet systems previously described are used. This work includes aerosol generation for respiratory inoculation of animals, use of the blendor, testing of aerosol generators, and similar operations. The open front hood is an adequate and safe device for most laboratory operations, but only if it is designed, installed, and operated properly. This point cannot be overemphasized. Motion picture studies of typical operations in an open-front hood using smoke simulants for aerosols showed that rapid motions by the worker, people walking past the face of the hood, and withdrawal of the worker’s arms from the inside of the hood all caused rapid dissemination of the aerosol from the hood to the surrounding room (Chatigny, 1960). The great value of this hood lies in its acceptability to the laboratory worker. It is a fair compromise between the safety engineer’s desire to afford every worker optimal protection and the laboratory worker’s desire to proceed with his task with minimal interference from the often mistrusted “mechanical gadgets.” The modular-type closed cabinets offer a great deal in improved protection and are less subject to accidental misuse. Their major deficiencies are very high cost, restricted work space, worker reluctance to use the equipment properly, and the difficulty of passing materials in and out without breaking the protective barrier. The latter quickly leads to a requirement for installing all laboratory equipment in the cabinet if the concept of full protection is carried to its logical conclusion,

3. Selection and Installation Both hood types described above are commercially available in a variety of models and materials. Air sweep type hoods are available in sizes up to 7 ft. long with attachable glove plates, gloves, blowers, filters, and air lock claves offered as accessories ( 1).3 a Numbers in parentheses refer to listing of commercially available safety devices given in Section IX.

FIG. 3. Closed-front modular safety cabinet-left : interior view, note recessed petri plate incubator-right : cabinet assembly attached to aerosol test chamber ( U. S. Naval Biological Laboratory photograph).

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Closed-front hoods or cabinets are available in small single units 4 ft. or less in length ( 2 ) or in modular sections which may be combined in various configurations desired ( 3 ) . They can be furnished with removable front glove plates, hinged windows, filters, blowers, UV lamps, and many other accessories including equipment for remote manipulations. Units with temperature and humidity controls, inert gas pressure systems, air lock entries, and a wide variety of accessories are available ( 3 ) . Cost of these hoods ranges from approximately $100 per lineal foot for the simple units to $2000 (or more) per foot for the more complex devices. Many of these modular hoods are also suitable for radioisotope work or for toxic chemical operations (2, 3 ) . It should not be necessary for the laboratory worker to consider the design of the hood, other than to make a selection as to size, type, and accessories. Unfortunately, manufacturers are not yet operating on a single set of standards, and some basic performance criteria must be specified by the user. Velocity of the inlet air at the front of the open hood and suction pressure against the gloves in the closed cabinet are most frequently employed as simple standards of performance. Closed-hood cabinets should not only be gastight but the negative pressure for normal operation should not exceed 1 in. water gage in order that the gloves may retain some flexibility and feel. Greater negative pressure distends the gloves, destroys feel, and makes various doors and ports difficult to operate. For the open hoods a single figure of air velocity is usually cited, although occasionally a varying range of 30 to 200 1.f.p.m. is given. Schulte and co-workers (1954), after examining many types of chemical fume hoods which have similar problems of contaminant capture and containment, recommended a range of from 80 to 100 1.f.p.m. for chemicals of moderate toxicity. Peterson (1959), in an attempt to rationalize the procedure for determining face velocities in chemical fume hoods, developed a hood performance factor comprising an entry coefficient based on work by Dalle Valle (cited by Peterson, 1959), a vapor control factor taking cognizance of the evaporation rates of solvents, and an environment factor which considered the air disturbance around the hood. Although, as indicated, these factors depended greatly on practical experience, if properly used Peterson’s procedure is of considerable value, particularly for hoods with a large frontal opening. Each open hood installation is a special case and it is inadequate, if satisfactory performance is to be attained, to consider only face velocity or suction pressure as a standard of performance. Tests with aerosols generated inside the hood of Fig, 2 indicated that an inlet velocity of 120 to 150 1.f.p.m. is required to effect capture of fine particulate material and to overcome the effect of people walking past the unit at moderate rates or performing operations which require reaching into and out of the hood,

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Exhaust air from the hood may be decontaminated by several methods before discharge (Van den Ende, 1943; Wedum, 1953; Williams and Lidwell, 1957). Decontamination can be accomplished either by collection or by destruction of the airborne contaminants. Destruction by thermal means is costly and cumbersome, but, in the final analysis, it is the only consistently successful method of achieving sterilization. Chemical destruction with vapor phase decontaminants such as triethylene glycol or hexylresorcinal is less satisfactory and is difficult to control. Van den Ende (1943), Krueger ( 1949), Williams and Lidwell ( 1957) and others described the use of gas-fired air incinerators. Decker et al. (1954) reported a penetration of 0.0001%or less for aerosols of Bacillus globigii spores through a commerically available ( 4 ) electrically operated air sterilizer. Air temperature was 575OF. for 3-second or 425OF. for 24-second retention times. Time-temperature studies for differing species of microorganisms have been few, and it is difficult to predict a minimal sterilizing temperature for each of the many different organisms which may be used. Bourdillon and co-workers (1948b) tested an electric air heater and observed a thermal death point of 437OF. for B. globigii spores retained in the heater for 0.4 seconds or longer. They indicated the desirability of using 482O to 572OF. as an exit air temperature to ensure the killing of large dust-coated particles or of those from heat-resistant strains and to compensate for variation in heat transfer to the flowing air. Prefiltration to remove large particles and a retention time of 1 second or less were suggested. A gas-fired air incinerator used to sterilize process air from aerosol experiments at the Naval Biological Laboratory is controlled to provide 750° to 1000OF. exit temperature, with a retention time of 1 to 3 seconds (Fig. 4 ) . More frequently, decontamination of the exhaust air is attained by collection of the airborne contaminants. Fibrous filters are the most popular, although electrostatic precipitators have been suggested and tested (Decker et al., 1951). Decker et al. (1952) proposed the use of spun glass filters utilizing two 0.5-in. layers of Aerocor P F 105 ( 5 ) in a housing directly mounted on the hood; they showed collection efficiencies of 99.68 to 99.95%using clouds of Serratiu indica and Escherichia coli T-3 bacteriophage as test organisms. Shape and size of the particles in the aerosols were described and data are indicative of the high collection efficiencies of such filters. Other commercially available unit-type filters ( 6 ) employing glass or glass-asbestos fibers with an advertised arrestance of 99.95%or greater for 0.3-p-diameter particles have been employed (Seifert and Callison, 1956) in bqcteriological hoods. This type filter was tested separately ( Bristol Laboratories, 1951), and efficiencies greater than 99.97%were consistently observed.

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FIG. 4. Contaminated-air-disposal incinerator and liquid wastes treatment equipment at U. S . Naval Biological Laboratory, Oakland, California ( U . S. Naval Biological Laboratory photograph).

The subject of filter design efficiencies and ratings is not within the scope of this chapter, but it is pertinent to outline some of the requirements of a filter for use in a bacteriological hood. 1. Reliability of the filter should be high. A power failure or air flow slowdown or occasional wetting should not cause blowout, malfunction, or significant efficiency loss. This requirement alone would rule out the

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use of electrostatic precipitators which do not at this time have the necessary reliability, 2. Collection efficiency should be not less than 99.95%for 0.3- to 5.O-pdiameter particles when tested with dioctyl phthalate smoke or with microorganisms in the manner described by Decker et al. (1952). If the filter is to be employed in virus work, tests with bacteriophage particles may be indicated. 3. A pressure drop of 1in. water gage at rated flow with a clean filter is suggested. This resistance is easily overcome by most small quiet lowpressure exhaust fans which are most economical in initial cost and operation. 4. The filter should be in a housing or assembly that will permit tight sealing, ready decontamination, and easy replacement. A prefilter is indicated if a heavy dust load is anticipated. 5. Finally, each filter installation should be checked in place. This is absolutely necessary if one is to be certain of the filter performance. Leakage past high efficiency filters, each of which is tested by the manufacturers, is usually due to the mounting of the filter, not the filter material. The exhaust fan or blower for the hood is usually supplied by the hood manufacturers. It must overcome air pressure losses from the hood-filter combination and the system duct work, The fan should provide the air flow desired at the opening of the hood, with due regard to pressure drops across clean and dirty filters. For a convertible hood, usable either with or without gloves, the blower should be sized automatically to limit the negative pressure inside the hood to 1 in. of water gage when the flow is substantially restricted, and to provide 50- to 100-1.f.p.m. air velocity through any openings occurring either normally or by accidents in the course of use. Finally, the blower materials should withstand attack from chemicals which may be used to decontaminate the hood. The hood is a basic safety device in the laboratory and, as such, can reduce or perhaps eliminate the necessity for development of many other minor devices designed to reduce airborne infection hazards. It should be selected not only with attention to the specific points noted above, but also with regard to the more obvious factors of suitable size, construction materials, accessibility, accessories, and, most important, the attitude of the workers who will use it.

B. THECULTURE-SHAKING MACHINE While the inoculation and the harvesting of the shaker flask culture may be done in a simple hood, the shaking operation itself is another problem. Reitman and Wedum (1956) described a ventilated enclosure

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for a flask-shaking machine. Figure 5 shows a similar enclosure used at the Naval Biological Laboratory to house a commercial rotary shaking machine ( 7 ) . It is not force ventilated but is provided with temperature controls, UV irradiation, view port lights, and a disinfectant spray device. The operator always looks through the view port to ascertain condition of all flasks on the machine before opening the door. The shaking machine and the air circulating fan in the box are automatically stopped

FIG. 5. Culture-shaking machine in safety cabinet (U. S . Naval Biological Laboratory photograph).

by the act of opening the door. The carriage tray may be kept moistened with liquid disinfectant, and a spray tube permits washdown of the interior without opening the doors in the event of flask breakage. To reduce the chance of such breakage, thick-wall Pyrex or plastic flasks are employed and individual clips to hold each vessel are installed on the machine. Since the act of shaking a culture flask produces aerosols (Reitman and Wedum, 1956), the plug or cap is considered heavily contaminated and the interior of the vessel assumed to be filled with highly concentrated aerosol. To prevent escape of this aerosol, parchment paper is tied or taped over the cotton plug or cap. Alternatively, vented canisters as described by Decker et al. (1952) may be employed. This device was

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designed to enclose a single culture vessel inside a can bearing a fibrous glass filter in the sealed top. In use, the canisters are clamped to the shaking machine. All shake culture vessels should be opened under a safety hood, C. THE CENTRIFUGE Centrifugation of infectious agents is an operation not readily performed in a simple safety hood. For a continuous flow centrifuge, there is little choice; whole area protection as described by Hed6n (1958) must be built around this device. The very nature of the process is such as to produce massive aerosols which must be contained. For a small container centrifuge, screw-cap tube holders are the simplest safety device. The containers should preferably be made of corrosion-resistant metal and should be loaded and unloaded under a safety hood. As with the shaking machine, they should be used in conjunction with plastic or heavy-wall tubes. Care must be taken to ensure that the screw caps or threads on the vessel do not become contaminated; Whitwell et al. ( 1957) showed that contaminated screw-cap containers frequently discharged aerosols from the fluid on the washer of the cap during centrifugation. Screw-capped centrifuge tube containers are available from at least one manufacturer ( 8 ) . Unfortunately, the containers have been made of aluminum which is not a material of choice for virus work in which strong caustics may be required as disinfectants. Other tubes of stainless steel with compression-type caps are available ( 9 ) in a limited variety of sizes. If there is room in the laboratory and funds are available, the centrifuge can, as Reitman and Wedum (1956) suggested, be modified so that the entire top area is enclosed in a hood (10). Less costly, but nonetheless effective, enclosures have been described by Gibson (1955) who enclosed a centrifuge in a small closet with UV irradiation and Lind (1957) who constructed an enclosure for the centrifuge and installed a view window in the top of the machine. D. THEBLENDORAND SIMILARDEVICES The high-speed blendor has been described as a prolific source of aerosols and various modifications of this device have been proposed. Smadel ( 1951 ) suggested addition of a vaccine-stopper-type harvesting port and a hermetically sealed top and recommended careful inspection before use. Reitman et aZ. (1953) and Hilleman and Taylor (1958) designed blendor bowls that would be leakproof in operation and could be unloaded without undue hazard. Both devices were well designed but would appear susceptible to rapid wear and damage of the bearings on the high-speed impeller shaft. Reitman et al. (1954a) modified the

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original bearing system. A commercial version of that described by Reitman et al. (1953, 1954a) is available at a considerably higher cost than the standard bowl usually furnished with the blendor (11).In a simpler vein Lamanna (1960) described a heat-sealed flexible plastic film enclo-

FIG. 6. Flexible autoclavable plastic film enclosures for blendor and grinders. ( D ) shows autoclaving pack ( U. S. Naval Biological Laboratory photograph-copyright by Journal of Laboratoy and Clinical Medicine, 1959).

sure for the blendor which could readily be fabricated in the laboratory (see Fig. 6 ) . Although no data were given on aerosol release after blending, it is obvious that such enclosures must be opened inside a safety hood. This procedure does correct the aerosol hazard but the problem of decontamination of the blendor drive remains. Although the bowl

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may be autoclaved, the base of the machine must either be left contaminated or be decontaminated chemically. All the devices noted above should be used with supplemental cooling of the bowl to prevent destruction of bearings and to minimize thermal effects on the product. The preparation of enzymes, cellular components, etc., from masses of microbial material by means of ultrasonics, colloid mills, ball mills, jet mills, and other devices provides obvious opportunities for generation of aerosols comparable to those offered by the blendor. Although little attention has been given to the hazard created by these operations, the containment requirement is no less severe. Lamanna et al. (1959), recognizing the safety problem when using milling devices, used sealed, flexible plastic tubes containing glass beads and the cultures in a modified centrifuge and in a roller device to provide a crushing action that produced no aerosolization of the product. Ultrasonic disintegrators should be used only in a safety hood or an enclosure. Colloid mills and other larger rotating spindle machines should have mechanical seals (12) added to the spindles and, even so, operation in a gastight safety hood is a practical necessity.

E. THEPIPETTE Pipetting, a simple, routine, almost universal operation, has received considerable attention because of the possibility of ingestion of microorganisms when mouth suction is used. As a simple precaution, the proximal ends of pipettes frequently are plugged with cotton, but this precaution has never been wholly effective and many workers have devised means to avoid oral pipetting. Wedum (1950) described several nonautomatic devices, most of which employed a rubber bulb or piston pump with finger-operated valves. Other methods included a tube connected to the pipette through a glass bulb containing a cotton filter, or a fine rubber tube connected to a suction line through a liquid trap containing disinfectant. Haber (1956) suggested the use of a Luer syringe connected to the pipette with rubber tubing, as did Dern and Pullman (1950) who examined the desirability of using the syringe as a pipette. They noted the obvious desirability of eliminating mouth operation of the pipette and suggested that the filter technique previously proposed should be used only where the agent involved is of low risk when ingested. With some disregard for possible contamination of the water supply, Dybkaer (1957) proposed that pipettes be operated from a water suction pump through tubing and a thumb-operated control valve. Many types of mechanical pipettes are available and, as Wedum et al. (1956a) suggested, these should be selected to deliver only by gravity if used outside a hood. One recently introduced type (13) delivers by gravity or by force

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through a vernier wheel and is useful for large and small volumes. Many other models suitable for use in a hood are available ( 14). At the Naval Biological Laboratory simple rubber medicine dropper bulbs have been used routinely for 15 years to operate 1.0-ml. pipettes to an accuracy of k . 0 5 ml., and the ease of operation never ceases to surprise the neophyte or the experienced worker who is convinced that such a simple expedient must either be inaccurate or difficult. For

FIG. 7. Pipetting operation in open-front safety hood. Note pipette bulb, discard pan, worker garb ( U . S. Naval Biological Laboratory photograph).

economy, with due regard to safety, the pipettes, selected to deliver 1.0 ml. discharged, are not plugged with cotton. Pipetting pathogens should always be done in a hood and care should be taken to avoid forcibly blowing out the final drops and to discharge the pipette with the tip held against the side of the tube or bottle. Contaminated pipettes should be discarded into a shallow pan of germicidal solution immediately after use. They should be autoclaved subsequently in the disinfectant before removal for cleaning and reuse. Finally, a disinfectant-wetted towel should be used to cover the work area to minimize the danger of splash from dropped culture contaminants. Figure 7 shows a typical safe pipetting operation as described here. The pipette

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shown is not that usually employed but is a “dropping” pipette used in the technique described by Miles et al. (1938).

F. THE SYRINGEAND NEEDLE AND THE INOCULATING LOOP 1. The Syringe and Needle The use of a syringe fitted with a sharp needle is a hazardous operation that requires great care. Glass syringes should be carefully inspected for chips or cracks before use, and the needle should be inspected for burrs

FIG. 8. Mouse inoculation in two-sided open front safety hood. Note towel on work surface, worker garb (U. S . Naval Biological Laobratory photograph).

and sharpness. The syringe should be the Luer-lock type to prevent accidental demounting of the needle under pressure. It should be filled carefully to minimize air bubbles and frothing of the inoculum. The needle should be inserted into an alcohol-wetted cotton pledget or cotton plug while expelling the air from the chamber. If the inoculation site is tough and resistant, as might be found in certain animal hides, it may be desirable to lubricate the plunger with sterile glycerin to effect a tight seal between the barrel and plunger of the syringe. All animal inoculations should be done in a safety hood (see Fig. 8) or in an isolated room with the worker in full protective garb, including

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surgeon’s long-sleeved gown, gloves, cap and transparent face masks. Double gloves are recommended for the hand holding the animal. The inoculation site should be swabbed with tincture of iodine or other disinfectant before and after the inoculation. After use the contaminated needle and syringe should be discarded separately into shallow pans of appropriate germicidal solution, removing the plunger from the syringe and thereby filling the syringe with germicide. Merger (1957) suggests that animal inoculations should always be done with the hand “behind the needle to avoid punctures. This seems feasible for work with mice, but if monkeys or larger animals are employed, some difficulty may be encountered and the further protection described above is indicated. 2. The Inoculating Loop Aerosols are created when a hot metal inoculating loop is dipped into liquid culture or agar culture and when the culture-wetted loop is put into a sterilizing flame. Solotorovsky et al. (1953) and Winner and Quiney (1953) described a hooded or shielded gas burner so designed as to enclose the loop being flamed and reduce the chance of spatter and aerosolization. Another device was suggested by Frisby (1954) who used a closed-end steel tube about 7 in. long, supported in the flame of a Bunsen burner. The loops were sterilized by insertion into the hot but draft-free tube. Special burners designed for this purpose are now available commercially ( 15). After flaming, the loop should be allowed to cool before making a start into a culture or dipping into liquid medium. A safety hood should be employed for all culture transfers.

G. THE INCUBATOR Most of the almost universally employed bacteriological incubators in use at this time are direct electric-heated thermal convection ovens. As such, they do not generate air currents with velocities sufficient to aerosolize spilled materials. In recent years the use of forced convection ovens with improved load capabilities has become more common. These ovens, with circulating and exhaust blowers, cause very rapid dissemination of any spilled or overgrown material. Blower fans in these incubators should be connected to stop automatically when the access door is opened. Because the most frequent accident with these machines is dropping or tipping culture vessels or petri plates when loading or unloading the machine, it is desirable to use metal containers for all materials. Darlow (1960) described the simple tests of dropping contaminated glass and plastic petri plates to the floor. He observed heavy

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aerosolization from the glass plates but very little from the plastic plates. Use of the latter in many situations appears desirable.

H. THESTEAMPRESSURE STERILIZER Every microbiology laboratory should be equipped with at least one steam-operated sterilizer designated specifically for decontamination of wastes. Wedum and co-workers (1956a), in considering the design of an infectious disease laboratory, recommended the use of a double-door sterilizer between the laboratory or a n h a 1 room and a clean preparation area and suggested its use for “Carboxide” gas decontamination. Steam sterilizers are commercially available in sizes and shapes to fit almost any enclosure, and the proper use of such equipment is well known (Perkins, 1957). At least two precautions are indicated: First, the vents from the autoclave chamber, usually drained into an open cup drain in the Iaboratory, should be connected directly to the waste line and vented through an asbestos or glass fiber filter clear of all occupied spaces. Second, full use should be made of automatic controls which will indicate a “safe” condition only when the sterilizer has been operated through a full cycle at sterilizing temperature. The machine should be loaded with all material in small containers to permit good heat penetration. Sterility indicators should be placed in the areas of most dense packing, in unfilled air voids, and in other thermally insensitive locations. Large trash cans filled with waste or animal bedding cannot be sterilized in a reasonable period, and such waste should be processed in small cans or in the animal cages. Operation of autoclaves is frequently a haphazard affair, and as a result there is more hazard than is warranted from this device. The use of automatic control and double-door, pass-through units can eliminate possible confusion caused by the need to leave notes or to maintain logs of start and stop time. The thermal recorder furnished with such units provides evidence that the cycle has been completed and it can be a valuable epidemiological tool should an infection occur in any of the workers handling material which has purportedly been sterilized.

I. ANIMALEXPOSURE EQUIPMENT ( RESPIRATORY ) The obvious hazard afforded by the various techniques for inoculation of animals via the respiratory route and for subsequent maintenance of the infected animals has forced investigators to design and test protective equipment for each particular operation. The general approach taken by most workers is that of containment of the hazard and decontamination of all exit wastes. Judgment as to the degree of hazard has varied widely,

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Rosebury (1947) described an extensive system for aerosol exposure and maintenance of animals. Similarly, the apparatus described by Leif and Krueger (1950) to permit quantitative exposure of small animals to aerosols of infectious agents demonstrated a high regard for the risk. This equipment, modified from that described by Reyniers (1943; cited by Leif and Krueger, 1950) for housing germfree animals, included an airtight stainless steel tank within which was housed an airtight aerosol generation and animal exposure apparatus. Arm-length rubber gloves attached to ports in the tank permitted manipulation of objects in the tank to which access was gained through disinfectant-flushed claves. Air discharged from the process and from the tank was sterilized directly in a gas flame. In contrast, Henderson (1952), whose apparatus in one form or another has been very widely used for studies in airborne infections, indicated that “discretion is advisable” when working with pathogens flowing through an unsealed aerosol chamber mounted on an open work bench. For worker protection, military type gas masks fitted with bacterial filters, surgical gown, gloves, and head covering were worn. Exhaust air from the aerosol generation device was filtered serially through 2 resin-impregnated merino wool filters with a penetration for 0.5-p-diameter particles of 1 X lo-% (sic). Hexylresorcinol vapors were generated in the room when vegetative organisms were employed. It was stated that no workers contracted infection as a result of handling the apparatus according to instructions. Wolochow et al. (1957) described a somewhat different system in which only nominal precautions were taken to contain the aerosols but where the worker was thoroughly enclosed in a ventilated flexible plastic suit (see Fig. 9 ) in a manner similar to that employed by workers rearing germfree animals ( Reyniers, 1959). This technique offers the advantage of greater ease in handling monkeys and other difficult animals and appears somewhat less subject to misuse than that described by Henderson ( 1952). Supplemental protective devices including UV irradiation, ventilating air filtration, process air incineration, and closed animal-holding cages were used with this animal exposure and holding system. The foregoing are offered as examples of several different approaches to the problem of safety during the exposure of animals to airborne pathogens. Other devices for this operation have been described, some using simple methods (Middlebrook, 1952; Piggott and Emmons, 1960) in an effort to contain the process, others using somewhat more elaborate equipment (Wells, 1940; Weiss and Segeler, 1952; Gogolak, 1953) and still others (Gremillion, 1959) using the full security procedures similar to those described by Leif and Krueger ( 1950).

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FIG.9. Worker wearing ventilated personnel suit (partial) in animal inoculation procedure ( U . S. Naval Biological Laboratory photograph).

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For highly infectious agents or agents of unknown infectivity, the utmost precautions are warranted. For work with those of lower infectivity, the use of a safety hood in conjunction with adequate protective garb including respiratory filters is desirable. Needless exposure to aerosols of apparently innocuous microorganisms or other materials is not a good practice. Incidents of induced hypersensitivity ( Leif, 1960) and unexpected serological reactions ( Reitman et al., 1955) have often followed such exposure.

J. ANIMAL-HOLDING DEVICES Equipment for housing laboratory animals infected with human pathogens must be designed with due regard to the well being of the animal in accordance with established principles for humane animal care. Adequate information on this requirement is widely available ( Worden, 1947; Farris, 1950). In addition, the equipment must provide containment of the infection hazard. Horsfall and Bauer (1940) described a system of portable animal cage housings which could be connected to a suction manifold through which ventilating air was drawn. The housings or cubicles measured 24 X 24 x 20 in., were made of Monel metal, and were equipped with inlet and outlet air filters and tight fitting doors. This system, designed and used successfully to eliminate cross infection between animals, was extremely successful in accomplishing this purpose and has served as a prototype for development of many other holding systems. Several changes have been made to render thiscage system suitable for housing animals infected with human pathogens. Rosebury (1947) added air-lock claves and thermal exhaust air sterilization and also fitted rubber gloves to the cage in an attempt to achieve full security. Leif and Krueger (1950) employed pressure-tight cylindrical tanks 24 in, long and 30 in. in diameter with flanged openings, one arm-length rubber glove for handling the animals, filtered air inlet, and air exhaust to an incinerator. In use, these tanks were bolted to similarly sealed animal exposure devices, and an unbroken mechanical barrier was thus maintained, In other approaches to containment, Decker et al. (1952) described a small stainless steel mouse cage using inlet and outlet air filters of glass fiber mat. Solotorovsky et al. (1953) used rectangular screw-cap glass jars with inlet and outlet cotton plug filters for housing tuberculosis-infected mice. The jars were kept on shelves equipped with an overhead area exhaust duct that collected all room air in the vicinity, passing it thence to an incinerator, For a somewhat different purpose, Van Bruggen (1952) successfully mounted cages in special hoods with a filtered exhaust to capture radioactive carbon dioxide exhaled by animals which had received compounds containing carbon-14.

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Gremillion ( 1959), on the other hand, proposed that the exposure of animals to highly infectious agents and subsequent holding of these animals be done entirely within gastight safety cabinets. Not surprisingly, many of these animal-holding equipment designs have borrowed extensively from the germfree animal methodology. The problems of contamination containment are reversed but analogous, and it is obvious that methods satisfactory for maintaining germfree animals could be adapted with little change to holding infected animals. Accordingly, advances in the technology of germfree research are of interest to investigators dealing with problems of protection against accidental laboratory infection. For example, Miyakawa ( 1959) described an extensive system of pressure-tight stainless steel tanks with remote control “hands” for tending the germfree animals, temperature and humidity control, and ventilating air sterilizing systems. Reyniers ( 1959) described extensions of the familiar germfree tank system that included “walk in” size tanks, portable tank units, air filters, and air sterilizing apparatus. Gustafsson (1959) designed a series of rectangular tanks with all glass tops, fixed glove ports on the sides, and dip tanks for access. These tanks, approximately 36 x 60 x 30 in. high, were sterilized in a 1,dr ge steam sterilizer. Trexler ( 1959) demonstrated inexpensive animal cage enclosures made of flexible vinyl plastic film with air locks for entry and glass fiber air filtration systems. Holding devices for small animals exposed to pathogenic aerosols over the whole body should include cages with solid sides and bottom, or cage housings similar to those described by Horsfall and Bauer (1940) to house animals singly to eliminate cross infection. A representative, commercially available (16), cage system, meeting the first criterion, is shown in Fig. 10. Such cages should be ventilated through high-efficiency inlet and outlet filters and the exhaust air sterilized by heat or decontaminated by filters. As the degree of infectiousness dictates, animal caretakers should wear protective clothing and respiratory protection or, alternatively, the entire operation should be housed in closed-hood systems. If data are available to indicate the maximum survival time of the agent on surfaces, animal fur, or fomites, the open top cages may be stored on racks fitted with UV lamps after a suitable holding period on a ventilating manifold. Ultraviolet intensity should be 200 microwatts per square cm. ( pw./cm.2) or higher at the top of the cage (Wedum, 1953; Phillips et al., 1957), and care should be taken to ensure that the animals are not in the direct rays. Wire mesh or dust-free bedding should be employed in the cages. Animals inoculated by intranasal instillation or orally should be considered to be similarly contaminated and treated accordingly. No evidence has been shown to indicate immediate, heavy contamination of animals from inoculation by a needle and syringe (intraperitoneal,

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subcutaneous, intercerebral, etc. ) provided the inoculation site is wiped with a disinfectant. A delayed, low-level contamination from feces and exudates is expected, and the use of solid side and bottom cages and UV cage rack irradiation is suggested. Again, workers should wear protective clothing. Housing infected monkeys is a problem because these animals are so very difficult to handle. They should be caged individually, and all transfers should be done in small handling cages. Wolochow et al. (1957) have

FIG. 10. Solid-side and bottom-ventilated animal cages attached to suction manifold (photograph courtesy Parco Co., Falls Church, Virginia).

described monkey cages, each suitable for short-term holding of one 5- to 81b. monkey. These cages, 20 x 20 X 28 in. high, were provided with view windows, filtered ventilating air inlets and outlets, bottom waste trays, and access ports and were exhausted through a vent system.

K. P R O T E ~ VCLOTHING E It is essential that a complete change of outer garments and shoes be made for all work in the infectious disease laboratory. A simple cotton wash suit similar to those worn by surgeons is inexpensive and is suitable for both male and female employees. Light coveralls are useful for main-

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tenance workers and others whose tasks do not require that they come in direct contact with infectious agents. Within the laboratory, the use of long-sleeve surgical gowns or other overgarment, caps, and surgeon’s gloves worn to cover the cuffs of the gowns is strongly recommended when infectious agents or infected animals are used. This garb immediately identifies the “contaminated worker from his co-workers, provides direct protection against spills or hand contamination, and provides a protective covering which can be discarded and autoclaved at the end of the operation. Adams et al. (1959) described a very simple, inexpensive, wrap-around surgical gown that is considerably easier to use than the conventional gown ( 17). In this study, pertaining to hospital operating room contamination, they observed that all outer clothing becomes contaminated with the microbial flora of the area after exposure of only a few minutes, and that if sterility was to be maintained in the operating theater, many changes of outer garments were required in the course of a work day. Although the gown described has short sleeves, it should be possible to fabricate it with long sleeves for use with surgical gloves. Shoes are included as clothing, and an extra pair of shoes reserved for use in the laboratory is required. Inexpensive plastic or cloth shoe covers are currently available (18) and may be worn directly over street shoes. They are satisfactory for laboratory visitors but not for routine laboratory worker usage. When inoculating animals with a syringe and hypodermic needle, doubled surgeon’s gloves should be worn (see Fig. 8 ) . Gloves must obviously be tested for leaks prior to use. If the worker must handle animal cages or other possibly sharp-edged objects, canvas gloves or heavier “canner’s” (19) gloves may be worn over the rubber gloves to prevent tears. Gloves should be put into a suitable liquid germicide immediately after use. If they are not excessively soiled, they may be permitted to soak for an extended period in the disinfectant, and subsequently washed, dried and prepared for re-use without autoclaving. All used laboratory clothing should be discarded into a closed container and subsequently sterilized. Heavily contaminated clothing should be wetted with a germicide and sterilized immediately after use. Separate dressing rooms, complete with showers, clothing discard containers, and germicide sprays, are an obvious necessity. Certain circumstances may warrant the use of additional laboratory “clothing.” If UV lamps are employed eyeglasses complete with side enclosures should be worn. For handling infected small animals, heavy, clear plastic face masks (20) covering the forehead and face to the chin should be worn. Monkey handlers in many laboratories use these face masks and heavy, gauntlet-type, leather gloves in addition to the labora-

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tory garb previously described. Monkey handlers, even in the normal animal house of at least one large biologicals manufacturing company, are required to wear wire mesh face masks and leather gauntlets in addition to conventional laboratory clothing ( Gausch, 1960). In animal exposure operations, fermentor rooms, and other areas of potentially heavy contamination it is necessary to provide a higher level of protection, particularly for the respiratory tract. Wedum et al. (1956a) suggested the use of commercial respirators, standard military gas masks, and ventilated personnel hoods, Henderson ( 1952) indicated the desirability of wearing a respirator for protection during aerosolization of pathogens but gave no data as to the desired effectiveness of such a device. Gauze surgical masks and felt filter respirators were tested by Abramson (1956) by exposing masked rabbits to aerosols of Mycobacterium tuberculosis. Average efficiencies of 13.8%were computed for the gauze masks and 71.4%for the felt filter masks. Guyton and co-workers (1956), using aerosols of B . subtilk var. niger, tested several types of these gauze masks on the workers and noted maximum efficiencies of 39%. Efficiencies of 99.9% or greater were observed for several commercial dust respirators and industrial masks (21). Recent developments in high efficiency filters permit excellent protection with these respirators without undue discomfort to the wearer. Such devices should be used with some caution. Although the tested efficiencies may be very good, they rarely are fitted exactly (Guyton and Lense, 1956) and, if the hazard is severe, protective standards of 99+% for the filter element become nearly meaningless. The full-face gas mask with a bacterial filter element presently available from safety supply houses ( 22) affords considerably better protection, protects against eye damage from UV lamps and, in recent forms, is reasonably comfortable for short work periods. It is an excellent device to have available to the laboratory workers for decontamination work in the event of a major accident. Full protection from aerosols, animal bites, abrasions, and UV light over extended work periods is readily afforded personnel by a ventilated suit (23) (see Fig. 9 ) . Wolochow et (12. ( 1957) describe the use of such a suit in an animal exposure operation, and H e d h (1958) noted that a ventilated vinyl suit is used in his laboratory-pilot plant when infectious agents were processed. The hood, or head piece, should be made of clear, 0.012- to 0.020-gage vinyl plastic with good low-temperature flexibility. The suit body should preferably be made of vinyl-coated nylon material to resist snags and tears. Approximately 15 c.f.m. of clean, fresh, oil-free air at temperatures between 60° and 80°F. should be supplied by air hose or by tank. Decontamination of the suit can be done successfully

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with suitable liquid germicides, provided the liquid is washed and scrubbed carefully over all surfaces and allowed to remain on the fabric for some time. Barrett (1959) described the successful use of 2%peracetic acid in decontamination of a built-up vinyl plastic suit worn by a worker tending germfree animals. This acid appears to be a widely applicable disinfectant but is extremely corrosive and difficult to use. Rubber gloves, not ordinarily considered an article of clothing, should be mentioned at this point. In the infectious disease laboratory great faith must be placed in the security afforded by the rubber gloves, yet these protective devices receive very frequent insults. Surgeon’s gloves, usually of pure gum latex, are susceptible to damage from heating, from germicides ( quaternary compounds are most deleterious), from UV irradiation, and from cuts and tears from animal cages, broken glassware, autopsy instruments, and other sources. If rough work is required, additional hand cover is desirable. Neoprene or plastic base gloves are available (19) and should be used. Cotton overgloves can be worn when tending animals. Arm-length gloves used in hoods are again used in areas of great risk and are subject to much abuse. Gloves for this purpose must be selected for exceptional tear and puncture resistance, resistance to decontaminating chemicals, and resistance to UV and ozone. Several synthetic rubbers (neoprene, as an example), properly compounded, offer good results in these respects (24). Other compounds which may offer better characteristics (Hypa101-1,~ Viton?) have not yet been employed for this purpose.

L. THEUSEOF PLASTICS IN LABORATORY SAFETYDEVICES The recent years have seen a rapid increase in availability of special equipment for the laboratory worker. The increased use of plastics in the laboratory is perhaps indicative of these rapid changes: petri plates, test tubes, flasks, beakers, funnels, and similar equipment are now available (25) in polystyrene, polyethylene, T e f l ~ n ,and ~ other plastics. Many millions of polystyrene petri plates have already been used generally with little indication of unfavorable effect on culture growth and with at least one report (Darlow, 1960) of improved safety by virtue of their nonshattering characteristics. Sterile tissue culture flasks with a surface especially prepared to retain the tissue mat are now available. Molded throw-away syringes with hypodermic needle attached are also useful because the needles are always sharp, the syringe will not crack or chip, and the needle will not separate from the syringe under pressure, The packaging of these assemblies provides a safe holder for the needle until the unit is to be used. E. I. DuPont Co., Inc., Polychemicals Department, Wilmington, Delaware.

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Molded plastic glassware substitutes for the laboratory are becoming available in increasingly wide varieties and at lower costs. Some of the more complex items will soon be available in autoclavable plastics which may be re-used a number of times. Additional simple devices, such as 1- and 10-ml. pipettes] intranasal pipettes, Erlenmeyer flasks, and other commonly used items will undoubtedly be manufactured for throw-away usage. The use of such apparatus on a use and throw-away basis is often economical when the full costs of cleaning and preparing glassware are examined; the corollary benefit of improved laboratory safety by virtue of handling ease and reduced breakage may be of even greater value. Some investigators may wish to use glass laboratory ware to resist chemical attack or to guarantee the absolute neutrality of the container for trace element work. Ray (1957) suggested that breakage of frequently used glassware and the hazard of flying glass from explosions could be reduced if the glass vessels were dipped in a plastisol solution producing a flexible vinyl coating, For more meticulous requirements in the laboratory the use of heavy-wall Pyrex (26) should always be considered. Such flasks, tubes, and beakers are extremely rugged and, on the basis of longer life expectancy, are often more economical than the standard weight glassware. Flasks selected for use on culture-shaking machines should be tested periodically with polarized light to detect strains in anticipation of cracking and actual failure. Heat-sealable, flexible, plastic films, usually vinyl or polyvinyl, have been found useful for many applications in the laboratory. Phillips et al. (1955) described an inexpensive plastic safety work hood useful for many operations and requiring only limited space. It was equipped with attached rubber gloves, an exhaust blower, and an air-lock entry port (27). Trexler and Reynolds (1957), attempting to design low cost enclosures for germfree animals, described a very useful flexible film enclosure with obvious applications to containment of infected animals. Plastic lined rooms have also been constructed (Trexler, 1959) and used for gnotobiote maintenance. Lamanna ( 1960) used heat-sealable, flexible] polyvinyl sheeting to enclose several usually hazardous operations, in an effort to develop safe methods for the laboratory without resorting to some of the more expensive devices which have been described here. As Trexler (1959) has commented, the weaknesses of the plastic films are the lack of resistance to heat and to tears or punctures. Such failings can be overcome, Some polyvinyl films may be autoclaved, although some loss in transparency is observed. The tear-puncture problem can be reduced by fabricating all but the view window portion out of vinyl-coated nylon material which is light and extremely durable. The potentialities of such materials are great when one considers the possibility of enclosed

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animal cage racks with access gloves at strategic points, simple zipup enclosures for centrifuges, shaker cabinets, and other hazard-producing devices. V. Decontamination In the course of the daily work in the infectious disease laboratory, the contamination of the atmosphere, the work materials, and the operator may be widespread. Even for an operation performed with the most careful precautions, equipment must be cleaned up after the operation. For an accident or spill, decontamination may be required for an entire room or for a building and its contents. In either event, more than one method of decontamination may be needed. Conventional methods of decontamination have been adequately discussed elsewhere ( McCulloch, 1945; Reddish, 1957); however, it is pertinent to discuss here a few points peculiar to the infectious disease laboratory, and to examine some recent developments in gaseous phase decontamination which have proven effective for decontamination of large spaces and of sensitive equipment.

A. THERMAL DECONTAMINATION Heating is the simplest and most widely used 'method of sterilization and is also the most effective. Time and temperature relationships required for killing of most agents have been published ( Perkins, 1957), and special applications for air sterilization have been discussed in a previous section. This method should be used whenever possible. B. LIQUIDDECONTAMINANTS A wide variety of liquid decontaminants is available. These materials have been evaluated for many applications ( McCulloch, 1945; Reddish, 1957 ) . It is beyond the scope of this work to detail the selection of decontaminants and procedures for their use for every conceivable microorganism and situation, but it is strongly suggested that the efficacy of every liquid disinfectant proposed for use in the laboratory be evaluated for each agent and for each different application, Optimal organism recovery techniques should be employed; consideration should be given to the amount of solid organic matter present, the temperature, the acidity ( p H ) , and other variables which may make published data inapplicable. Liquid germicides are excellent as discard tray solutions for glassware and small apparatus pending sterilization in an autoclave. It is desirable that the germicide selected for this purpose does not etch the glass or leave a residue. However, residual effect which has been observed for

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several disinfectants (Klarmann et al., 1953; Lester and Dunklin, 1955) can be used to advantage in decontaminating animal cage racks, work tables, floors, and entire areas. The synthetic phenolic-base disinfectants most useful in this respect (“0-Syl” and “Amphyl”) do not leave gummy film, as do creosol and pine oil, and remain effective on a wider variety of surfaces, Germicides applied expressly for the residual effect should be noncorrosive for the materials treated. Liquid germicides for personnel decontamination should be selected not only for specific action against the agents involved but, additionally, for minimal ill effect on the skin and mucous membranes. It may be desirable to have special germicides, germicidal soaps, or germicidal lotions (Murray and Calman, 1955) available to the workers if the liquid germicides required for routine use in the laboratory are irritative or allergenic.

C . GAS OR VAPORPHASEDECONTAMINANTS Contaminated safety hoods, delicate equipment, and large areas frequently do not lend themselves to use of heat or liquid germicides. The use of gaseous disinfectants such as ethylene oxide, formaldehyde vapors, and, more recently, p-propiolactone ( BPL) , has provided a reasonably satisfactory solution for such problems. In order to reduce flammability, ethylene oxide is customarily employed in combination with carbon dioxide (28) (10%ethylene oxide, 90% CO,) available in standard 200-ft.3 cylinders or with a halogenated hydrocarbon diluent (29) (11%ethylene oxide, 89% halogenated hydrocarbon) available in low-pressure storage canisters of the throw-away aerosol bomb type. Ethylene oxide, in these mixtures, is noncorrosive but slow acting; average sterilizing times vary between 4 and 8 hours. Considerable absorption occurs in rubber, leather, and some plastics. Ethylene oxide is sporicidal at concentrations of 450 to 1000 mg. per liter with exposure times of 5 and 2 hours, respectively (Lloyd and Thompson, 1956). There is evidence that presence of moisture in the air is essential (Kaye and Phillips, 1949). Air temperatures of 85O to 110OF. are desirable for best efficacy. The former (CO,) mixtures must be uscd in closed containers, and the usual practice is to use modified steam pressure sterilizers. Schley et al. (1960) described simple methods for applying the latter halogenated hydrocarbon mixture in polyethylene bags, autoclaves and simple metal drums (oil drums). These mixtures also, thereby, eliminate the flammability hazard that exists with pure ethylene oxide and the high pressure occurring with the carbon dioxide diluted material. Formaldehyde-steam vapors are most useful for decontaminating rooms, safety hoods, and ventilator filters. Wedum et al. (1956a) suggests that 1 ml. of 37%formaldehyde solution be vaporized for each cubic foot

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of volume enclosed and allowed to act for 24 hours. Vaporization may be achieved with a commercial vaporizer or simply by boiling Formalin with water on an electric hot plate. Since high relative humidities (so%+) are required for effective action, it may be necessary to vaporize additional water. Air temperature should be in excess of 70°F. Hoods, vent ducts, filters, and even rooms usually may be satisfactorily sealed with masking tape and sealing compounds on doors and windows. Filters in safety hoods should be provided with loose dampers on the exit side to permit concentration buildup along with some leakage of these vapors through the filter. Filters may be decontaminated in place without closing up the exhaust duct by vaporizing 1ml. of the 37%solution per cubic foot of air per minute for 30 or more minutes (Wedum et al., 1956a). Disadvantages of formaldehyde are: ( 1) Its persistent residual paraformaldehyde, which covers walls and surfaces; the slow depolymerization of paraformaldehyde can affect culture growth in such an enclosure; ( 2 ) an uncertainty of decontamination efficacy due to its very poor penetrating power ( Committee on Formaldehyde Disinfection of the U. S. Public Health Laboratory Service, 1958; Hundemann and Holbrook, 1959). Recently BPL has also been suggested for wide area decontamination (Hoffman and Warshowsky, 1958). It is effective against a wide variety of viral, bacterial, and fungal agents, both in aqueous solutions and in the vapor phase (Hartman et al., 1955; LoGrippo et al., 1955; Dawson et al., 1959, 1960). Its microbicidal efficacy in the vapor phase is, in general, on the order of 25 times greater than that of formaldehyde and 4000 times greater than that of ethylene oxide. It is effective at temperatures of 10°C. or below but requires high humidity. Allen and Murphy (1960) observed that in a closed container at 25OC. in an equilibrium concentration of 13.2 mg. BPL per liter of moisture-saturated air, sterilization time varied from 60 minutes for dry spores of B. subtilis var. niger to 5 minutes for Pseudomonas aeruginosa. Hoffman and Warshowsky ( 1958) found that in very large volumes up to 75,000 cubic feet (i.e., a building), concentrations of approximately 5 mg. per liter of air for 2 hours at U 0 C . and 80%relative humidity (R.H.) completely eliminated contamination with B. subtilis spores. For optimal action, the BPL should be used with relative humidities of 70%or higher at a temperature of 24°C. or higher, although it is effective at temperatures down to 10°C. or even lower. BPL is a valuable substitute for ethylene oxide and formaldehyde as a vapor phase disinfectant. It is an active agent, leaves no residue when used in its pure form (97%) (Spiner and Hoffman, 1960), is apparently not corrosive, is easily disseminated, and can be air-washed clean in a

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relatively short period. However, caution must be exercised in its use, and a need for further testing is indicated before it should be employed universally. Disadvantages to its use include the fact that it is a known carcinogen in subirritating doses (Roe and Glendenning, 1957) and that extended contact with the liquid can cause erythema and vesication (Spiner and Hoffman, 1960). Its penetrating power is similar to that of formaldehyde but is not as good as ethylene oxide, and its efficacy should be tested carefully before routine use for decontamination in filter mats or fabrics requiring deep penetration. Generation of the vapors ean be done simply by elevating the temperature, boiling off the vapor, or dispersing it with spray-type devices. This discussion of vapor phase decontaminants has been concerned largely with the effects of these vapors for surface decontamination. Their effects on airborne contaminants have not been as well delineated. For terminal decontamination, each of those listed is usable and should be selected on the basis of the action desired. For decontamination of airborne contaminants in habited spaces, a selection of less toxic vapors is indicated, and considerable energy has been expended in this direction. Glycols, hypochlorites, hexylresorcinollevulinic acid, a-hydroxycarboxylicacids, and many other substances have been used with varying degrees of success (“Heating Ventilating Air Conditioning Guide,” 1958; Bourdillon et al., 1948a; MacKay, 1952; Morris, 1960). The general difficulty and even nuisance of this practice would seem to make it desirable only in extreme circumstances. In most laboratories the changeover rate of the air in any room ventilated in accordance with modern practice would appear to be as effective as such vapors in reducing the level of airborne contamination. The maintenance of 50%R.H. in the rooms may also be very effective in reducing the number of viable airborne bacteria. Dunklin and Puck (1948) showed that approximately 504%R.H. provided a very rapid kill of many species of bacteria in aerosols, Filtration and UV irradiation can be employed to decontaminate recirculated air. The principal value of these vapor decontaminants would seem to lie in protection against fungi and spores which are resistant to action by UV irradiation, Corroborating data for the agent in question are indicated.

D. ULTRAVIOLET IRRADIATION Ultraviolet irradiation from low-pressure mercury tubes is useful in the infectious disease laboratory against both surface and airborne contamination, It has been employed in safety hoods, walk-in incubators, room air locks, on animal cage racks, in ventilating ducts, and in irradia-

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tion of room air. Ultraviolet lamps can be used successfully provided consideration is given to certain factors: (1) The rays have poor penetrating power. Dusts, liquids, or almost any direct radiation shield nullify the sterilization against surface contamination, and a layer of dirt or dust on the UV tube itself will reduce the output appreciably. (2) Air temperature and velocity affect both the UV output of the tubes and the action on airborne microorganisms (Luckiesh, 1946; Nagy et al., 1954) and apparent detrimental effects of high humidity have been noted (Koller, 1939; Stenderuk, 1950). ( 3 ) The effectiveness of UV varies widely for the various species of microorganisms (Koller, 1952), but it is greatest against vegetative species, The most useful applications of UV in infectious disease laboratories are for control of contamination ( 1) in equipment housings (shaking machines, centrifuges, safety hoods, incubators, etc.) and ( 2 ) in animalholding rooms. When used inside a safety housing, the selection of the UV tube is more frequently a problem of fitting a tube into the housing than that of achieving an irradiation intensity level. Surface intensities up to 75 pw./cm.2 at surfaces can readily be achieved. Care must be taken to ensure that workers are not exposed to such intensity without protective clothing. Door interlock switches and warning signs are needed. Because ozone output from the lamps is deleterious to rubber products and can affect cultures, it is usually desirable to use low ozone-type lamps. In the animal room the installation of UV light fixtures on small animal cage racks reduces the possibility of pathogenic microorganisms being disseminated from infected animals. Phillips et al. (1957) installed UV tubes to direct rays across the tops of cages with closed sides and bottoms at an intensity of 250 pw./cm.2. Upper-level UV, ventilation duct irradiation, and direct irradiation are all helpful in reducing contamination in animal rooms. Installation in ventilating systems and data for upper-level room irradiations have been discussed by Luckiesh (1946), Harstad et al. (1954), and Nagy et al. (1954). Shechmeister ( 1957) reviewed the literature to that date. Practical application data are available (Buttolph and Haynes, 1950; Buttolph et nl., 1950). Working levels of irradiation intensity desired for area decontamination in the infectious disease laboratory have not always been clearly defined. Wedum et al. (1956b) suggested a level of 20 or more pw./cm.2 at the floor level in air-lock entries to contaminated rooms and 10 pw./cm.2 in laboratory rooms. In a different application, Hart (1960) maintained a level of 18 to 30 pw./crn.? and achieved very effective prevention of

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wound infection in hospital surgery. Wolochow et al. (1957) employed UV at 30 pw./cm.2 in a hood housing a device for exposing monkeys to aerosols of microorganisms and used 20 pw./cm.2 at waist-level height in an animal-holding room. The figures given above are those for installations wherein there is usually only short-term personnel exposure, or when workers are dressed in suitable protective clothing. For longer exposures, the Council of Physical Therapy, American Medical Association has specified a maximum intensity of 0.5 p w . / ~ r n for . ~ 7 hours or less and 0.1 pw./cm.2 for continuous exposure ( cited by Shechmeister, 1957). Hart ( 1960), discussing some 25 years’ experience with applications of UV light, reported that simple plastic eye shields have given good protection and no erythema has been noted either on patients or on medical workers subjected for prolonged periods to the much higher intensities listed above. It is probable that in routine use the minimal protection afforded by a pair of protective goggles or a modified face mask as described by Buttolph and Haynes (1950) is warranted.

VI. laboratory Design The opportunity and requirement for the design of a laboratory built especially for the study of infectious diseases is rare. More often, existing facilities must be employed and modified to fit this type of work or, in new construction, the laboratory is only one component of a larger facility. To this end, these remarks are generally concerned not with the general problem of laboratory design and operation for which there are many excellent precedents, but rather with a few of the specific problems of infectious disease laboratory design and operation which affect the safety of the work. Wedum et al. (19564 discussed in considerable detail the design requirements for an infectious disease laboratory and proposed several basic plans. Laboratory designs included facilities for small animal inoculation and holding, dishwashing, media preparation, personnel showers, clothes change rooms, laboratories, and offices. Closed safety cabinets, equipment for respiratory challenge of animals, ventilated animal cages, and other specialized safety equipment were described. Solotorovsky d al. (1953) described a laboratory installed primarily for studies employing tubercle bacilli but suitable for work with other agents creating a “higher hazard of airborne infections.” The facilities and operating procedures described were somewhat less extensive than those proposed by Wedum et nl. (1956a) but did include similar precautionary tcchniques.

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The remarks below are drawn from these and other writings, and from some personal experience in modifying existing structures for safe use as infectious disease laboratories. A. LOCATION AND TRAFFIC PATTERN If possible, the laboratory should be completely separated from receiving areas, classrooms, or other areas to which there is ready public access. Entry to the laboratory should be via a noncontaminated or “clean” reception room or office area or employees’ dressing room. With the exception of emergency exits, direct access from public corridors to laboratories or infected animal-holding rooms should be avoided. Service entrances, waste disposal or animal receiving areas should open into a clean area connected to the laboratory via double-ended sterilizers and doubledoor air locks to restrict entry. Within the laboratory, every attempt should be made to provide each worker or work group with a workspace that is a “dead end,” with no through traffic. Individual rooms, open-sided cubicles, even well-divided peninsula-type benches all offer this isolation in varying degrees. If a glassware preparation and service department is provided, it should be separated from the laboratory and animal rooms by double-ended, automatically controlled sterilizers. Two sterilizers may be needed to permit decontamination of animal wastes and carcasses separately from laboratory wastes and glassware. A solid hearth animal cremator (30) should be installed near the laboratory to incinerate carcasses. B. ROOM FINISHES The type of room finish should be selected to facilitate both containment of hazardous contamination and eventual chemical decontamination of the entire area. Simplicity of furnishings and trim is essential and false ceilings, recessed pipe chases, and concealed spaces should be avoided unless these can be sealed tightly. In new buildings, plaster and cement finishes offer excellent seals. Existing rooms can be sealed with tapes, elastic caulking compounds (31), or plastic sealers. Recent developments in polyester, isophthalic, and epoxy plastic resin finishes offer excellent materials for this purpose. By their use in conjunction with glass fiber cloth reinforcement applied to wood or brick surfaces, it is possible to achieve the equivalent of monolithic construction. In particular, epoxy resin paints (32) which are chemically resistant, can be applied with selected pigments to almost any clean surface as a finish coat. The initial cost of this room finish is approximately five times

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that of an ordinary paint cost, but the durability of this finish is such as to make it economically justifiable. Other flexible epoxy materials are also available for sealing wood floors and for finishes on concrete floors. If a resilient floor is desired, application of sheet vinyl covering provides a durable watertight surface which is easily cleaned and decontaminated. C. SERVICES Water, vacuum, and waste services require special consideration. Other services, including gas, air, steam, and refrigerants, require only neat installation and normal load factors for design. All building mechanical equipment should be installed external to the laboratory proper to reduce decontamination problems and to facilitate servicing, Openings through which pipes, conduits, and ducts enter the laboratory space should be sealed to prevent leakage of contaminated material into service chases, hollow walls, or other areas difficult to decontaminate. The water supplies to the laboratory should be isolated from the building supply and the potable water by an open break tank to prevent possible back siphonage. Spring-loaded faucets are helpful in reducing water usage and subsequent waste treatment problems. All liquid wastes from the laboratory should be presumed to be contaminated. If wastes from animal rooms can be handled separately in the animal room to reduce solid content of the sewage, the remaining laboratory wastes can usually be chemically decontaminated. The microbial agents used in the laboratory will determine the choice of chemical. Thermal treatment at 200°F. for 30 seconds has been suggested by Wedum et al. (1956a), who notes that if the pathogenic sporeformer Bacillus anthrucis is present, a temperature of 260OF. for 10 minutes may be necessary for sterilization in a continuous flow system. Such treatment has been successfully employed at the Naval Biological Laboratory for several years (see Fig. 4). Installation and operating costs have been high and severe corrosion of components has made frequent repairs necessary. Simple batch pasteurization tanks are suggested for use in small laboratory installations. For new construction, corrosion-resistant waste piping is suggested. Careful routing and identification of all waste system vent pipes and separation of the laboratory waste pipes from the building sanitary waste piping is mandatory if maintenance personnel are to be protected from inadvertent exposure to contaminated material. Vacuum systems serving the laboratory should be equipped with high efficiency (99.95+%) air filters (33) located in the pipelines prior to their exit from the laboratory work area. These should not be used as substitutes for traps or additional filters on each apparatus connected to the vacuum system but should be used as secondary safeguards. Air from

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the vacuum pumps should not be discharged into occupied spaces; routing it to the ventilation exhaust system is an acceptable method of disposal. D. VENTILATION SYSTEMS In addition to meeting conventional ventilation requirements, the laboratory ventilation system should provide a flow of air from the potentially least contaminated toward the most contaminated areas and a safe discharge of all exhaust air. The tendency to specify various levels of pressures for this purpose should be avoided, since a pressure drop to a negative pressure across a closed door is nonexistent when that door is opened, and the confidence engendered by a heavily engineered zone pressure system is misplaced. Laboratory rooms should be individually ventilated with 100%fresh air at 10 to 15 changes per hour or more; the design of the system should include the effect of the air volume exhausted through safety hoods. Ventilating air supplied to the laboratory should be filtered in order to reduce incoming contaminants. A slight excess (ca. 10%) of forced exhaust over input for each room will provide constant inward flow. Open vent grills or louvers on doors between rooms within the laboratory will permit air flow and will reduce the pressure change occasioned when the doors are used. Animal rooms should receive at least 20 air changes per hour, with careful attention given to air distribution in order to eliminate drafts. Air conditioning is desirable and, if used, will permit sealing all windows closed. These air change rates, which are somewhat higher than normal, are desirable not only for good ventilation but also for rapid clearance of airborne contaminants in the event of accident (Morris, 1960). Although 100%fresh air supply systems are the most desirable for this application, recirculation systems can be employed, provided that care is taken to provide decontamination of the air being recirculated and that air from a potentially highly contaminated area is not recirculated to an area of low potential contamination, Animal-holding rooms should be ventilated entirely with fresh air if at all possible, There is no requirement for routine filtration or other decontamination of all exhaust ventilating air from the laboratory rooms provided safety hoods are employed for hazardous operations previously described, However, for precautionary protection of workers outside the infectious disease laboratory, the room air exhaust should be extended clear of all other air inlets, buildings, or occupied areas. Exhaust air from animal rooms should be filtered at the exhaust grills in the animal room to remove dust and hair, and should be given further treatment with UV or filters as is suggested for safey hood exhausts if a highly infectious agent is employed, or if the animals were inoculated via the respiratory route.

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VII. Safety Programs and Administration The described safety devices would be of very little value if their use were considered to be a substitute for safe procedures and careful techniques. Because of the inherent hazards of the work, both to the individual and to his co-workers, the definition of what comprises safe procedures and careful technique must be carefully spelled out in safety programs, safety standards, or standardized technical operating procedures. These provisions, at the minimum, are a set of regulations which provide the basis for safe action; at the maximum, they are a complete set of operating instructions in a job analysis for each operation. To be effective, a safety manual should encompass industrial safety practices, standard operating procedures, standards for protective garb, organization rules and regulations applicable to the laboratory, standard germicides for use in the laboratory, first aid and medical care regulations, and general rules for procedures in the laboratory. To compile such a manual is a formidable task, and to obtain compliance by the users is nearly as difficult. Some organizations solve both problems by establishing employee safety committees to draw up and administer such standard manuals with participants representing all working levels in the organization. Safety manuals used in public health laboratories, hospitals, government laboratories, and similar public institutions are frequently available and are useful as models in establishing a program to fit the specific needs of the users. Every worker should be familiar with the standard operating procedures of the laboratory at which he is employed. A short indoctrination course is most helpful to new employees before they begin work in the laboratory. At the Naval Biological Laboratory a 2-week course in “Hot L a b techniques is required of every new employee. The course includes actual practice in making culture dilutions using pipette and safety bulb, inoculating animals, procedures for discarding glassware, and open front work hood operating techniques. Lectures, demonstrations, and motion pictures are also used to familiarize the worker with other special procedures employed in this laboratory. Every laboratory engaged in work involving infectious agents should have a medical officer available as a consultant or staff member, Black et al. (1953) and Fish and Spendlove ( 1950) described the value of preemployment and periodic physical examinations, X-rays, and blood tests in two large research establishments working with human pathogens. In addition to these functions, the medical officer on call must be available for corrective action following accidents with infectious material or for consultation with any laboratory employee who reports illness. Adequate

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medical records are important to both employees and management since medical records and histories of illnesses and of accidents (regardless of their immediate outcome) are frequently the basis for settlement of workmen’s compensation claims. In many states, eligibility for workmen’s compensation medical care and disability benefits depends on establishment of the employee’s occupation as a proximal cause of the disability. Immunization of laboratory personnel with the most effective products available is mandatory. Smadel (1951) listed several viral vaccines and rickettsia] vaccines used at the Army Medical Service Graduate School and suggested that such vaccines should be employed whenever available for the particular agent under study. The practical application of any set of safety guides, rules, or regulations must inevitably rest with the laboratory worker and his supervisor. No guide can possibly cover all the minutiae of the daily work, and in the final analysis each worker must learn to think safety and instinctively to act safely. The supervisor, to encourage this attitude, must set an example and exercise control in maintaining good safety standards. As a representative of management, he has the legal and moral responsibility to take all reasonable action to provide a safe place of employment. Wedum (1959) discussed an analysis of the progress of an intensive safety program in a large research laboratory. He concluded that after installation of all possible safety devices, controls and programs, further progress in accident prevention could be made only when the research scientist made accident prevention a part of each research plan.

VI I I. Discussion Many of the safety measures described in this chapter have been available for 10 to 20 or more years. Some organizations have made extensive use of them and have actively promoted the usage of safety equipment and techniques. Still other organizations have used a permissive approach in which the laboratory worker sets his own individual standard of safety with little or no direction or guidance. Laboratory management bears both a moral and a legal responsibility to ensure a safe work environment. Somers and Somers (1954), discussing workmen’s compensation, pointed out that although “legal doctrine has long held that injury could be considered as arising out of employment if it could fairly be traced to the contributing proximate cause,” the emphasis has shifted from the word proximate to the word contributing. The recent history of workmen’s compensation cases in the courts has made it clear that it is sufficient that the employment need be considered only a contributing cause.

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Heinrich (1931; cited by Somers and Somers, 1954) observed that approximately 88%of industrial accidents (in a group of 75,000 studied) were caused primarily by the unsafe acts of workers, Similar conclusions could be drawn from histories of accidents in the microbiology laboratory, Unsafe work practices stem, in part, from the reluctance on the part of the laboratory staff to employ many of the various safety measures available. The objections usually stated are (1) the safety devices and techniques are cumbersome and awkward, and slow the course of the work; (2) no real hazard exists; ( 3 ) no persons other than the laboratory worker himself are fully aware of the details of the work and are capable of advising safe practices; (4)use of safety procedures will increase the cost of the work. It appears that management bears substantially the responsibility for the safety of the worker, but the worker, himself, is usually the controlling agent in the application of any safety measures. Although both the laboratory worker and the administration are vitally interested in safe operations, widely differing points of view on methods of implementation of safe procedure may prevail. Perhaps the best way to arrive at a goal which is mutually satisfactory would be for the worker, as the most qualified participant, to originate the safety measures and for management to provide whole-hearted support in advice and assistance. Variations on this theme have been noted previously here and elsewhere, To work toward this ideal safety implementation technique, two basic requirements should be met. First, management or administration should establish and publish clearly defined policies on safety; they should be willing to provide such safety devices as may be needed, and should support and encourage the workers’ demands for improved safety by recognizing the worth of any time spent in such activities as well as providing the competent assistance of industrial hygienists or safety engineers. Secondly, the laboratory worker should have a basic training in safe procedures, a clear understanding of management’s policies on safety, and, most obviously, the desire to work safely. It is unrealistic to suppose that a short indoctrination course, or a reading of a safety manual, will change work habit patterns that have been learned and practiced, in some cases, for many years. To be most effective, training in safety procedures should accompany training in all microbiological laboratory procedures at all levels of education. This suggestion is not new; rather, it is a reiteration of the plea heard from nearly every industrial safety worker. It is, in the final analysis, the means which can bring the greatest success in reduction of all laboratory accidents.

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IX. Commercially Available Safety Devices Type of equipment 1. Open-front hoods

Supplier

Kewaunee Mfg. Co., Adrian, Michigan; Hamilton Mfg. Co., Two Rivers, Wisconsin; S. Blickman, Inc., Weehawken, New Jersey 2. Closed-front hoods (single Controlled Atmosphere Mfg. Co., Jacksonville, Florida; Air-Shields, Inc., Hatboro, Pennsylunit) vania; Microchemical Specialties Co., Berkeley, California; S. Blickman, Inc., Weehawken, New Jersey Kewaunee Mfg. Co., Adrian, Michigan; Controlled 3, Modular hoods Atmosphere Mfg. Co., Jacksonville, Florida; Air-Shields, Inc ., Hatboro, Pennsylvania 4. Electric air sterilizer H. E. Trent Co., Philadelphia, Pennsylvania Owens-Corning Fiberglas Corp., Toledo, Ohio 5. Filter materials glass mat 6. Filters-unit type high Mine Safety Appliances Co., Pittsburgh, Pennsvlvania; American Air Filter Co., Inc., Louisville, efficiency Kentucky; Cambridge Filter Co., Cambridge, Massachusetts 7. Culture-shaking machine, New Brunswick Scientific Co., New Brunswick, New Jersey rotary 8. Screw-cap centrifuge tube International Equipment Co., Boston, Massachuset ts holders 9. Centrifuge tubes (compresBeckman/Spinco Division, Palo Alto, California sion seal) 10. Centrifuge enclosure S. Blickman, Inc., Weehawken, New Jersey Fisher Scientific, New York, New York 11. Safety blendor bowl Garlock Packing Co., Palmyra, New York; Crane 12. Mechanical rotary seals Packing Co., Chicago, Illinois 13. Gravity- or force-operated Arthur S. LaPine and Co. (pumpette), Chicago, Illinois pipettors 14. Pipettors and micro Alfred Bicknell Associates, Inc., Cambridge, pipettors Massachusetts; A. S. Aloe Co., St. Louis, Missouri; Fisher Scientific Co. (propipette). New York, New York Braun-Knecht-Heimann Co., San Francisco, 16. Microb-incinerator California 16. Ventilated animal cages Parco Co., Falls Church, Virginia 17. Surgeon’s gown (modified) Apasco Corporation, Wolfeboro, New Hampshire Apasco Corporation, Wolfeboro, New Hampshire; 18. Shoe covers Falcon Plastic Products Mfg. Co., Culver City, California B. F. Goodrich Co., Cleveland, Ohio; M. L. Snyder 19. Canner’s gloves and Son, Philadelphia, Pennsylvania 20. Plastic face masks and Mine Safety Appliances Co., Pittsburgh, Pennsylpersonnel hoods vania; Braun-Knecht-Heimann Co., San Francisco, California; Chicago Eye Shield CO., Chicago, Illinois

186

MARK A. CHATICNY

Type of equipment

Supplier -

21. Dust respirators

22. Gas masks 23. Plastic ventilated suit 24. Arm-length rubber gloves

25. Plastic laboratory ware 26. Pyrex lahoratory ware 27. Flexihle plastic safety cabinet 28. Ethylcne oxide-carbon dioxide safety mixture 20. Ethylenc oxide-fluorocarbon safety mixtures 30. Animal incinerators 31. Elastic caulking compound 32. Epoxy resin finishes

33. Filter, ceramic

Mine Safety Appliances Co., Pittsburgh, Pennsylvania; American Optical Co. (type R-2000, with R25 filter), Safety Products Division, Southbridge, Massachusetts; Chicago Eyc Shield Co., Chicago, Illinois Mine Safety Applianres Go. (with bacterial filter), Pittsburgh, Pennsylvania Snyder Mfg. Co., Inc., New Philadelphia, Ohio; Gordon B. Holcombc Co., San Francisco, California; Dayton Safety Supply, Dayton, Ohio Charleston Rubber Co., Charleston, South Carolina; Erskine Healy Co., Rochester, New York; Wilson Rubber Co., Akron, Ohio Falcon Plnstic Products Mfg. Co., Culver City, California; Braun-Knecht-Heimann Co., San Francisco, California Corning Glass Works, Corning, New York The Torsion Balance Co., Clifton, New Jersey Union Carbide and Carbon (Carhoxide), New York, New York American Sterilizer Co. (Cry-OXIDE), Erie, Pennsylvania Joseph Goder, Inc., Chicago, Illinois A. C. Horn Co., Inc. (Hornflex), Bell Gardens, California; nicks-Armstrong and Pontius (D A P), Dayton, Ohio Glidden Co. (Nupon), San Francisco, California; Shell Chemical Corp. (Epon), New York, New York; Walter N. Boyscn Co. (Copon), Oakland, California Selas Corp. of America, Dresher, Pennsylvania

X. Motion Pictures and Filmstrips on laboratory Safety Devices and Procedures 1. Care of laboratory animals (filmstrip), U. S. Public Health Service, 1953. 80 fr., color, 35 mm. and disk: 1 s., 16 in., 3334 r.p.m., 10 minutes. 2. Using animals in the laboratory (filmstrip), U. S . Public Health Service, 1953. 68 fr. color, 35 mm. and disk: 1 s., 16 in., 33% r.p.m., 10 minutes. 3. Infectious hazards of bacteriological techniques (motion picture), U. S. Public Health Service in cooperation with Biological Laboratories, U. S. Army Chemical Corps, 1951. 13 minutes, sd., color, 16 mm. 4. The centrifuge (filmstrip), U. S. Public Health Service in cooperation with Biological Laboratories, U. S. Army Chemical Corps, 1955. 86 fr., color, 35 mm. and disk: 1 s., 16 in., 3334 r.p.m., 12 minutes. 5. The high-speed blender (filmstrip), U. S. Public Health Service in cooperation

MICROBIOLOGY LABORATORY SAFETY

6.

7.

8. 9.

10.

11. 12. 13. 14. 15.

16.

187

with Biological Laboratories, U. S. A m y Chemical Corps, 1952. 81 fr., color, 35mm. and disk: 1 s., 16 in., 3Y! r.p.m., 13 minutes. The hypodermic syringe (filmstrip), U. S. Public Health Service in cooperation with Biological Laboratories, U. S. Army Chemical Corps, 1952. 95 fr., color, 35 mm. and disk: 1 s., 16 in., 33% r.p.m., 12 minutes. The inoculating needle (motion picture), U. S. Public Health Service in cooperation with Biological Laboratories, U. S. Army Chemical Corps, 1951. 10 minutes, sd., b&w, 16 mm. The inoculating needle (filmstrip), U. S. Public Health Service in cooperation with Biological Laboratories, U. S. Army Chemical Corps, 1951. 102 fr., color, 35 mm. and disk: 1 s., 16 in., 33% r.p.m., 9 minutes. The lyophilizer (filmstrip), U. S. Public Health Service in cooperation with Biological Laboratories, U. S. Army Chemical Corps, 1951. 76 fr., color, 35 mm. and disk: 1 s., 16 in., 3354 r.p.m., 8 minutes. The pipette (filmstrip), U. S. Public Health Service in cooperation with Biological Laboratories, U. S. Army Chemical Corps, 1951. 85 fr., color, 35 mm. and disk: 1 s., 16 in., 33% r.p.m., 9 minutes. Chemical techniques: Pipettors (motion picture), U. S. Department of the Navy, 1955. 5 minutes, sd., color, 16 mm. Handling and use of glassware (filmstrip), U. S. Public Health Service, 1952. 82 fr., color, 35 mm. and disk: 1 s., 16 in., 33% r.p.m., 8 minutes. Safety measures in tuberculosis laboratories (filmstrip), U. S. Public Health Service, 1954. 37 fr., color, 35 mm. and disk: 1 s., 16 in., 33% r.p.m., 9 minutes. Laboratory methods for airborne infection, Part 1: The cloud chamber (motion picture), U. S. Public Health Service, 1957. 28 minutes, sd., color, 16 mm. Methods and instruments used in the study of airborne diseases (motion picture), U. S. Navy, 1957. 9 minutes, sd., color, 16 mm. (Note: available from U. S. Naval Biological Laboratory, Oakland 14, California. ) Ratproofing (motion picture), U. S. Public Health Service, 1954. 10 minutes, sd., b&w, 16 mm.

Distribution data and availability of all of the motion pictures and filmstrips listed above are catalogued in the “Film Reference Guide for Medicine and Allied Sciences” (June, 1960). This catalogue is listed as U. S. Public Health Service Bulletin PHS487. One exception (No. 15) is available as noted. ACKNOWLEDGMENTS The assistance of Miss Doris Clinger, without whom this review would not have been possible, is gratefully acknowledged. This work was sponsored by the Office of Naval Research under a contract with the Regents of the University of California. Reproduction in whole or in part is permitted for any purpose of the United States Government.

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188

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190

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Oxidation of Aromatic Compounds by Bacteria MARTINH. ROGOFF] United States Bureau of Mines, Pittsburgh Coal Research Center, Pittsburgh, Pennsylvania

I. Introduction

................................................... 193 . . . . 195

11. A Central Metabolic Pathway for the Degradation of Aromatic Rings.

A. Key Intermediates.. ........................................... 195 B. General Mode of Oxidation of Aromatic Compounds by Bacteria. . . . . . 201 111. Oxidative Metabolism of Polynuclear Aromatic Compounds by Bacteria. . . 201 A. Naphthalene and Some Naphthalene Derivatives. . . . . . . . . . . . . . . . . . 201 B. Higher Polynuclear Aromatic Hydrocarbons. ....................... 204 C. Alternate Pathways and End Products ............................ 206 D. Considerations in the Biochemistry of Degradation of Polycyclic Aro207 matic Compounds.. .............................................. IV. Oxidative Metabolism of Benzenoid Compounds by Bacteria.. . . . . . . . . . . 208 A. Degradative Oxidation of Some Benzoic Acids.. . . . . . . . . . . . . . . . . . . . 208 B. Oxidative Metabolism of Phenylalanine, Mandelic Acid, and Their p-Hy210 droxy Derivatives. ................................................ V. Degradation of Miscellaneous Aromatic Compounds. . . . . . . . . . . . . . . . . . . . 212 A. Metabolism of the Indole Nucleus.. .............................. 212 B. Degradation of Aromatic Herbicides.. ............................ 213 C. Degradation of Nicotinic Acid.. ................................. 216 VI. Future Experimentation. .......................................... 217 VII. Summary ....................................................... 218 References ...................................................... 219

1. Introduction The degradation of aromatic compounds by microorganisms constitutes an essential step in the “carbon” cycle, in regard to oxidation of both natural products and aromatic compounds added to the environment through the activity of man. The latter includes aromatic compounds present in the effluent water of industrial wastes, synthetic compounds such as the hormone herbicides or the sulfonated aromatic detergents; the burden of their detoxification or degradation is added to the existing natural processes. A study of the means by which bacteria cleave the aromatic ring appeared quite early with the report by Emmerling and Abderhalden (1903) of a strain of Micrococcus chinicus which oxidized quinic acid by aromatization to protocatechuic acid. Work on aromatic oxidations in the

’Present address: Bioferm Corporation, Wasco, California. 193

194

MARTIN H. ROGOFF

next two decades was concerned mainly with the degradation of benzene derivatives by microorganisms (Stormer, 1908; Fowler et al., 1911; Wagner, 1914; Gray and Thornton, 1928). This early work can be found in an excellent review by Happold (1950a) which deals with the biochemistry of the oxidation of aromatic rings by bacteria. Evans and associates ( 1951) summarized the then-known intermediary pathways by which soil bacteria metabolize mononuclear aromatic compounds. Early work with polynuclear aromatic compounds (other than lignin) appeared in the series of papers by Tausson (1927, 1928a, 1929), who reported bacterial attack on naphthalene and phenanthrene. Oxidation of the polynuclear aromatics has not been extensively studied until quite recently; work in the field has been reviewed by Evans (19%) and briefly by Fernley and Evans ( 1958). The ability to utilize aromatic compounds as sole source of organic carbon is not universal among microorganisms. In the light of present knowledge, microbial attack on the aromatic ring appears to be dependent on induced enzyme formation. Since the presence of other oxidizable carbon compounds may prevent the induction of the enzymes necessary for aromatic degradation, the practical criteria imposed for attack are (aerobic) growth in a mineral-salts medium with an aromatic compound as the sole source of carbon and energy. Bacteria capable of growth under these conditions have been isolated from soil, sewage, feces, and sea water. Among these are representatives of the families Coccaceae, Mycobacteriaceae, Pseudomonadaceae, Spirillaceae, Bacteriaceae, and Bacillaceae (Gray and Thornton, 1928; Bernheim, 1941, 1942). Of these the soil pseudomonads appear, by far, the most active. Certain of the higher fungi have been shown to be capable of attack on aromatics including strains of Aspergillus (Kluyver and van Zijp, 1951), Penicillium (Hockenbull et al., 1952), Oosporu (Landa and Eliasek, 1956), and Neurosporu (Gross et al., 19%). The activities of soil and wood-rotting fungi in lignin degradation are well known (Henderson and Farmer, 1955). As regards aromatics, however, attention to the activities of the molds has been directed more towards their synthetic and “transforming” abilities rather than towards their degradative talents. In recent years metabolic studies, consisting mainly in isolation of intermediates and determination of induced enzyme patterns, have yielded some fairly definitive knowledge about the way in which aromatic compounds are metabolized by bacteria. Sufficient information has been obtained to allow us to view the field somewhat broadly and to visualize certain general metabolic pathways through which these aromatic degradations are carried out. The material dealt with in this chapter is concerned with central paths and means by which bacterial enzymes

OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

195

manipulate and cleave the aromatic ring with the formation of hydroxylated aromatic intermediates, and the further cleavage of these hydroxylated compounds to aliphatic intermediates which, at some stage, can enter the main terminal respiratory cycles of the cell.

It. A Central Metabolic Pathway for the Degradation of Aromatic Rings

A. KEY INTERMEDIATES The variations thus far observed in the oxidation of aromatic compounds by bacteria are few. In general, bacterial enzymes cleave the aromatic ring by the insertion of oxygen between the atoms forming the C-H bonds of two adjacent ring carbons, so that two hydroxyl groups are present on the ring in an ortho relationship; the carbon-carbon bond between the hydroxyls is subsequently split, Benzenoid compounds containing various substituent groups undergo enzymatic manipulations so as to reach this ortho-hydroxylated state. Thus phenol is hydroxylated to catechol, which is cleaved. Similarly, polynuclear aromatic compounds undergo the same type of reactions with stepwise oxidation of their rings until benzenoid compounds are reached. The oxidative pathways may be visualized as being analogous to a funnel from whose tip issues the final aromatic residue to undergo ring cleavage. This point of convergence in numerous cases of aromatic ring oxidation is the compound catechol ( 1,2-dihydroxybenzene) . Dependent on substitution in the original aromatic molecule undergoing oxidation an alternate compound, protocatechuic acid (3,bdihydroxybenzoic acid), may be reached. Most known pathways of microbial attack on aromatics are directed toward, and arrive at, these two key intermediates.

1. Metabolism of Catechol and Protocatechuic Acid The key intermediates undergo ring cleavage and further transformation to form aliphatic compounds (succinate, acetate) which can enter the terminal respiratory cycles. The efforts of a number of workers have elucidated the steps involved in these reactions; the pathway by which catechol is degraded is seen in Scheme 1. Catechol is split between the hydroxyl groups to form cis@-muconic acid. Tausson ( 1928a) had suggested that the benzene nucleus was split to form muconic acid, by phenanthrene-oxidizing bacteria, and then oxidized to carbon dioxide. Evans and Happold (1939) excluded tmnqtruns-muconic acid as a possible intermediate on the basis of the inability of their phenol- and catechol-oxidizing Vibrio 0/1 to grow on or to metabolize the compound. Hayashi and Hashimoto ( 1950) tentatively identified as ck,cis-muconic

196

MARTIN H. ROCOFF

acid a product of the oxidation of catechol by a purified Pseudomonas enzyme. Stanier and Hayaishi ( 1951) used the acid obtained by Hayaishi and Hashimoto as a substrate for a crude enzyme preparation which catalyzed the oxidation of catechol to p-oxoadipic acid. The preparation did not yield the 0x0 acid from the muconic acid which excluded it from being a true intermediate. It had been shown by Elvidge et ul. (1950) that a cis,truns-muconic acid they had prepared chemically was virtually indistinguishable from the cis,&-muconic acid, and that simply boiling

, COOH COOH H20

Migration of Double Bond

CH

Catechol

ck&-Muconic Acid

CH~.COOH+CHZ,COOH I

C H 2 . COOH Acetic Acid (As AcstylCoA)

Succinic Acid (As SuccinylCoA)

SCHEME 1. Oxidative metabolic pathway of catechol by microorganisms.

the cis,&-acid inverted it to the &,truns-form. Apparently the treatment used by Stanier and Hayaishi to purify the &,&-acid in their microbiological studies must have inverted the samples. The picture was clarified by the work of Evans and Smith (1951) who demonstrated that the pure cis,&-isomer was metabolized by intact cells, and was nonoxidatively converted into p-oxoadipic acid by a crude catechol-oxidizing, cellfree preparation; the cisJtruns-and truns,trans-isomers were inactive. It was found by Evans’ group (Evans et ul., 1951) that the lactone 7-carboxymethyl-A@-butenolide,related to cZs,cis-muconic acid, could be converted to p-oxoadipic acid by the same cell-free enzyme preparation, The conversion of cis&-muconic acid to 8-oxoadipic acid is carried out by a lactonizing enzyme and a Iactone-splitting enzyme; these have been

OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

197

separated by Sistrom and Stanier (1954a, b). The lactonizing enzyme, which requires Mg++ or Mn++ions for activity, catalyzes the reversible interconversion of cis,&-muconic acid and the ( ) -1actone; it can also convert the cis,trans-isomer into an equilibrium mixture with the (-)lactone, but more slowly than with the natural substrate. Evans and Happold (1939) studying phenol breakdown by a Vibrio found a compound which appeared transiently in the medium and which gave positive Rothera and Gerhardt reactions characteristic of a p-0x0 acid. The compound was isolated by Kilby (1948) and identified as p-oxadipic acid. Thus the steps in the degradation between cis,cismuconic acid and p-oxoadipic acid are complete, the last compound in the chain, however, being the first isolated and identified, The final step before the entry of the compounds into the terminal respiratory cycles was shown by Kilby (1951) to be a C,-C, split of p-oxoadipic acid to succinate and acetate. The enzymology of the C,-C, split was worked out by Katagiri and Hayaishi (1957). They prepared a cell-free enzyme system from a Pseudomonus spp. which metabolized p-oxoadipic acid in the presence of coenzyme A ( CoA) and succinyl coenzyme A ( SucCoA) , In the primary reaction catalyzed by a specific thiophorase SucCoA acts as a CoA donor and p-oxoadipyl-CoA is produced. In a second reaction p-oxoadipyl-CoA is cleaved in the presence of a specific thiolase to yield SucCoA and acetyl-CoA. The SucCoA is utilized again as a CoA donor. Protocatechuic acid is also degraded to p-oxoadipic acid; the steps in this degradation are not so well known (Scheme 2) as those of the catechol path. A cell-free, protocatechuic, acid-oxidizing enzyme preparation obtained from Neurospora crassa was used by Gross et al. (1956) to demonstrate the following sequence of reactions: Protocatechuic acid + &,cis-p-carboxymuconic acid + ( -) -p-carboxymuconolactone + poxoadipic acid. From experiments using [2 :6-C14]-protocatechuic acid as a substrate, it was demonstrated that the keto group carbon was derived solely from carbon-6 of protocatechuic acid. A Pseudomonus cellfree enzyme system also gave p-oxoadipic acid; however, it was found that the keto group carbon was randomly derived from carbon-1 and carbon-6 of protocatechuic acid. In addition the Pseudomonas system was not active on either the ( ) - or ( - ) -p-carboxymuconolactone; this compound is not an intermediate in the Pseudomonus system, which converts protocatechuic acid to p-oxoadipic acid. Recently Ribbons and Evans (1959) reasoned, from tracer results, that in the Pseudomonas system an intermediate with a symmetrical structure is formed before p-oxoadipic acid. They considered butanolido-p,y-y',fl-butanolide ( dilactone ) or its open-chain counterpart p,p'-dihydroxyadipic acid as possibilities; on these, dehydrase action would produce p-oxoadipic acid

+

+

198

MARTIN H. ROGOFF

with the keto group carbon equally derived from the p- and p'-carbons. The dilactone was synthesized and used as a substrate for both the Neurospora and Pseudomonas systems; only the Pseudmonas system produced P-oxoadipic acid from the dilactone. It is probable that the dilactone ( or p,p'-dihydroxyadipic acid) is the intermediate preceding FOOH

5

ICHI

OH

FWH Neurospora

COOH

'COOH

OH

/3 -Carboxyrnuconic

fcid I

Protocotechuic Acid

'I

Pseudomonos

p

-Carboxymuconolactone

I

OH

P

0'-Oihydroxyodipic Acid

SCHEME2. Oxidative metabolic pathways of protocatechuic acid by microorganisms.

p-oxoadipic acid in protocatechuic acid metabolism by these pseudomonads. 2. Salicylic Acid

Catechol is reached directly in the oxidation of phenol; it apparently is formed by a single oxidative decarboxylation step from benzoic acid (Parr et al., 1949; R. A. Evans et al., 1949; Sleeper and Stanier, 1950). When a polycyclic aromatic compound is undergoing degradation, the last remaining aromatic ring will have a side chain attached, the residuum of prior ring splitting. Removal of this side chain is necessary in order to reach catechol. In this event, salicylic acid (0-hydroxybenzoic

OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

199

acid), assumes the role of a key intermediate in oxidations involving more than one condensed ring. The compound immediately precedes catechol in the oxidative path and readily undergoes oxidative decarboxylation to form catechol (Walker and Evans, 1952; Roof et al., 1953). Salicylic acid is an intermediate in the oxidation of the polycyclic aromatics naphthalene ( Strawinski and Stone, 1943), phenanthrene ( Rogoff and Wender, 1957a), and anthracene (Rogoff and Wender, 1957b). Oxidative decarboxylation of salicylic acid to catechol appears to be the main oxidative pathway for the former. However, in a recent paper, Yano and Arima (1958) report that several strains of Pseudomonas show an alternate pathway for salicylic acid degradation via gentisic acid (Scheme 7 ) . It is relevant to mention that Roof et al. (1953) reported two strains of gram-negative aerobic bacteria which were able to oxidize salicylate, gentisate, and 2,3-dihydroxybenzoate. a. Substituted Salicylic Acids, When a naphthalenic compound undergoing oxidation bears a substituent group (CH,, C1, Br) on one ring, oxidation is usually directed at the unsubstituted ring. The salicylic acid derivative produced will then bear the substituent group of the starting compound. Thus Walker and Wiltshire ( 1955) isolated 3-chlorosalicylic acid as an intermediate from culture fluids of a gram-negative bacterium utilizing l-chloronaphthalene as a source of carbon. Rogoff and Wender ( 1959) studying 1- and 2-methylnaphthalene oxidation by soil pseudomonads isolated the intermediate 3-methylsalicylic acid from culture fluids of a strain oxidizing l-methylnaphthalene; 4-methylsalicylic acid was recovered if 2-methylnaphthalene was provided as the substrate. The methyl substituent is apparently removed after ring cleavage since resting cells of the Pseudomonas strains, grown on an appropriate methylsalicylic acid, were able to oxidize the corresponding methylcatechols. b. Higher Homologs of Salicylic Acid. Oxidation of only two members of the polycyclic aromatic hydrocarbon series above naphthalene have thus far been studied in any detail; these are phenanthrene and anthracene. These compounds are completely degraded by soil pseudomonads to carbon dioxide and water. Attack is initiated by cleavage of an end ring with stepwise degradation of the remaining rings (Rogoff and Wender, 1957a, b ) . Thus from phenanthrene-oxidizing cultures these investigators were able to isolate the intermediate l-hydroxy-2-naphthoic acid; anthracene-oxidizing cultures yielded 3-hydroxy-2-naphthoic acid. These acids are the next highest homologs to salicylic acid in the polycyclic series. Sequential induction experiments showed that these acids are afterwards metabolized through salicylic acid via the catechol pathway. It is then seen that end-ring fission in the bacterial degradation of POlyCy~li~ aromatic hydrocarbons probably occurs by means of reactions

'

200

MARTIN H. ROGOFF

similar to those evidenced in cleavage of the benzene ring. Higher homologs of salicylic acid appear as intermediates following degradation of the end ring with salicylic acid itself as the intermediate representing the benzene derivative preceding catechol.

3. Dihydronrenediol Intermediates Boyland and Levy (1935), and Young (1947) observed that naphthalene and anthracene (and perhaps some of the carcinogenic polynuclear aromatic hydrocarbons, a supposition subsequently proved correct ) were attacked in the mammalian body by dihydroxylation with the formation of dihydroarenediols. These compounds are postulated as precursors of the phenols obtained from the acid-treated urines of animals dosed with polycyclic aromatic hydrocarbons (Smith, 1950). On the basis of the mode of attack in the mammalian organism Parr et al. (1949) visualized the oxidation of aromatic compounds by bacteria to proceed with the aid of either H,O, or free OH radicals to form dihydroarenediols, with subsequent reactions to reach the ortho-dihydroxy compounds ( catechol ) . Stanier (1948) had postulated that oxidative attack on the ring might be precipitated by a partial or complete saturation of the double bonds by water addition, a process which would destroy the resonance of the benzene ring, allowing dehydrogenations to occur on the less stable products. A dihydroarenediol intermediate was first isolated from a bacterial oxidation of an aromatic compound by Walker and Wiltshire ( 1953). These investigators isolated ( ) -trans-1,2-dihydro-1,2-dihydroxynaphthalene and salicylic acid from cultures of an aerobic gram-negative rod oxidizing naphthalene. These compounds were confirmed as intermediates by the demonstration that they obeyed the criteria of sequential induction. They also studied breakdown of halogenated naphthalene derivatives (Walker and Wiltshire, 1955) and obtained 8-chloro-l,2-dihydro-1,2dihydroxynaphthalene from the oxidation of l-chloronaphthalene by a suitable organism. Colla and co-workers (1957) studied the oxidation of phenanthrene and anthracene by strains of Flavobacterium and isolated 3,4-dihydro-3,4-dihydroxyphenanthrenefrom phenanthrene-oxidizing cultures. They also obtained evidence for formation of a “diol” intermediate, probably the l,&isomer, during anthracene attack by the culture. Dehydrogenation of a dihydroarenediol would yield a product with an ortho-dihydroxy configuration, and, indeed, Fernley and Evans (1958) demonstrated that cells of their pseudomonads grown on naphthalene were sequentially induced to oxidize both trans-1,2-dihydro-l,2-dihydroxynaphthalene, and 1,2-dihydroxynaphthalene.The latter compound was also oxidized by a crude cell-free extract of naphthalene-grown cells

+

OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

201

with coumarin as the major product. Thus end-ring attack on a polycyclic compound appears to occur in a manner analogous to that of oxidation of the benzene ring with catechol formation. Isolation of a dihydroarenediol intermediate from bacterial oxidation of a benzenoid compound has not yet been accomplished, although such compounds have been reported as formed in the mammalian organism (Parke and Williams, 1958). It is probably not amiss to assume that cleavage of benzenoid compounds involves a “dihydrodiol” type intermediate at some stage during bacterial oxidation. B. GENERAL MODEOF OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

From the preceding discussion it can be seen that bacterial oxidation of aromatic compounds, in general, proceeds through a series of intermediary “type” compounds. When the substrate is polynuclear, reactions

SCHEME 3. General mode of ring splitting (schematic).

equivalent to the addition of 1 mole of H,Ozoccur with the formation of a dihydroarenediol. This type of compound undergoes dehydrogenation to a compound with two hydroxyl groups in the ortho configuration, and the ring is cleaved between them. The salicylic acid “type” intermediate is found as the compound preceding catechol; its carboxyl group represents the residuum of prior ring splitting, or side-chain oxidation. This general mode of ring splitting is summarized in Scheme 3. 111. Oxidative Metabolism of Polynuclear Aromatic Compounds by Bacteria

A. NAPHTHALENE AND SOMENAPHTHALENE DERIVATIVES

Our current knowledge of the intermediary microbial metabolism of naphthalene is summarized in Scheme 4. Strawinski and Stone (1943) had identified salicylic acid in napthalene cultures of a pseudomonad. Walker and Wiltshire ( 1953) isolated ~-trans-1,2-dihydro-l,2-dihydroxynaphthalene in addition to salicylic acid and established their roles as intermediates by sequential induction experiments. Fernley and Evans (1958) studied the degradation of naphthalene by a soil pseudomonad. They isolated coumarin, and detectable amounts of o-hydroxy-trans-cinnamic acid ( o-coumaric acid), o-hydroxyphenylpropionic acid (melilotic

202

MARTIN H. ROGOFF

acid), and salicylic acid, The naphthalene-grown cells were sequentially induced to oxidize tra~-l,2-dihydro-l,2,dihydroxynaphthalene, 1,2-dihydroxynaphthalene, and salicylic acid; oxygen uptake was observed on coumarin, o-coumaric acid, and melilotic acid, but the rates were not sufficiently high to warrant assumption of their roles as intermediates. Experiments with a crude cell-free extract of naphthalene-grown cells

1 ,2-Dihydro-1 ,I-dihydroxynophthoI ens

Naphthalene

I

02 (air)

1,Z-Dihydroxynaphthalene

1,2-Nophthoquinone

I

PHydroxy-ck cinnamic Acid

J

\

Coumarin

THROUGH

-

CATECHOL

CH=CH

‘COOH o-Hydroxy-W ;innontic Acid

Melilotic Acid

Salicylic Acid

SCHEME4. Degradation of naphthalene by soil bacteria.

demonstrated that during the oxidation of 1,2-&hydroxpaphthalene approximately 3 moles of oxygen were consumed and 1 mole of carbon dioxide was liberated; coumarin was the major product isolated, The gas exchange data are in agreement with the view that o-hydroxy-cis-cinnamic acid (isolated as coumarin) is produced by oxidative decarboxylation of the postulated precursor o-carboxy-cis-cinnamic acid. Negative results were obtained by resting cells tested on two o-carboxycinnamic acids

203

OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

which had the trans-configuration. The cells were also inactive on o-carboxyphenylpropionic acid, phthalide, acetic acid, o-carboxybenzoylacetic acid, and phthalic acid. Oxidation of the methylnaphthalenes was reported by Strawinski (1943) who found the compounds metabolized with more difficulty by soil bacteria than the parent compound naphthalene. Although less is known of the intermediary metabolites in methylnaphthalene attack by bacteria, recent evidence has shown that the degradative pathways (Scheme 5) appear to parallel the naphthalene path (Scheme 4).

2-Methylnophtholena

7-Msthyl-l,2-di hydro-l,2dihydroxynophtholsne

7-Methyl-2.nophthol

I

"CH3

i

J3H3 CIFAVAGE

HOOC

2-H ydroxy-l-methylbenzoic Acid

SCHEME 5.

~

AND FURTHER OXIDATION

\ 4-Methylcotechol

Degradation of 2-methylnaphthalene by soil bacteria.

+

Canonica and associates ( 1957) isolated ( ) -trans-7-methyl-172-dihydro-1,2-dihydroxynaphthalenefrom culture fluids of a strain of Pseudom o w oxidizing 3-methylnaphthalene. The same culture fluids also yielded 4-methylsalicylic acid. Extraction of culture fluids of the same strain oxidizing l-methylnaphthalene yielded 3-methylsalicylic acid, Treccani and Fiecchi ( 1956) found that resting cells of the 2-methylnaphthalene-grown organism could oxidize 4-methylsalicylic acid, but not the 3-methyli~omer. They concluded that the metabolism of the 3- and 4-substituted isomers was different. Rogoff and Wender (1959), using a soil pseudomonad, isolated the same methylsalicylic acids from oxidation of the appropriate methylnaphthalenes. These investigators demonstrated, by sequential induction experiments, that the methylsalicylic acids and their respective methylcatechols were intermediates in the methylnaphthalene oxidations, The intermediates with the methyl group ortho to the hydroxyl were, however, oxidized more slowly than those with the methyl group in the meta or para position. The effect, apparently steric, was most marked in

204

MARTIN H. ROGOFF

the case of 3-methylsalicylic acid, a possible explanation of Treccani and Fiecchi’s observation, The point of interest in the studies is that cells grown on naphthalene, or either of the methylnaphthalenes, were induced to oxidize salicylic acid and catechol and their methyl derivatives (Rogoff and Wender, 1959). This suggests that the same enzymes elaborated for oxidation of the parent compounds (salicylic acid, catechol) are involved in the oxidation of the methyl derivatives; the presence of the methyl group on the benzene nucleus apparently does not confer a specificity requirement on the enzymes involved. When the naphthalene nucleus bears a chlorine substituent in place of a methyl group the characteristics of the oxidative pathway are not altered. Walker and Wiltshire (1955) studied the degradation of l-chloronaphthalene by a soil organism and identified 8-chloro-1,2-dihydro-1,2dihydroxynaphthalene and 3-chlorosalicylic acid as intermediates in the oxidation, Their findings were confirmed by Canonica et a2. (1957) who are isolated ( ) -trans-7-chloro-1,2-dihydro-1,2-dihydroxynaphthalene and 4-chlorosalicylic acid from Pseudomow cultures oxidizing 2-chloronaphthalene.

+

B. HIGHER POLYNUCLEAR AROMATICHYDROCARBONS According to Tausson ( 1928a) naphthalene, biphenyl, anthracene, and phenanthrene are readily attacked by many soil bacteria; he described two bacterial species that oxidized phenanthrene (Tausson, 1928b). Sisler and ZoBell (1947) noted that certain marine bacteria assimilated phenanthrene and anthracene somewhat faster than naphthalene or 1,2benzanthracene; 1,2,5,6-dibenzanthracene was assimilated only one-fourth as fast. Tausson (1928b) investigated the pathway of bacterial oxidation of phenanthrene. Basing his postulate on the rates at which three strains of phenanthrene-oxidizing organisms assimilated suspected intermediate compounds, he proposed that the phenanthrene molecule was split across the 9,lO-bond with formation of two molecules of o-hydroxybenzyl alcohal. His proposed path was as follows: Phenanthrene + o-hydroxybenzyl alcohol + o-hydroxybenzaldehyde + salicylic acid + catechol + muconic acid + carbon dioxide and water. Rogoff and Wender (1957a) observed that soil pseudomonads did not attack phenanthrene at the 9,10-bond, as postulated by Tausson, but that attack was directed at an end ring. These investigators isolated l-hydroxy2-naphthoic acid from phenanthrene-oxidizing cultures and demonstrated by sequential induction experiments that salicylic acid and catechol were also intermediates in this oxidative pathway. Similar experiments with an anthracene-oxidizing strain demonstrated end-ring attack on this corn-

OXIDATION OF AROMATIC COMPOUNDS BY BACXERIA

205

pound (Rogoff and Wender, 195%) with the formation of 3-hydroxy-2naphthoic acid, and sequential induction of anthracene-grown cells to salicylic acid and catechol. Recently Colla et al. (1959) isolated 3,4dihydro-3,4-dihydroxyphenanthreneand salicylic acid from cultures of a Flavobacterium oxidizing phenanthrene. They also obtained a dihydroarenediol intermediate from anthracene dissimilation. This intermediate

Phenanthrene

Anthrocens

[m] I

I

\

3,4-Dihydro-3,4.dihydroxyphsnanthrens

1.2-0 i hydro-1,Zdihydroxy-

i

anthracene I I

OH

v

COOH

or>0'"

1-Hydroxy2.naphthoic Acid

0

'

I

0

I

1

3-Hydroxy-2-naphthoic Acid

0

/

0'

OH THROUGY S C H E M L

Salicylic Acid

Cotechol

SCHEME6. Dissimilation of phenanthrene and anthracene by soil bacteria.

has not been identified but it is proposed that it bears the 1,2-configuration. Current knowledge of the dissimilation of phenanthrene and anthracene is summarized in Scheme 6. The finding by Colla et al. of the dihydroarenediol indicates that end-ring attack on phenanthrene and anthracene may well occur in a manner analogous to that of naphthalene attack by soil bacteria (Scheme 4) through cleavage of 3,4-dihydroxyphenanthrene and 1,2-dihydroxyanthracene, respectively, the ultimate products in the path being salicylic acid (or a higher homolog).

206

MARTIN H. ROGOFF

C. ALTERNATEPATHWAYS AND ENDPRODUCTS In certain cases, intermediates in the oxidative pathways may undergo enzymatic or nonenzymatic reactions, dependent on the organism used and the conditions in the culture medium, with the formation of compounds which do not lie directly in the oxidative paths, or which accumulate as end products which are not further utilized by the organism. Murphy and Stone (1955) reported that naphthalene oxidation by a strain of Pseudomow resulted in the formation of 1,2-napthoquinone (Scheme 4), eventually in toxic amounts. It was also found that 1,2naphthoquinone was produced by a nonenzymatic conversion of 12dihydroxynaphthalene, now a known intermediate in naphthalene dissimilation (Fernley and Evans, 1958). Stanier ( 1950) had proposed that ortho-quinones were intermediates which occurred just prior to ring splitting, resulting from an enol-keto rearrangement of ortho-hydroxyl groups; rupture of the ring was proposed as occurring between the adjacent carbonyl groups. Murphy and Stone (1955), however, demonstrated that this was not the case with the 1,2-naphthoquinone they isolated. The products isolated by Fernley and Evans (1958) from naphthalene-oxidizing cultures, o-coumaric and melilotic acids (Scheme 4), have been proposed by these workers as resulting from the known biochemical reactions of isomerization and reduction, and that the compounds may be metabolized further by as yet unknown routes. Whether these compounds are true intermediates has not yet been satisfactorily established. During studies of the oxidation of %methylnaphthalene by soil pseudomonads under nonaerated conditions, Rogoff and Wender ( 1959) reported the isolation of 2-naphthoic acid in addition to the “normal” intermediate 4-methylsalicylic acid. The compound represents an alternate pathway for 2-methylnaphthalene dissimilation involving oxidation of the methyl group, although 2-naphthoic acid was not further metabolized by these bacteria, nor was it active in sequential induction experiments. Treccani (personal communication ) feels that the compound 2-naphthoic acid isolated is formed by chemical oxidation of 2-naphthylalcohol (an intermediate in an alternate pathway involving the latter compound and 2-hydroxy-4-carboxymethylbenzoicacid) under acid conditions in the medium. Another instance of methyl group oxidation prior to ring splitting is seen in the oxidation of p-cresol, which is transformed to protocatechuic acid through p-hydroxybenzyalcohol, p-hydroxybenzaldehyde, and p-hydroxybenzoic acid (Dagley and Patel, 1955). As a result of chemical reaction of an intermediate in 2-methylnaphthalene degradation, Treccani and Fiecchi ( 1956) isolated 7-methyl-2-naphtho1, a product

OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

207

apparently produced by the dehydration, under acid conditions, of the intermediate 7-methyl-1,2-dihydro-l,2-dihydroxynaphthalene. Dehydration of the dihydroarenediol intermediates to their monohydroxy derivatives under acid conditions has often been noted in studies of the metabolism of polycyclic aromatic compounds in mammals (Parke and Williams, 1958 ) . D. CONSIDERATIONS IN THE BIOCHEMISTRY OF DEGRADATION OF POLYCYCLIC AROMATICCOMPOUNDS The conversion of polycyclic aromatic hydrocarbons to phenols in the animal body presumably occurs through the dihydroarenediol intermediates. The position of the hydroxyl groups of the resulting phenols is most often not at centers of highest reactivity in the parent molecule, but at sites of secondary reactivity. Pullman and Pullman (1955) have proposed that most polycyclic aromatic hydrocarbons contain two regions whose characteristics are important in determining the chemical behavior of the compounds. These are the K region, a type of bond such as is found between the 9- and 10-carbons of phenanthrene and an L region containing carbons with a reactivity similar to those of the 9- and 10carbons of anthracene. Hydroxylation in the animal body at bonds other than these reactive sites, that is at sites of secondary reactivity, is held to be due to attachment of the K region of the hydrocarbon to a tissue, thereby blocking hydroxylation at this site. In the addition complex of hydrocarbon and tissue, sites of secondary reactivity are activated and hydroxylation occurs at these newly activated carbons (Pullman and Baudet, 1954). When polycyclic aromatic hydrocarbons containing K or L regions are degraded by soil pseudomonads these regions are not the positions which undergo attack (Scheme 6 ) ; primary attack of phenanthrene occurs at the 3,4-bond and anthracene is probably attacked at the 1,2-bond. Rogoff (1957) pointed out that in phenanthrene degradation by Pseudomom spp. bonds other than those directly attacked were involved in the oxidation. Blocking the 9-position ( K region) of phenanthrene with a methyl group prevented attack on the compound. When the bacteria were grown on naphthalene, which does not contain a bond of high reactivity, the rate of phenanthrene oxidation was considerably lower than if the cells had been grown on 2-methylnaphthalene or phenanthrene, both of which contain electron dense regions. In addition, naphthalene-grown cells absorbed considerably less phenanthrene, on a molar basis, from solution than did phenanthrene-grown cells; the latter absorbed phenanthrene or naphthalene in approximately equimolar amounts. These findings give some indication that electron dense regions

208

MARTIN 11. ROCOFF

in polycyclic aromatic hydrocarbons may well play a role in orienting hydroxylation preceding ring splitting of the compounds by bacterial enzymes, much in the same manner as has been found in mammalian systems. Future research to establish the relevance of electron dense regions to polycyclic attack by bacteria is indicated.

IV. Oxidative Metabolism of Benzenoid Compounds by Bacteria A, DECRADATIVE OXIDATION OF SOMEBENZOIC ACIDS

Oxidation of the benzoic acids by bacteria shows, within very narrow limits, the variations possible for dissimilation of a variety of substrates. In the oxidation of benzoic acid by soil pseudomonads, catechol is reached in what is apparently one oxidative decarboxylation step (R. A. Evans et al., 1949; Sleeper and Stanier, 1950). This was demonstrated by sequential induction experiments and by the isolation of catechol from cultures in which these substrates were being utilized. Tracer experiments, carried out by Sleeper (1951) with benzoic acid labeled with C]' in the carboxyl or 1-carbon positions, demonstrated that catechol formed by the bacteria from the carboxyl carbon-labeled benzoic acid was inactive; that formed from the l-carbon-labeled compound retained the specific activity of the parent compound. It was also noted that all the activity of the catechol derived from the benzoic-l-U4 acid resided in the 1- and 2-carbons. When the carboxyl-labeled benzoic acid was completely metabolized, the radioactivity of the carboxyl group was found almost exclusively in respiratory carbon dioxide, while an appreciable percentage of the activity of the l-carbon-labeled compound showed up in the bacterial cells and the supernatant liquid. Thus a direct decarboxylation is indicated. The oxidation of benzoic acid by various strains of Azotobacter was studied by Voets (1958). Using the sequential induction technique with benzoate, catechol, or salicylate as substrates he found, in agreement with the investigators of the Pseudomows systems, that catechol, but not salicylic acid, was an intermediate in benzoic acid oxidation by A. chroococcum and A. beyerinckii. However, cells of A. vinelandii grown on benzoic acid were able to oxidize salicylic acid with no lag period. This strain difference was supported by the work of Wang and Tchan (1948) who found salicylic acid produced by a benzoic acidoxidizing strain of Azotobacter. Thus an alternate path for benzoic acid oxidation exists dependent on the strain used. Photochemical oxidation of benzoic acid by Rhodopseudomonas palustris under anaerobic conditions has been reported to proceed via the catechol path (Scher and Allen, 1960). The direct pathway from salicylic acid to catechol has been mentioned

OXIDATION OF AROMATIC C O M P O U N D S BY BACTJCRIA

209

previously. Recently, Yano and Arima (1958) demonstrated the conversion of salicylic acid to gentisic acid ( 2,6-dihydroxybenzoic acid ) by several strains of Pseudomonas. Previously Walker and Evans (1952), had isolated gentisic acid as an intermediate in the dissimilation of mhydroxybenzoic acid prior to ring splitting, a finding c o n h e d by Roof et al. (1953).Ring cleavage of gentisic acid might occur through a new COOH

Benzoic Acid

0”” COOH

\

THROUGH SCHEME 1

Salicylic Acid

0

HO

4H

\

Cntechol

FOOH PATHWAY

UNKNOWN

\ m- Hydroxy-

0

benzoic Acid

pHydroxy benzoic Acid

OH Protocatechuic Acid

SCHEME 7. Oxidative metabolism of the benzoic acids by soil bacteria.

pathway with maleylpyruvic acid as the possible product (Evans, 1956). However, Yano and Arima (1958) have demonstrated that m-hydroxybenzoic acid may also be oxidized via protocatechuic acid. These workers isolated two cell-free enzyme systems from strains of Pseudomonas. Their m-hydroxybenzoic acid hydroxylase “A” catalyzes transformation of the compound to protocatechuic acid; the “B” hydroxylase converts mhydroxybenzoic acid to gentisic acid. The enzymes require coenzyme I1

210

MARTIIW H. ROGOFF

( T P N ) , and probably TPNH as cofactors. The phenomenon may be due to strain difference; both enzymes have not yet been demonstrated to exist simultaneously in the same strain, Protocatechuic acid is also the product of p-hydroxybenzoic acid oxidation (Evans, 1947). Protocatechuic acid metabolism has been discussed (Scheme 2 ) . It has been shown, however, that p-hydroxybenzoic acid does not precede protocatechuic acid as an intermediate when quinic acid is oxidized to protocatechuic acid by species of Pseudomonas, Achromobncter, and by other soil bacteria (Rogoff, 1958). The oxidative pathways for the dissimilation of benzoic acid and its monohydroxy derivatives are summarized in Scheme 7 . The other benzoic acid whose metabolism by bacteria has received some attention is phthalic acid, Evans (1955), studied the breakdown of

0 \

COOH

H

COOH

HO

Phtholic Acid

O

~

C

\

4,s-Dihydroxyphtholic Acid

HOI O COOH I -

O Anoerobic O H COOH

Anoerobic

;;

\

Protocatschuic Acid

Io2

Through Scheme

2

SCHEME8. Phthalic acid metabolism by soil bacteria.

phthalic acid by a soil pseudomonad and identified 4,5-dihydroxyphthalic acid in growing cultures; the compound was found to be an intermediate in sequential induction experiments, as was protocatechuic acid. Ribbons and Evans (1959) reported isolation of protocatechuic acid from growing cultures. They prepared a cell-free enzyme system from phthalate-grown cells which would decarboxylate 4,5-dihydroxyphthalic acid under anaerobic conditions to protocatechuic acid. In the presence of oxygen the undialyzed preparation gave p-oxoadipic acid from protocatechuic acid; the dialyzed preparation produced p-carboxymuconic acid. Both compounds are normal intermediates in the protocatechuic acid path (Scheme 2 ) . The anaerobic decarboxylation differs from the oxidative decarboxylations previously discussed. Phthalic acid metabolism is summarized in Scheme 8. €3. OXIDATIVE METABOLISM OF PHENYLALANINE, MANDELIC ACID, AND THEIR p-HYDROXY DERIVATIVES

The oxidative metabolic pathways of these compounds will be considered together since all are benzenoid compounds having oxygenated

OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

211

side chains with some alteration of the side chain occurring before ring cleavage. Tyrosine ( p-hydroxyphenylalanine ) undergoes many reactions in the mammalian organism including decarboxylation, ring oxidation to the melanin pigments and adrenaline, and perhaps even ether formation to thyroxine. We are concerned here only with microbial oxidative metabolism of the compounds. In actuality, relatively little is known about the bacterial metabolic pathways for the dissimilation of phenylalanine and tyrosine although it appears that homogentisic acid is the key aromatic intermediate preceding ring cleavage of both compounds. A Pseudomonas spp., induced to utilize tyrosine, was found to contain the enzymes necessary to oxidize homogentisic acid (Suda and Takeda, 1950). These investigators also prepared a cell-free enzyme system which catalyzed the rupture of the ring. The enzyme was called homogentisicase, and required a ferrous iron cofactor. Jones et al. (1952) reported that Evans’ Vibrio 0/1 strain produced homogentisic acid from tyrosine. At about the same time several investigations of the degradation of tyrosine by molds were being carried out. Aspergillus niger was shown to produce homogentisic acid from phenylacetic acid (Kluyer and van Zijp, 1951), while Hockenhull et al. ( 1952) demonstrated that Penicillium chrysogenum partially degraded the acetic acid side chain to yield benzaldehyde prior to ring cleavage. A similar strain was shown by Isono (1953) to produce o-hydroxyphenylacetic acid from phenylacetate, and a mutant of this strain accumulated homogentisic acid when fed phenylacetate. A study of the adaptive patterns of Vibrio 0/1 by Dagley et al. (1953) indicated that oxidation of phenylalanine to homogentisic acid by this strain proceeded through phenylpyruvic and phenylacetic acids. The phenylhydrazone of phenylacetic acid was isolated from the culture fluids. This made it unlikely that tyrosine and p-hydroxyphenylpyruvic acids were intermediates in phenylalanine degradation. Demonstration of steps beyond homogentisate with microbial enzyme systems has not been reported. The above steps are summarized in Scheme 9. The metabolism of mandelic acid was described by Stanier (1948); Gunter ( 1953) established the pathway for p-hydroxymandelic acid. The enzymology of the conversion of mandelic acid to benzoic acid was studied by Gunsalus and co-workers ( 1953). These investigators prepared four soluble enzyme systems from a strain of Pseudomonns fluorescens which catalyzed the following transformations: ( 1 ) a racemase which converted D( - )-mandelic acid to L( )-mandelic acid, ( 2 ) a dehydrogenase which converted L ( ) -mandelic acid to benzoylformic acid, ( 3 ) a carboxylase which carries out the decarboxylation of benzoylformate to benzaldehyde, and ( 4 ) two dehydrogenases which carry out the reduction of coenzyme I (DPN) and TPN, respectively. Benzalde-

+

+

212

MARTIN H. ROCOFF

6-

hyde is converted to benzoic acid and then metabolized through the catechol path ( Scheme 1 ); p-hydroxybenzoic acid from p-hydroxymandelic acid oxidation is oxidized via the protocatechuic acid path (Scheme

2).

;COOH

Anabolic

\

dH Phenylolonine

Tyrorins

4

0

CH2.CO.COOH

OH

p-Hydroxyphsnyl. pyruvic Acid

Phenylpyruvic Acid

1

FH2.COO H

GHO PR00A0Cr THROUGH CATECHOL

H a o g e n t i r i c Acid

I

\

Phsnylocetic Acid

Bonzoldehyds

J

PATHWAY UNKNOWN pHydroryphenylacetic Acid

SCHEME 9. Bacterial oxidation of phenylalanine and tyrosine.

V. Degradation of Miscellaneous Aromatic Compounds A. METABOLISM OF THE INDOLE NUCLEUS

Tryptophan is most commonly metabolized by bacteria through elimination of the alanine side chain with the production of indole as an end product, The reaction is carried out by the enzyme tryptophanase (Happold, 1950b) which has a Vitamin B, requirement; the reaction products are indole, pyruvic acid, and ammonia, Certain members of the genus Pseudomonus, however, carry out the complete degradation of tryptophan. Two different degradative routes have been found. Stanier and Hayaishi (1951, 1952) report that most pseudomonads

OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

213

oxidize the compound through (kynurenine?) anthranilic acid and catechol ( aromatic pathway) while certain others use another route involving kynurenic acid ( quinoline pathway). Occasional strains carry out incomplete oxidations with anthranilic acid or kynurenic acid as end products. Metabolism of tryptophan has been reviewed by Evans (1956) and the metabolic paths are outlined by Umbreit ( 1952). The stepwise reactions between tryptophan and anthranilic acid closely resemble the analogous mammalian pathway. A new route for degradation of the indole nucleus which does not involve kynurenine or anthranilic acid was described by Proctor ( 1958). He found, in sequential induction studies, that cells of a Pseudomonus

Indolencetic Acid

3-Hydroxyindolo

~ E M E10.Oxidation of

3-Methylindole

Solicylic Acid

Cotechol

the indole nucleus through the salicylic acid pathway.

strain induced to oxidize indoleacetic acid were also induced to 3-methylindole, 3-hydroxyindole, salicylic acid, and catechol. Proctor also isolated 3-methylindole, salicylic acid, and catechol from culture fluids. The main features of the path are (1) decarboxylation of indoleacetic acid to 3methylindole, ( 2 ) replacement of the methyl group by a hydroxyl, and ( 3 ) ring splitbing and the formation of salicylic acid. The pathway as postulated by Proctor is presented in Scheme 10. The pathway is interesting in two regards; it implies degradation of heterocyclic compounds through the central metabolic paths, and the initial decarboxylation may be analogous to Ribbons' and Evans' anaerobic decarboxylation of 4,5-dihydroxyphthalic acid (Scheme 8). Proctor noted evolution of carbon dioxide prior to oxygen uptake during metabolism of indoleacetic acid by intact cells. B. DEGRADATION OF AROMATIC HERBICIDES The use of growth control substances, 2,4-dichlorophenoxyacetic acid in particular, as a regular farm practice has become quite common. The

214

MARTIN H. ROGOFF

aromatic weed control agents include the nitro-aromatics such as 4,6dinitro-o-cresol and the phenoxyacetic acids. These compounds are physiologically active and accumulation in toxic amounts in soil represents a potentially hazardous situation. The usual rates of application of the herbicides to field soils have not proven, for the most part, to be toxic to soil bacteria. In addition the soil microflora is potentially capable of detoxifying most of the herbicides currently in use. Once exposed to the herbicides subsequent exposure results in rapid disappearance of the compounds from the soil. 1. The Phenoryacetic Acid Herbicides Workers studying the oxidation of chlorophenoxyacetic acid noted the difficulty with which 2,4-dichlorophenoxyacetic acid ( 2,4-D ) was attacked by soil bacteria in mineral-salts media. Evans and Smith (1954) isolated a gram-negative rod which readily attacked p-chlorophenoxyacetic acid ( CPAA ) . They isolated 4-chloro-2-hydroxyphenoxyaceticacids and 4chlorocatechol from CPAA cultures; the compounds obeyed sequential induction criteria. Later, Evans and Moss (1957) isolated a p-chloromuconic acid from the action of washed cells on 4-chlorocatechol. The acid was thought to be the cis,trcins-isomer. Spectroscopic evidence was obtained which indicated formation of a related lactone, possible pchloromuconolactone. The p-chloromuconic acid isolated was not metabolized by the cells; Evans and Moss felt that inversion had occurred during the extraction process. The pathway for 2,4-D degradation was studied by Smith (1954). He obtained chromatographic evidence for the presence of 2,4-dichlorophenol, and 3,5-dichlorocatechol in 2,4-D cultures. Rogoff and Reid ( 1956) described a 2,4-D decomposing soil diphtheroid which oxidized 2,4-dichlorophenol, and Steenson and Walker ( 1957) presented evidence obtained by sequential induction experiments that this compound and also 4-chlorocatechol were intermediates in the pathway. Fernley and Evans (1959) obtained a product from 2,4-D degradation by a pseudomonad which proved to be a-chloromuconic acid. In contrast to the p-chloromuconic acid previously isolated from CPAA cultures ( Evans and Moss, 1957), 2,4-D grown cells readily metabolized both a synthetic a-chloromuconic acid and that same compound isolated from the culture fluid, It appears that the other chloro-substituent of the ring is eliminated prior to ring cleavage. Steenson and Walker’s finding that 4-chlorocatechol is an intermediate may indicate that either ring chlorine may be the one eliminated ( Scheme 11) . Sequential induction data obtained by Steenson and Walker ( 1957) indicate that 4-chloro-2-methylphenoxy-

OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

215

acetic acid ( MCPA ) is dissimilated through 2-methyl-4-chlorophenol. These-findings are summarized in Scheme 11. The sequential induction studies with the phenoxyacetic acids have demonstrated some interesting examples of cross adaptation which may point up the advantageous nonspecificity of action of some of the enzymes

0

O.CH2.COOH

D.CH2.COOH

tl

tl 2.4-Dichlorophenoxv. ocetic Acid

pChlorophenoxyacelk Acid

t

“‘0 OH

Cl

CI 2-Hydroxy-d.chloro~ phenoxyacetic Acid

2.4-Dichlorophenol

t

t

0” OH

C

I

~

O

t

-cI~

>

c1

tl

3,s-D ichlorocotechol

4-Chlorocatechol

I

I

i

1

C J C m k l H

o.Chlorornuconic Acid

\

II CH

7

I

0 CH

’C

fi.Chl~ra;uconic Cl

-I

FURTHER METABOLIZED WITH CHLORINE L I B E R A T I O N

SCHEME11. Degradation of the chlorophenoxyacetic acid herbicides by soil bacteria.

of the bacteria which act as scavengers in soil. Thus Steenson and Walker (1957) found that bacteria grown on 2,4-D, although unable to oxidize any of the other five possible isomers, could oxidize 2,4-dibromo-, 4-bromo-2-chloro,4-chloro-, and, to a lesser extent, 2-chlorophenoxyacetic acids. In another study (Steenson and Walker, 1958) they found that MCPA or 2-chloro-4-methylphenoxyaceticacid-grown Flavobacteriuin

216

h$ARTIN H. ROCOFF

were adapted to oxidize 2,4-D. An Achrornobacter strain when groyn on the intermediates 2,4-dichlorophenol or 2-methyl-4-chlorophenol was induced to oxidize 2,4-D and MCPA as well as the inducing compound. The situation may be similar to the previously mentioned ability of salicylic-acid-grown cells to oxidize the methylsalicylic acids ( Rogoff and Wender, 1959). 2. Aromatic Nitro Compounds Relatively little is known about the degradation of the nitrophenols. Evans and Simpson (1953) isolated a pseudomonad which grew in a mineral-salt medium with 0.02% o- or p-nitrophenol. They showed substitution of the nitro group by a hydroxyl group with elimination of the nitro group as nitrite ion. Jensen and Gundersen (1955) confirmed this with an atypical form of Corynebacterium simplex. These authors studied the ability of the organism to attack a great variety of nitrophenols and advanced a theory that breakdown of the compounds by this organism is predicated on a para-orientation of the hydroxyl and nitro groups on the benzene ring (Gundersen and Jensen, 1956). The p-nitro group may represent the point where enzymatic attack begins and the ring structure is broken.

C. DEGRADATION OF NICOTINIC ACID Many bacteria can utilize nicotinic acid as a sole source of carbon and energy; the Pseudomonas group is particularly active in this regard. Hughes (1952) found that the first product in the degradation of the compound by P. fluorescens was 6-hydroxynicotinic acid; he also prepared cell-free extracts which catalyzed the reaction, but would not further oxidize the product (Hughes, 1955). Behrman and Stanier ( 1957) using another strain of Pseudomonus fluorescms established the pathway by which nicotinic acid is degraded by these organisms to aliphatic metabolites (Scheme 12). Dependent on conditions in the reaction vessel, induced whole cells of the organism accumulated 2,s-dihydroxypyridine or pyruvic acid from nicotinic acid. Soluble fractions from cell-free preparations typically produced ammonia, carbon dioxide, formic, acetic, and lactic acids when either 6-hydroxynicotinic acid or 2,s-dihydroxypyridine was provided as substrate; one particular soluble fraction oxidized 6-hydroxynicotinic acid with the formation of 2,5-dihydroxypyridine. When the metabolism of 2,s-dihydroxypyridine by diluted fractions was arrested before completion, fumaric acid could be detected chromatographically in the reaction mixture. Chromatographic evidence showed that maleamic and maleic acids were converted to mixtures of fumaric and malic acids, and fumaric acid was partly converted to malic

OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

217

acid by the extract; fumaramic acid was not attacked, This indicated that N-formylmaleamic acid might be the immediate product of ring cleavage between the 5- and f3-carbons. This pathway is interesting in that ring cleavage of a heterocyclic compound occurs in a manner which seems analogous to cleavage of a benzenoid compound. Oxidation of rn-hydroxybenzoic acid, and occasionally salicylic acid, occurs through gentisic acid (Scheme 7), and tyrosine is oxidized through homogentisic acid (Scheme 9 ) . In these cases hydroxyl groups are introduced into the ring in a para relationship and

Nicotinic Acid

r

6-Hydroxynicotinic Acid

2,5-0ihydroxynicotine

1

t(-Fonylmalaamic Acid

Maleamic Acid

Moleic Acid

Fumaric Acid

SCHEME 12. Nicotinic acid oxidation by Pseudomow fluorescens.

ring cleavage occurs across a bond adjacent to one of the hydroxyls. Apparently the same type of reaction is found in the splitting of the pyridine ring. An anaerobic fermentation of nicotinic acid by an unidentified Clostridiurn was reported by Harary (1956) with acetic acid, propionic acid, ammonia, and carbon dioxide .as end products (Harary, 1957a). Later experiments (Harary, 1957b) showed that the bacteria were capable of a reversible, anaerobic oxidation of nicotinic acid to 6-hydroxynicotine. The formation of the same intermediate in both aerobic and anaerobic oxidation may indicate similar degradative pathways.

VI. Future Experimentation The elucidation of the pathways by which microorganisms degrade aromatic compounds has received much attention only in the last decade. Considering the host of organic aromatic compounds available, and to

218

MARTIN H. ROGOFF

which new compounds are added daily, a much greater effort in studying microbial transformation of aromatic compounds is needed before research in the field may become “fruitful.” Current interest in aromatic herbicides, insecticides, food preservatives, and physiologically active compounds places a mandate on more intensive study of aromatic, alicyclic, and heterocyclic ring metabolism by microbes. The most obvious application of the abilities of the ring-metabolizing bacteria would be in aromatic transformations analogous to those now carried out on the steroids. Isolation of potentially useful intermediates in the microbial dissimilation of some of the higher members of the polycyclic aromatic series might also prove a fruitful field. These higher members are interesting in that some of the compounds are carcinogenic. Other experimentation in directions not so readily apparent are in order. Production of 2-naphthoic acid from 2-methylnaphthalene by selective oxidation of the methyl group might conceivably open a path to more useful compounds from the parent compound than can be achieved by present chemical methods. Perhaps 2-naphthol might be obtained economically from the naphthalene dissimilation intermediate 1,2-dihydro-l,2-dihydroxynaphthalene. In another approach slow steps occur in certain oxidative paths. For example, steric hindrance by the methyl group of 2-hydroxy-3-methylbenzoic acid, results in slow utilization of both this intermediate and the starting compound, l-methylnaphthalene; there is no hindrance in the intermediate obtained from 2-methylnaphthalene and the latter is rapidly oxidized. It might be possible to separate a mixture of the methylnaphthalenes on this basis, a chemically difficult separation. Similarly, contaminating compounds in bulk chemicals ( thionaphthene in naphthalene; carbazole in phenanthrene ) might prove amenable to “microbial separation.” There are undoubtedly many more fields of experimentation open as seen in the numerous broken arrows and “pathway unknown” designations in the schemes. These few will suffice here.

VII. Summary The oxidative metabolism of aromatic compounds by bacteria consists of manipulation of the ring systems in such a way that the products of degradation may enter the main terminal respiratory cycles of the cell at some point. This is accomplished through a limited diversity of oxidative pathways. Cleavage of the ring is preceded by hydroxylations necessary to provide two hydroxyl groups on the aromatic ring. These are usually found in the ortho-position ( catechol, protocatechuic acid, 1,2-dihydroxynaphthalene)but in certain cases may appear in the paraposition ( gentisic acid, 2,s-dihydroxypyridine) , When in the ortho-posi-

OXIDATION OF AROMATIC COMPOUNDS BY BACTERIA

219

tion the ring is cleaved between the carbons bearing the hydroxyl groups; when they occur in the para-position, there is ring cleavage across a bond adjacent to one of the carbon atoms which bears a hydroxyl group. The compounds bearing the ortho- or para-oriented hydroxyl groups become key intermediates, diverse organic molecules being metabolized through such key compounds as catechol, protocatechuic acid, and gentisic acid, or their analogs, Polycyclic aromatic compounds are degraded by endring fission with the ultimate product of ring cleavage being salicylic acid or an analog; salicylic acid undergoes a single oxidative decarboxylation step to catechol. The intermediary type compound formed just prior to the o-dihydroxy intermediate in the oxidation of polycyclic aromatic hydrocarbons is the dihydroarenediol; compounds of this type have not been demonstrated in cleavage of the benzene ring. The oxidases which cleave benzenoid rings catalyze reactions in which an atom of molecular oxygen enters directly into the oxidized product (Hayaishi et al., 1955). These enzymes also appear to have a group requirement for ferrous iron.

REFERENCES Behrman, E. J., and Stanier, R. Y. (1957). J. Biol. Chem. 228, 923-945. Bernheim, F. (1941). J. Bacteriol. 41, 387. Bernheim, F. (1942). J. Biol. Chem. 143,383. Boyland, E . , and Levy, A. A. (1935). Biochem. J. 29,2679-2683. Canonica, L., Fiecchi, A,, and Treccani, V. (1957). Rend. ist. lombardo sci. 91, 119-129. Colla, C., Biaggi, C., and Treccani, V. (1957). Atti accod. nazl. Lincei Rend. Clnsse sci. fs. mat. e nat. 23, 66-69. Colla, C., Fiecchi, A., and Treccani, V. (1959). Ann. J. Microbiol. ed Enzimol. IX, 1-5. Dagley, S . , and P&l, M. D. (1955). Biochem. J. 60, xxxv. Dlgley, S., Fewster, M. E., and Happold, F. C. (1953). J. Gen. Microbiol. 8, 1. Elvidge, J. A., Linstead, R. P., Sims, P., and Orkin, B. A. (1950). J. Chem. SOC. pp. 2235-2241. Emmerling, O., and Abderhalden, E. ( 1903). Zentr. Bakteriol. Parasitenk. Abt. I1 10, 339. Evans, R. A., Parr, W. H., and Evans, W. C. (1949). Nature 164, 674. Evans, W. C. (1947). Biochem. J. 41, 373. Evans, W. C. ( 1955). Biochern. J. 61, x. Evans, W. C. ( 1956). Ann. Repts. P r o p . Chem., Chem. SOC. (London) 53,279-294. Evans, W. C., and Happold, F. C. ( 1939). J. SOC. Chem. 2nd. (London) 58, 55. Evans, W. C., and Moss, P. (1957). Biochem. J. 65, 8 pp. Evans, W. C., and Simpson, J. R. (1953). Biochem. J. 55, xxiv. Evans, W. C . , and Smith, B. S. W. (1951). Uiochmi. J. 49, x. Evans, W. C., and Smith, B. S. W. (1954). Biochem. J. 57, xxx. Evans, W. C., Smith, B. S. W., Linstead, R. P., and Elvidge, J. A. (1951). Nature 168, 772.

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Femley, H. N., and Evans, W. C. (1958). Nature 182,3734375. Femley, H. N., and Evans, W. C. (1959). Biochem. J. 73,22 pp. Fowler, G. J,, Ardem, E., and Lockett, W. T. ( 1911). Proc. Roy. SOC. B83, 149. Gray, P. H. H., and Thomton, H. G. (1928). Zentr. Bakteriol. Parasitenk. Abt. I I 73, 74-96. Gross, S . R., Gafford, R. D., and Tatum, E. L. (1956). J. Biol. Chem. 219,781. Gundersen, K., and Jensen, H. L. (1956). Acta Agr. S c a d . 6, 100-114. Gunsalus, C. F., Stanier, R. Y., and Gunsalus, I. C. (1953). J. Bacteriol. 66,548. Gunter, S. E. (1953). J . Bacteriol. 66, 341. Happold, F. C. ( 1950a). Biochem. SOC.Symposia (Cambridge, Engl. ) No. 5 , 8596. Happold, F. C. (1950b). Advances in Enzyniol. 10,51. Harary, I. ( 1956). Nature 177, 328. Harary, I. (1957a). J . Biol. Chem. 227, 815-822. Harary, I. (1957b). J. B i d . Chem. 227,823-831. Hayaishi, O., and Hashimoto, Z. (1950). Med. J . Osaka Univ. 2, 33. Hayaishi, O., Katagiri, M., and Rothberg, S. (1955). J . Am. Chem. SOC. 77, 5450. Henderson, M. E. K., and Fanner, V. C. (1955). J. Gen. Microbiol. 12,37. Hockenhull, D. J. D., Walker, A. D., Wilkin, G. D., and Winder, F. G. (19521. Biochem. J . 50, 605. Hughes, D. E. (1952). Bbchim. et Biophys. Acta 9,226. Hughes, D. E. (1955). Biochem. J. 60,303. Isono, M. (1953). J. Agr. Chem. SOC. Japan 27, 255. Jensen, H. L., and Gundersen, K. (1955). Nature 175, 341. Jones, J. D., Smith, B. S . W., and Evans, W. C. (1952). Biochem. J. 51, xi. Katagiri, M., and Hayaishi, 0. (1957). J. Biol. Chem. 226, 439-448. Kilby, B. A. (1948). Biochem. J. 43, v. Kilby, B. A. (1951). Biochem. J. 49,671. Kluyver, A. J., and van Zijp, J. C. M. ( 1951). Antonie uan Leeuwenhoek J. Microbtol. Serol. 17, 315. Landa, S., and Eliasek, J. (1956). Chem. Zisty 50, 1934. Murphy, J. F., and Stone, R. W. (1955). Can. 1.Microbiol. 1, 579-588. Parke, D. V., and Williams, R. T. (1958). Ann. Repts. Progr. Chem. (Chem. Soc. London) 55, 376-388. Parr, W. H., Evans, R. A., and Evans, W. C. (1949). Bbchem. J. 45, nix. Proctor, M. H. (1958). Nature 181, 1345. Pullman, A,, and Pullman, B. (1955). Aduances in Cancer Research 3, 117. Pullman, B., and Baudet, J. (1954). Compt. rend. SOC. biol. 238, 964. Ribbons, D. W., and Evans, W. C. (1959). Biochem. J. 73, 21 pp. Rogoff, M. H. (1957). Bacteriol. Proc. (Soc. Am. Bacteriologists) pp. 133-134. Rogoff, M. H. (1958). J. Gen. Microbiol. 10, 330-338. Rogoff, M. H., and Reid, J. J. (1956). J. Bacteriol. 71, 303-308. Rogoff, M. H., and Wender, I. (1957a). J . Bacteriol. 73, 264-268. Rogoff, M. H., and Wender, I. (1957b). J. Bacteriol. 74, 108-109. Rogoff, M. H., and Wender, I. (1959). J. Bacteriol. 77, 783-788. Roof, B. S., Lannon, T. J., and Turner, J. C. (1953). Proc. SOC. Exptl. BioZ. Med. 84, 38. Scher, S., and Allen, M. B. ( 1960). Bacteriol. Proc. ( SOC. Am. Bacteriologists) p. 67. 67. Sisler, F. D., and ZoBell, C. E. (1947). Science 106,521-522,

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Screening for and Biological Characterization of Antitumor Agents Using Microo r ga n is rns1 FRANK M. SCHABEL, JR.,

AND

ROBERTF. PITTILLO

Kettering-Meyer Laboratory, Southern Research Institute, Birnilngham, Alabama I. 11. 111. IV. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microbial Screening Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microbial Assay of Antitumor Agents.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological Characterization of Antitumor Agents. . . . . . . . . . . . . . . . . . . . . . . . Correlative Observations in Microbial and Mammalian Systems. . . . . . . . . . . Discussion and Future Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

223 229 232 234 243 251 253

I. Introduction How great is the need for chemotherapeutic agents effective against cancer in man? A few simple statistics can quickly give us an obvious answer to that question. In the United States today there are about 500,000 new cancer cases diagnosed each year, 250,000 cancer deaths each year, and about 750,000 cancer patients under treatment each year. If morbidity rates remain at the current level, about one in four Americans now living will eventually develop cancer. As frightening as these figures are, the prognosis of cancer, once diagnosed, does little to ease the mind. At the present time about two of six patients can be cured by conventional therapy ( surgery and/or radiation therapy) if treated quickly at the time of first detection, one of six will die because his tumor was not detected early enough and his disease had progressed beyond the point of successful conventional treatment at time of first detection, and three of the six will have cancers of types which cannot be controlled by conventional therapy. Thus, between one-half and two-thirds of cancer patients today are beyond the application of conventional therapy at the time their disease is first recognized. The majority of these cases are those in which the disease has metastasized to multiple sites beyond practical reach of surgical or ionizing radiation treatment when first detected. Current knowledge suggests that these patients might be effectively treated only by drugs selectively toxic for tumor cells that can be administered by some practical route and de' T h e work reported here that was conducted in the authors' laboratories was supported by Contract No. SA-43-ph-1809 with the Cancer Chemotherapy National Service Center, National Cancer Institute, National Institutes of Health. 223

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TABLE I LEADINGCAUSES OF DEATH IN THE UNITED STATESIN 1930,1945,AND 1958"

Cause of death

Deaths per 100,000 population

1930 1. Diseases of circulatory system 2. Infectious and parasitic diseases 3. Diseases of the nervous system and sense organs 4. Violent and accidental deaths 5. Disease of the genitourinary system 6. Cancer and other tumors 7. Diseases of the respiratory system 8. Diseases of the digestive system 9. Diseases of early infancy 10. Rheumatic] nutritional] endocrine gland, and other general diseases

237 137 112 105 104 102 96 86 50 35

1945 Diseases of the heart Diseases of the myocardium Cancer and other tumors Diseases of the nervous system and sense organa Diseases of the coronary arteries and angina pectoris Cerebral hemorrhage Accidental deaths Nephritis 9. Pneumonia, all forms 10. Tuberculosis

322 164 139 110 100 86 73 67 44 40

1958 Diseases of the heart Malignant neoplasms Vascular lesions affecting the central nervous system Accidents Diseases of early infancy Influenza and pneumonia General arteriosclerosis Diabetis mellitus Congenital malformation Cirrhosis of the liver

368 147 110 52 40 33 20 16 17 11

1. 2. 3. 4. 5. 6. 7. 8.

1. 2. 3. 4. 5. 6.

7. 8. 9. 10.

' Source: National Office of Vital Statistics.

livered by the blood and other body fluids to the metastatic tumor foci wherever in the body they may be. Table I lists the ten major causes of death in the United States in 1930, 1945, and 1958. It can quickly be seen that infectious disease mortality has sharply dropped in this period under the impact of success-

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ful chemotherapeutic drugs, Large numbers of the infectious disease deaths 20 to 30 years ago were among children and members of the younger age groups. Reducing deaths in this segment of the population advanced the mean age and brought more people into the older age groups with a resulting actual increase in death rates due to diseases more common in older age groups-heart disease, vascular lesions, cancer, etc. Cancer has always been an important cause of death; the impact of drug control of infectious disease with the resulting advancing of the mean age has merely served to increase its relative importance. Prior to about 1940 little effort was expended on cancer chemotherapy research. Cancer was considered to be an unlikely candidate for successful chemotherapeutic control because cancer cells, although thought to be biologically aberrant from normal cells, were still viewed as homologous to the host. A selective differential sensitivity of cancer cells to the toxicity of therapeutic drugs as compared to normal cells, the very keystone of rational chemotherapy from Ehrlich‘s time to the present, seemed too unlikely, in the case of cancer, to justify a serious search for anticancer drugs. The demonstration of the effectiveness of the sulfonamide drugs against certain bacterial diseases and the dawn of the “antibiotic era” in the late 1930’s stimulated great interest and activity in chemotherapy research, especially in relation to infectious diseases, resulting in the spectacular successes familiar to all. Cancer chemotherapy research still seemed a potentially unrewarding effort with little likelihood of success because the cancer cell, unlike the pathogenic bacterial cell, is not foreign to the host; and no significant humoral or cellular defenses, so important as adjuncts to successful chemotherapy in bacterial diseases, are known to be raised to limit the multiplication and ,spread of cancer cells. The 1940’s witnessed some discoveries which indicated that cancer cells could be selectively inhibited by chemotherapeutic agents, In 1940 encouraging results were reported with the palliative treatment of disseminated prostatic carcinoma of man with certain hormones ( Herbst, 1941; Huggins and Hodges, 1941; Huggins et al., 1949). The next major advance in cancer chemotherapy was a by-product of the wartime work on the pharmacology of the war gas, methyl-bis ( p-chloroethyl) amine hydrochloride-nitrogen mustard. Studies showed that nitrogen mustard and certain derivatives thereof were especially destructive to lymphoid tissue and rapidly dividing cells and suggested the possibility of their use in certain neoplasms of lymphoid character. This led to studies both in experimental animal neoplasms and human neoplasms which showed that nitrogen mustard had significant activity against Hodgkin’s disease and certain cases of lymphosarcoma in man (Gilman and Philips, 1946).

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By the late 1940’s the stage was set for intensive searches for drugs that might be useful for the treatment of human neoplastic disease by the three major factors just described: 1. The sulfonamide-antibiotic successes stimulated renewed interest in searching for practically usable chemotherapeutic agents for a number of diseases, including cancer, currently beyond practical control by drugs. 2. Drug control of mortality from many of the major bacterial and protozoal diseases had increased the relative importance of human cancer as a cause of death. 3. Limited success with drug treatment of certain types of human cancer suggested to some investigators, particularly the partisans of chemotherapy, that the apparent rational obstacles to drug control of cancer were not insurmountable, at least in some forms of the disease. Rational chemotherapy is based on the presumption, with few exceptions little more than an article of faith, that a metabolite essential for life for the target cancer cell is not essential for the host cell; or if it is essential for the host cell, the requirement for, or rate of utilization of, this metabolite is smaller in the host cell than in the target cancer cell. Drugs which interfere with the formation or utilization of the metabolite will selectively inhibit the target cell without unacceptable damage to the host, resulting in effective chemotherapy. To the present time the sensitive target metabolite has not been described for any neoplastic cell, either animal or human; searches for anticancer drugs have been and for the most part still are being made empirically. In searching for anticancer drugs the chemotherapy investigator faces a much more difficult problem, both from the standpoint of procedure and of interpretation, than he faces in searching for antimicrobial agents. In a search for antimicrobial agents, it is usually possible to isolate the disease-producing microorganism from naturally infected individuals and screen large numbers of candidate drugs against it in simple in vitro systems. Drugs found to be active in in vitro trials can then be tested in vivo against the same organism in experimental animals with reasonable expectations that those effective against the experimentally produced disease in animals will have a comparable effect against the same disease in man. No such straightforward procedure is currently available to the cancer chemotherapy investigator. A considerable number of human tumors can be grown successfully in laboratory animals and compounds screened for carcinostatic activity against them. These procedures and results to date have been recently reviewed. Since this particular experimental tool is not the subject of this presentation, it will suffice here to quote Dr. Woolley from that discus-

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sion-"The full usefulness of the heterologous transplantation of human tumors for therapy evaluation cannot be assessed at the present timesince this is a new and only partially-developed area" (Stock, 1958). The procedure holds promise but is not as yet practically established. The use of human cancer patients for screening of previously unselected compounds is not practical because of: ( a ) the marked variability in the natural history of the disease in man and the great difficulty in obtaining objective reproducible results especially with drugs of slight to moderate activity, ( b ) the unrealistic demands of direct screening in man for generally unavailable quantities of drugs to be tested, and ( c ) the moral obstacles to using biologically uncharacterized drugs in man, the gravity of the prognosis notwithstanding. The cancer chemotherapy investigator has used spontaneous or induced and generally transplantable neoplasms of laboratory animals as his test systems for chemotherapy trials. A large number of leukemias and lymphomas, sarcomas and carcinomas of mice, rats, hamsters, rabbits, and other usable laboratory animals are available for this purpose and have been widely used in screening for carcinostatic agents. Such systems have serious disadvantages. Among these are the fact that as screening tools they are expensive, requiring extensive facilities for handling experimental animals if even modest numbers of candidate drugs are to be tested and, more important, the inability to compare directly the drug response of experimental tumors in laboratory animals with that of analogous human tumors or even of any human tumor. A few years ago an attempt was made, under the auspices of the American Cancer Society, to compare the response of seventy-four biological systems to twenty-seven compounds representing: ( a ) known antitumor compounds, ( b ) compounds not known to possess antitumor activity but possessing inhibitory activity in biological systems other than tumor systems, and ( c ) several presumed inactive congeners of active carcinostatic agents. The seventy-four biological systems included a representative spectrum of animal tumors, animal viruses, bacteriophages, various bacteria and fungi, differentiation and developmental systems in a slime mold, the frog and chick embryos, the fruit fly Drosophila, and twenty-one biochemical indices of synthetic processes in four animal tumors and in rat spleen using four different isotopically labeled precursors. It was hoped that such a study would help to answer two very practical questions: 1. Does tumor growth inhibition by a compound reflect an effect on a property or function which is more characteristic of tumors than of other biological systems, or is it an expression of an action on a property or function common to many diverse biological systems? If the latter

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is the case, can nontumor systems be found which might be more useful, rapid, and economical than experimental tumor systems in screening for new anticancer agents? 2. To what extent does the study of inhibition of one experimental tumor have applicability to other experimental tumors? The results of this study were published in 1955 (Gellhorn and Hirschberg, 1955) with the following conclusions, in our opinion, still valid today: “1. The results confirm and extend previous observations on the variability of response among experimental neoplasms to chemotherapeutic agents, There is no single tumor which could be expected to select all useful agents; and, therefore, a spectrum of tumors provides a greatly improved screening system. “2. There is no evidence for the existence of any nontumor system which could replace a tumor system as a screening tool for carcinostatic agents. This conclusion applies to the microbiological, developmental, and biochemical synthetic systems studied here.” The great unanswered question regarding any biological system in use or seriously considered for use as a screening system for anticancer agents is “Does it predict the anticancer activity of new drugs for all or any type of human cancer?” With the possible exception of one or two transplantable animal neoplasms which probably do predict the response of acute lymphatic leukemia in man to certain classes of drugs, this question cannot be answered for any of the multitude of biological systems, including experimental tumor systems, which have been used in cancer chemotherapy screening to date. Since 1955 one new in vitro screening system has been under investigation in a number of laboratories as a possible anticancer-agent-predicting system. This system quantitatively determines the cytotoxicity of drugs for mammalian cells in cell culture. Eagle and Foley (1956) reported that a number of agents known to have an antitumor effect in animals or man were highly cytotoxic against several human and animal cells in cell culture, while several compounds without antitumor activity failed to show significant cytotoxicity against these same cells in uitro. These observations suggested to these investigators that cytotoxicity in cell cultures might be a useful primary screen for the detection of potential antitumor agents. These same workers have extended their observations to a series of two hundred compounds selected to include known anticancer agents as well as agents highly active as inhibitors in other biological systems but not known to possess antitumor activity (Eagle and Foley, 1958).In this group of two hundred compounds there were sixty-eight with reported in vivo antitumor activity in two or more

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experimental tumor systems. By using a 50%or greater inhibition of growth of the cell cultures as a cutoff point, 79% of these sixty-eight active antitumor compounds were inhibitory at concentrations in the cell culture medium of 1.0 x gm./ml. Of ninety-one compounds in the series with no reported antitumor activity, nineteen (21%) were cytotoxic at concentrations in the cell culture medium of 1.0 X gm./ml. or less. This apparent favorable correlation between cytotoxicity in cell culture and antitumor activity in vivo has been the basis for an in vitro screening trial in cell culture by the Cancer Chemotherapy National Service Center ( CCNSC ) using randomly selected organic synthetics and antibiotics which were also being evaluated for in oivo anticancer activity in experimental animals. The results of this comparative study are not yet available.

II. Microbial Screening Systems As stated previously, screening for anticancer agents in tumor-bearing animals is demanding of time, space, labor, and money. In addition, comparative studies indicate that a screening system capable of predicting all or most of the anticancer agents useful against all of the many various types of human cancer would have to consist of an unknown but probably large number of different animal neoplasms. If this be true, in vivo screening in animals would fail on several counts to meet Foley’s definition (Foley et al., 1958) of an ideal screening system“The ideal method . . . must not only be simple, rapid, inexpensive, and adaptable to large-scale operation, but should detect the maximum number of antitumor agents with a rate of ‘false positives’ sufficiently low not to invalidate the use of the method as a screening procedure.” Microbial systems are “simple, rapid, inexpensive, and adaptable to large-scale operation.” Whether microbial systems can be selected which will “detect the maximum number of antitumor agents with a rate of ‘false positives’ sufficiently low” remains to be seen. The utility of microbial systems in cancer chemotherapy research has already been amply demonstrated. The first clinical use of aminopterin and amethopterin in human leukemia (Farber et al., 1948) was suggested on the basis of observed antimetabolite activity in Lactobacillus casei and Streptococcus faecalis (Hutchings et al., 1947; Seeger et al., 1947). The observation that 8-azaguanine inhibition could be reversed by guanine was first observed in Escherichia cold and Staphylococcus aureus (Roblin et al., 1945). When 8-azaguanine was first tested for antitumor activity in uiuo, Sarcoma 180 was used and the compound was found to be inactive (Stock et al., 1949). In later studies a purine antagonistsensitive tumor (E0771) was used, and 8-azaguanine was shown to

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p.,AND

ROBERT F. PITTILLO

have profound carcinostatic activity (Kidder et al., 1949). It is now well known that Sarcoma 180 is relatively insensitive to purine antagonists and this fact emphasizes the necessity of using in vivo systems with varying antimetabolite sensitivities in screening for anticancer agents. It is possible that false negatives are as great a problem in in vivo screening using animal neoplasms as in any other biological system. Other anticancer agents which have proved to be effective in inhibiting the growth of experimental tumors which were first suggested on the basis of their inhibitory activity against bacteria are 2,6-diaminopurine (Hitchings et al., 1948), 6-thioguanine (Elion et al., 1951), 2,4diamino-5-( 3',4'-dichlorophenyl ) -6-ethylpyrimidine ( Hitchings et al., 1952), 6-mercaptopurine ( Elion et al., 1951), actinobolin (Pittillo, unpublished observations), the actinomycins ( Waksman, 1960), and puromycin (Porter et al., 1952). Several studies have been made to compare the sensitivity of different microbial systems to known biological inhibitors including anticancer agents. In the American Cancer Society study referred to earlier (Gellhorn and Hirschberg, 1955), the following microorganisms were tested: Escherichia coli, Serratia marcescens, Bacillus subtilis, Staphylococcus aureus, Mycobacterium smegmatis, Mycobacterium phlei, Lactobacillus casei, Tomlopsis utilis, Saccharmyces cerevisiae, Kloeckera brevis, Penicillium notatum, Aspergillus fumigatus, Streptomyces griseus, and Streptomyces antibioticus. Later the CCNSC supported a comparative study of the inhibitory activity of one hundred and eighty compounds, including many known to have anticancer activity and others known to be toxic in other biological systems but not known to have any antitumor activity, against the following microorganisms: Escherichia coli (two different strains), Lactobacillus arabinom (in two different media), Leuconostoc citrovorum, Lactobacillus fermenti, Candida albicans, Saccharomyces carlsbergensis, Streptococcus faecalis ( in two different media), Lactobacillus casei (in two different media), Tetrahymena pyriformis, Glaucoma scintillans, Colpidium campylum, and Neurospora crassa (Foley et al., 1958). In the American Cancer Society-supported study (Gellhorn and Hirschberg, 1955), the positive correlation between microbial inhibition and activity in other biological systems with the twenty-seven selected compounds was poor. In the CCNSC-supported study (Foley et al., 1958), the results indicated that 951% of the compounds considered to possess antitumor activity in in vivo tumor systems could be detected on the basis of their microbial inhibitory activity, using as few as four selected microbial systems. The false positive (60-652) and false negative ( 5 % ) results were noted, but the investigators pointed out that

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“since a similar situation may prove to exist when effectiveness in animal tumor assays is compared with effectiveness in human neoplasia, such results do not detract from the potential usefulness of microbiological screens.” The Warburg theory ( Warburg, 1956), which proposes that cancer results when a normal cell adopts an anaerobic metabolism as a means of survival after injury to its respiratory mechanisms, is still the subject of considerable research and controversy. Based on the premise that anaerobic microorganisms might respond more like tumor cells than do aerobic organisms because of the presumed greater similarity of their metabolism to that of tumors, Bradner (1958; Bradner and Clarke, 1958) compared ninety compounds on which antitumor data were available (against Sarcoma 180 and Adenocarcinoma 755) for inhibitory activity against ten species of anaerobic bacteria. The over-all correlation between inhibition of these bacteria and inhibition of tumor growth by these compounds was poor. The number of compounds tested was small, the spectrum of anaerobic bacteria tested was limited, and the tumor systems used for comparative purposes were undesirably few. DiPaolo and Rosenfield (1958) studied the inhibition of several species of Clostridium and a number of respiratory-deficient, induced mutants of five species of Saccharomyces. The sensitivity of these microorganisms to known anticancer agents did not indicate that they would be any more useful than common aerobic organisms in screening for potential anticancer agents. Gause and co-workers have worked with mutants of bacteria, yeasts, and flagellates characterized by impaired oxidative metabolism induced by exposure to ultraviolet light, camphor, trypaflavine, or 9,lO-dimethyl-l,2-benzanthracene and have observed that these organisms are more sensitive to the inhibitory activity of some anticancer agents than the parent organisms from which the mutant strains were derived ( Gause, 1958; Gause et al., 1959). The authors conclude that such mutant microorganisms may be helpful in screening for new anticancer agents since these may be microbiological equivalents of cancer cells-“that is, biochemical mutants of microorganisms with deficient respiration and probably with some other alterations in cell metabolism which are specific for malignant growth.” Okami et al. (1958) used this same reasoning in studying the activity of a number of anticancer agents against several yeasts and respiratorydeficient mutants derived from them. Whether this interesting concept will be of practical usefulness in screening for anticancer agents can be determined only after adequate experimental trial. If the controversy over the Warburg theory should be

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resolved in support of it, more extensive screening for antitumor agents in anaerobic bacteria would be indicated. While microbial systems have many practical advantages as potential screens for anticancer agents and have actually provided first observations leading to the recognition of the anticancer activity of a number of our most useful agents, to our knowledge a well-integrated program to actually test their predicting potential for anticancer agents in a random screening program has not been undertaken as yet. The CCNSC is currently screening a limited number of randomly selected organic synthetics against Escherichia coli, Lactobacillus cmei (in two different defined media ) , Streptococcus faecalis, Tetrahymena geleii, and Saccharomyces cerevisiae. The agents tested in this screen will also be tested in the in vivo antitumor screens of the CCNSC and presumably some of the agents showing marked inhibition of one or more of the microbial systems will be tested for tumor-inhibiting activity in man, whether or not they show antitumor activity in the in vivo animal antitumor screens of the CCNSC. It will be important to test drugs that are active inhibitors of microbial systems but are inactive in the in wivo antitumor screen for activity against a wide spectrum of tumors, including some tumors in man in order to evaluate the predicting capacity of microbial systems for anticancer agents. Demonstration of the presence or absence of correlation between microbial systems and animal tumor systems would be of little help, so long as the predicting ability of animal tumor systems for activity against one or more human tumors is in doubt.

Ill. Microbial Assay of Antitumor Agents The fact that most antitumor agents will also inhibit some microorganisms has been of considerable practical value, especially in the assay of antitumor antibiotics. In reference to the search for new antibiotics, it has been said “ . . The crucial problem is assay, the prompt and precise estimation of how much of the desired substance is present. In no sector of antibiotic research is this problem more vexing than in the quest for an antibiotic effective against neoplasms” ( Ehrlich, 1960). Historically, the search for antibiotics effective against transplantable neoplasms in rodents was begun by evaluating all currently available purified antibiotics for activity against some suitable tumor in mice, usually Sarcoma 180 (Reilly et al., 1953). A few of these antimicrobial antibiotics, such as actinomycin, cycloheximide ( Actidione) , and oxytetracycline (Terramycin ) , showed minimal inhibition of Sarcoma 180. Although work of this nature failed to unearth a potent anticancer antibiotic, the fact that antibiotic substances, specifically antimicrobial sub-

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stances, were able to inhibit tumors encouraged the search for new antitumor antibiotics, Subsequently, screening programs were established in which crude culture filtrates were tested for antitumor activity in duo. In many cases beers selected for antitumor tests had previously shown inhibitory activity in some microbial system, Isolation of these antitumor antibiotics could, therefore, be followed microbiologically. Those which did not inhibit microbial systems posed special isolation problems, and intensive searches were made for in uitro biological systems sensitive to the active antitumor principle. Azaserine was the first antibiotic discovered to have marked activity against rodent tumors (Stock et al., 1954). Crude beers of the azaserineproducing culture also inhibited a number of microorganisms, among them E . coli, and the antitumor activity of initial fractionation samples was found to accompany the anti-E. coli activity. Further purification of azaserine revealed, however, that E . coli inhibition was not due solely to azaserine, and a deliberate search was instituted for a microorganism that would specifically measure the azaserine content of beers and fractionation samples. Such a tool was found in the yeast Kloeckera breuis NRRL Y-915 and a precise, sensitive, disk-tray, agar-diffusion was developed (Kohberger et al., 1955). A similar situation occurred with the antibiotic 6-diazo-5-oxo-~-norleucine ( DON). Comparative assays on beer and fractionation samples both in mice bearing Sarcoma 180 and in a variety of microbial systems established that the DON activity could be accurately measured by agar-diffusion assay employing Tomlopsis albida NRRL Y-1400 ( Ehrlich et al., 1956). Two bacteria were used for assay purposes in the early stages of the fractionation of alazopeptin-containing beers. An original crude concentrate that was active against Sarcoma 180 in mice showed a high degree of activity against both Coynebacterium xerose LL No. 46 and Salmonella gallinarum LL No. 10. Although definitive correlation of activity between the bacterial and the tumor systems was not proved, the bacterial assays were employed until the alazopeptin was purified to the stage where spectrophotometric assay could be used (DeVoe et al., 1957). The isolation of the antitumor antibiotic sarkomycin was accomplished by the simultaneous use of tumor and microbial assays-in this instance, a cup-plate assay using Micrococcus pyogenes var. aureus 209-p (Umezawa et al., 1954; Hooper et nl., 1955). Mitomycin-containing beers were found to inhibit the Ehrlich ascites tumor in mice and also bacteria (Hata et al., 1956; Sugawara and Hata, 1956). The isolation and characterization of the various mitomycins were made possible by the simultaneous application of bacterial, tumor, and chemical assay (Wakaki et al., 1958). A number of antimicrobial antibiotics possessing carcino-

234

FRANK M. SCHABEL, JR., AND ROBERT F. PITTILLO

static activity were originally isolated as antimicrobial agents, and their antitumor activity was first demonstrated with highly purified or crystalline products. These include actinobolin, cycloheximide, and actinomycin. Actinobolin is assayed against Sarcina lutea PCI lOOlW (Pittillo et d., 1959). Cycloheximide responds satisfactorily in a well-defined assay using Saccharomyces pastorianus ATCC 2366 as the test organism (Ford and Leach, 1948). Antibiotics of the streptovitacin group were first discovered in spent cycloheximide beers (Field d al., 1959). Crude streptovitacin was clevoid of antibacterial activity but significantly inhibited Trichomonns vaginulis. Although its activity against the yeast Sacchnromyces pnstorianus was eventually used for the refinement of purification procedures, the original isolation of streptovitacin A was accomplished with the aid of the Walker 256 adenocarcinoma in rats (Sokolski et nl., 1959). Microbial assays with Bacillus subtilis were used to isolate diazomycins A, B, and C from crude beers possessing carcinostatic activity against mouse tumors (Rao et al., 1960). Streptonigrin was isolated from beers showing activity against Adenocarcinoma 755 in mice and human tumor type HS No. 1 in rats by using B. subtilis and M . pyogenes var. aweus assays (Rao and Cullen, 1960). The difficulties associated with the isolation of natural products such as antitumor antibiotics using in uivo systems are involved and expensive, and those who have attempted it are impressed with these difficulties. Therefore, the demonstration of a microbiological assay system which can be used to follow the isolation and purification of natural products with antitumor activity is of great practical importance, and all workers involved in this type of isolation look for microbial assay systems. Indeed, it is our opinion that isolation of antibiotics with antitumor activity would go forward more rapidly if assay systems were available for many of the antibiotics with promising antitumor activity that have been detected in our antitumor screening program in recent years.

IV. Biological Characterization of Antitumor Agents Cancer in man is not one disease but a host of diseases, and it is unlikely, therefore, that we will find a single drug, a panacea, which will control all of the different types of human malignant neoplasias. Currently available anticancer chemotherapeutic agents are able to control temporarily only a portion of the leukemias and lymphomas, some of the hormone-sensitive carcinomas of the breast and prostate, and a few tumors of low incidence such as choriocarcinoma, Wilm’s tumor, and perhaps a few others. Many organic chemists are actively engaged in synthetic work altering the structures of known anticancer agents in

MICROBIAL TOOLS FOR ANTITUMOR RESEARCH

235

attempts to increase the antitumor activity and/or reduce the undesirable toxicity of known agents and thereby improve and extend their utility. While their efforts have been moderately successful in a few cases, most cancer chemotherapy investigators would agree that the discovery of new classes of anticancer agents holds one of the greatest promises of achieving their goal of chemotherapeutic control of cancer. Barring the discovery of new classes of anticancer agents, it is possible that we may be able to circumvent the causes of our failure to cure with some of the known anticancer agents, especially the antileukemic drugs. Human leukemia that initially responds to treatment with antileukemic drugs ultimately develops resistance to the drug and treatment fails. If we had drugs that were selectively active against drug-resistant leukemic cells, we might be accomplishing leukemia cures today. We believe that the application of microbiological procedures and special microbiological systems to the problem of the discovery of new classes of anticancer agents and the discovery of drugs selectively toxic for drug-resistant neoplastic cells is potentially very useful. For some time we have been using bacteria in a variety of ways in our cancer chemotherapy research program at Southern Research Institute (Schabel, 1958). We have a primary, rodent-tumor screening laboratory operating under contract with the CCNSC. With few exceptions the materials submitted to our in vivo antitumor screen are completely uncharacterized biologically. About 80%of our total primary screening activity is with crude antibiotic beers and about 20%with synthetic organic compounds. New agents that possess carcinostatic activity in vivo against mouse neoplasms are evaluated bacteriologically in an attempt to relate them to presently known agents both from the standpoint of identity and the possible mode of action. The essential feature of this evaluation consists of testing tumor-inhibitory materials for inhibitory activity against a spectrum of drug-resistant and drug-sensitive strains of bacteria (see Table 11). This spectrum is far from complete in terms of including either culture strains resistant to all known antitumor substances or a sufficiently broad spectrum of diverse microbial types to detect inhibitory activity of the majority of antitumor materials. However, it does include organisms which are resistant to different types of antitumor agents such as antibiotics ( azaserine, actinobolin, actinomycin) , antimetabolites ( amethopterin, 6-mercaptopurine, %fluoroadenosine), and alkylating agents (nitrogen mustard), All these microorganisms are grown on chemically defined media (Anderson, 1946; Flynn et al., 1951; Hutchison, 1958). Antitumor materials (crude beers, purified antibiotics, or synthetic compounds) are tested as solutions which are applied to paper

236

FRANK M. SCHABEX, JR., AND ROBERT F. PITIlLLO

TABLE II BACTERIA USEDIN STUDYING POTENTIAL ANTICANCER AGENTS Bacteria E. coli, ATCC 9637b /AZA /DON /FPA /ETH /ACB /MIT /NET /AMP /SNA /6TG /FRM /HN2 /FA8 /FAD E. coli, B96 E. coli, B /6MP /ACB 8.faecalis, ATCC 8043 /ACB /ACM /AMP /6MP /AZG

Compound to which strain is specifically resistant None Azwerine Diazo-0x0-tnorleucine p-Fluorophenylalanine Ethionine Actinobolin Mitomycin C Netropsin Amethopterin Sulfanilamide 6-Thioguanine N-Methylformamide Nitrogen mustard 2-Fluoroadenosine 2-Fluoroadenine None None 6-Mercaptopurine Actinobolin None Actinobolin Actinomycin D Amethopterin 6-Mercaptopurine 8-Azaguanine

Degree of resistance' 10,000 x 10,000x

1,ooox 10 x

> 100 x 100x 50 X

10 x 10 x 10 x 2x 100 x >1 ,OoOx >l,OOOX -

100x > 100 x

>500 X > 100x 10,000 x 500 X

1,ooox

Degree of resistance in relation to the parent drug-sensitive line from which it was derived. b Diagonal line( / ) is followed by the name of the compound to which organism is resistant.

disks and placed on the surface of agar pour-plates, heavily seeded with the individual bacteria in the spectrum. After suitable incubation, the plates are examined for inhibition of bacterial growth around the paper disks. Inhibition of the parent drug-sensitive line of an organism and lack of inhibition of a drug-resistant line of the same organism suggests two possibilities: ( a ) structural similarity or identity between the test drug and the agent to which the organism is resistant; or ( b ) similar or related biological mode of action between the test drug and the agent to which the organism is resistant. Table I11 shows examples of the behavior of a group of tumor-inhibitory crude antibiotic beers against the bacterial spectrum, These

237

MICROBIAL TOOLS FOR ANTITUMOR RESEARCH

TABLE 111 THE ACTIVITYOF A SELECTEDGROUPOF ANTIBIOTICMATERIALSON SPECTRUMOF BACTERIAAND RESISTANTSTRAINS’ “Crude” beers

A

Antibiotics

975 400 693 187 011 427 071 ACMb ACBc DON

Bacteria

E. coli, ATCO 9637 /AZA

/DON /FPA /ETH /ACB /MIT /NET /AMP

/SNA /6TG /FRM /HN2 /FAS /FAD E. coli, B96 E. coli, B /6MP /ACB S. faecalis, ATCC8043 /ACB /ACM / A M /6MP /AZG

+

o

o

+

+ $ $ + + + 0 + + + + + + +

O 0 0 O O O 0 O O O O O O O 0 0 O O

0 0 0 O 0 0 O 0 0 0 0 O 0 0 0

0 0 + + + + + + + + + + + +

+ + + +

0 O 0

0 0 0

+

+ +

+

t

+

+ + + +

O O 0 0 O O 0 0 O O O 0 0 0 O 0

+

+ + + +

+

+

+

+ + + +

+ +

+ +

0

0 0 0 0 0 0 0 0 0 0 0 0

+ + + + + + + + + + + + + + + 0

O O O O O +

O

0 0 0 0 0 0

+

+ + + + + + + + + +

+

o

+ + + + + +

0 0

+ + + +

+ O 0 + P O + O + +

O

+ +

o

+

+

Symbols: = inhibition, 0 = no inhibition. * Actinomycin D. Actinobolin. Azaserine. ’See Table I1 for explanation of abbreviations.

+ +

+

+

+ +

o o

+ + 0

+ O

+

+ + + + + + + + + + + 0

+ 0

+

+ + +

AZAd

+ o + + + + + + + + o + + + 0 0

o

0 0 0

+ 0 0 O

+ + + + + + + + + + + + + + + 0 0 0 0 0 0

’

data indicate to us that beer 975 contains netropsin or a material similar in structure and/or biological mode of action to netropsin since it was a potent inhibitor of all E. coli strains except the one resistant to netropsin. Similarly, beer 400 appears to contain actinomycin or an actinomycin-like material since the actinomycin-resistant strain of Streptococcus faecal& was resistant to this beer. Beer 693 seems to contain actinobolin,

238

FRANK M. SCHABEL, JR., AND ROBERT F. PITTILLO

although perhaps in low concentration as indicated by the failure of this beer to inhibit E. coli. Beer 187 appears to contain a diazoketonetype inhibitor; this beer failed to inhibit both the azaserine and DONresistant strains of E. coli but had marked activity against the rest of the spectrum. Beer 011 is of great interest since it appears to inhibit in a manner similar to amethopterin. To our knowledge no naturally occurring folic acid analogs have been reported. Since no cross resistance to beers 427 and 071 was observed with any of the drug-resistant bacteria, they are of special interest. These observations suggest the possibility that they may contain materials whose biological modes of action are novel. Obviously, data of this sort cannot be used to identify definitively the antibiotic content of a given beer. At best they serve to give us some idea as to the nature of the inhibitor as related to the behavior of known antitumor agents. Similar observations have been made with synthetic compounds. We are also seeking synthetic compounds with new biological modes of action, and studies with microbiological systems are useful in selecting such compounds. A few examples of interesting observations with synthetics against the bacterial spectrum are listed in Table IV. Compound 947 inhibits all the bacteria except the netropsin-resistant E. coli. The fact that it may have a similar mechanism of action to netropsin detracts from its interest, but being a synthetic compound it may be useful in studying the biological mechanism of action of netropsin. Compound 817 may be a new folic acid antagonist since only the amethopterin-resistant S. faecalis is cross resistant to this compound. It is likely that it differs from amethopterin in its biological mode of action since most of the strains of E . coli studied are inhibited by similar concentrations of amethopterin. Compound 981 inhibits all strains of E. coli studied except the one resistant to ethionine; and since it has no other unique characteristics, we would defer further work with it in favor of compounds of greater theoretical interest. Compound 403 failed to inhibit the actinobolin-resistant strain of E . coli B. Although the fact that it may have a mechanism of action related to actinobolin detracts from its importance as a chemotherapeutic agent, this compound may prove useful in elucidating the mechanism of action of actinobolin. Compound 605 is of great interest because none of the drug-resistant bacteria are cross resistant to it. This suggests that it may have a biological mode of action different from amino acid antagonists, purine antagonists, folic acid antagonists, nitrogen mustard, and antibiotic anticancer agents represented by this spectrum of resistant bacteria. If it represents a new class of

239

MICROBIAL TOOLS FOR ANTITUMOR RESEARCH

TABLE IV THEACTNITYOF CERTAIN ANTITUMOR SYNTHETIC MATERIALSIN SPECTRUM OF BACTERIA AND RESISTANT ST RAINS^

A

Synthetic materials Bacteria

947 817 981 403 605

E . coli, ATCCb 9637 /AZA /DON /FPA /ETH /ACB /MIT /NET /AMP /SNA /6TG /FRM /HN2 /FAS /FAD E. coli, B96 E . coli, B /GMP /ACB S. faecalis, ATCC8043 /ACB /ACM /AMP /6MP /AZG

Symbols:

+ + + +

o o o

+ + +

o o o

+ + +

+ +

( l o o + o + o +

O

+

O

+

+

O

+

O

+

+

O

+

O

+

+ + + +

O O O O

+ + + +

O O O O

+ + + +

+

O

+

O

+

o + +

+

+ +

o o o

o

o o

+ + +

+

+ +

+ +

o o o

o

+ +

0

+ + +

+

+ +

+

0

+

+

O

O

+

+ +

+ +

O O

O O

+ +

+ +

+ o

o o

o o

+ +

Amethopterin 2-Fluoroadenosine

+ + + ++ +0 + 0 +0 + + + + + + + + + + +0 + +

+ + + ++ ++ + + ++ + + 0 0 0

+ + + 0 0

+ 0 0 0

+ = inhibition, 0 = no inhibition.

* See Table I1 for explanation of abbreviations. anticancer agents, it is of great theoretical and potential significance. Obviously, the mere fact that a strain of bacteria is resistant to two different agents, either synthetic compounds or antibiotics, does not establish that both are inhibiting identical metabolic reactions. Therefore, we are interested in determining the sensitive metabolic area in bacteria inhibited by any new anticancer agent. A new agent that fails to inhibit specific drug-resistant bacteria may differ somewhat in its inhibitory activity from the agent to which the bacteria are resistant. In this case the new agent will be of less interest than one with an ap-

240

FRANK M. SCHABEL,

JR.,

AND ROBERT F. PITIlLLO

parent completely novel mechanism of action but will often warrant further work. Preliminary studies designed to establish the metabolic area inhibited by new agents are carried out by attempting to reverse the bacterial inhibition with mixtures of metabolites. Once a new antitumor material has been shown to have significant inhibitory activity for one or more of the bacteria, attempts are made to determine the metabolic area which is being inhibited. The antitumor materials, in a concentration sufficient to inhibit completely the microorganisms, are incorporated into heavily seeded, agar pour-plates. Filter paper disks impregnated with various mixtures of metabolites are placed on the surface of the inhibited cultures and observed, after suitable incubation, for growth around one or more of the metabolite-containing disks. In initial tests, the following metabolite mixtures are used: purine mixture, containing 80 pg. each per disk of adenine, guanine, xanthine, and hypoxanthine; pyrimidine mixture, containing 80 pg. each per disk of uracil, thymine, and cytosine; amino acid ‘‘A mixture containing 80 pg. each per disk of glycine, serine, histidine, alanine, threonine, leucine, isoleucine, sarcosine, valine, norvaline, and a-aminobutyric acid; amino acid “ B mixture containing 80 pg. each per disk of aspartic acid, glutamic acid, glutamine, lysine, arginine, ornithine, citrulline, cystine, cysteine, methionine, homocysteine, and betaine; amino acid “ C mixture containing 80 pg. each per disk of phenylalanine, tyrosine, tryptophan, proline, and hydroxyproline; and a vitamin mixture cantaining 10 pg. each per disk of folic acid, leucovorin, ascorbic acid, thiamine, riboflavin, pyridoxal, biotin, nicotinamide, p-aminobenzoic acid, BIZ, choline, and calcium pantothenate. Inhibitors which are reversed by metabolite ( s ) are further studied by more precise, quantitative procedures. The spreadplate procedure described by Foster and Pittillo (1953) has been particularly useful. In Table V are shown the results obtained in metabolite reversal experiments with some of the beers and synthetics listed in Tables 111 and IV. Beer 071 apparently contained a novel antibiotic since it inhibited all of the drug-resistant bacteria (Table 111).The reversal data indicate that this antibiotic is interfering with amino-acid metabolism. The fact that the amino acids phenylalanine, tyrosine, glutamine, glutamic acid, and aspartic acid reverse bacterial inhibition caused by the antibiotic coupled with the earlier observation (see Table 111) that this antibiotic inhibits the strains of E . coEi resistant to p-fluorophenylalanine, glutamic acid-y-hydrazide, and ethionine appears to indicate a novel mechanism of inhibition. The observations with beer 187 against the resistant bacteria (Table 111) indicated that it contained an azaserine-like or DONlike material. This was confirmed by the reversal obtained with the

TABLE V THE

EFFECTOF

CERTAIN

METABOW

ON THE INHIBITION OF

BACTERIA CAUSED BY

SELECTED ANTITUMOR

AGENTS‘

Metabolites

Material Beer Oil

Concentration (pg./ml. or dilution 1: -)

Bacterial strain used

10 20 30

E . coli, ATCC 9637

10 20

E . coli, ATCC 9637

Amino acids Purine Pyrimidine 0 mixture “A” “€3” “C” mixture -

-

-

-

I-

Beer 187

Compound 403

40 3

Compound 605

3

10 30 a

Svmbols:

-

E . coli, B

-

E . coli, ATCC 9637

-

10

30

-

-

+ + +-

+

-t

+ ++

Vitamin mixture

Active individual metabolites

-

None Tyrosine, phenylalanine Tyrosine, phenylalanine, glutamine, glutamic acid, aspartic acid

-

Guanine, xanthine, hypoxan thine None

+ + +

+ = growth, i.e., reversal; - = no growth.

Adenine

242

FRANK M. SCHABEL, JR., AND ROBERT F. PITTJLLO

purine bases, guanine, xanthine, and hypoxanthine, which also reverse azaserine and DON in bacteria (Bennett et uZ., 1956; Maxwell and Nickel, 1957). Compound 403, thought possibly to be related to actinobolin in its historical mechanism of action, was found not to be a reversible inhibitor, an observation indicative of a novel mechanism of action since bacterial inhibition by actinobolin can be reversed by metabolites ( Pittillo and Schabel, unpublished observations). On the other hand compound 605, presumed to be a new type of inhibitor on the basis of its outstanding activity in the bacterial spectrum (Table IV), was found to be reversed by adenine. The importance of finding drugs selectively toxic for drug-resistant neoplastic cells has already been mentioned. In bacteriological examination of new antibiotic and synthetic materials we sometimes find agents which are either selectively toxic for some one or more resistant strains but nontoxic for the parent cultures or inhibitory to the parent strain but with greatly increased toxicity for some derived resistant strains, Table VI lists typical examples of this kind. These results indicate that antibiotic beer 644 might be useful against actinobolin-resistant tumors; antibiotic beer 666, since it is active against a number of drug-resistant bacteria, probably should be widely evaluated against a broad spectrum of both drug-resistant and drug-sensitive tumors; and antibiotic beer 949 might be useful against azaserineresistant tumors. Synthetic compound 794 might be useful against azaserine-resistant tumors, compound 330 against amethopterin-resistant tumors, and compound 130 against azaserine-resistant tumors. We know of no N-methylformamide-resistant experimental or human tumors, but compound 130 should be tested in combination with N-methylformamide against tumors sensitive to N-methylformamide or other active antitumor carbamates. The fact that a new antitumor agent fails to inhibit a drug-resistant microorganism is suggestive but by no means conclusive that the biological mechanism of action of these agents in mammalian cells would be the same. We do not have mammalian tumor cells resistant to such a spectrum of antitumor agents as can be achieved with bacteria and while studies of this type fail to establish a biological correlation between inhibition or cross resistance by a new agent against drug-sensitive and drug-resistant bacteria as compared to drug-sensitive and drugresistant cancer cells, nevertheless, these observations give us information concerning new materials that we cannot obtain in any other way at present and at least suggest possible biological modes of action of new agents.

243

MICROBIAL TOOLS FOR ANTITUMOR RESEARCH

TABLE VI THE SELECTIVE TOXICITYOF CERTAINANTITUMOR AGENTS FOn RESISTANT BACTERIA Antitumor agents

Bactcriu

E . coli, ATCC 0637r /AZA /DON /FPA /ETH /ACB /MIT /NET /AMP /SNA /6TG /FRM /HN2 /FAS /FAD E. coli, BOG E. coli, B /6MP /ACB S . ,fnPcnlis, ATCC 8043 /ACD /ACM /AMP /GMP /AZG

Antibiotic beers" 644 666 94!)

0" 0 0 0 0 0 0 0

0 0 0

0 0 0 0 0 0 0 0 0 1.0 0 0 0 0

0 1.7 0 1.5 1.5 0 1.4 1.8

1.2 1.7 0

1.6 1.8 1.4 0

1.2 1.4 1.1

1.1 0 0 1.4 1.4 0

0

0 1.0 0 0 0 0 0 0 0 0

0 0 0

0 0

0 0 0 0 0

0 0 0 0 0

Synthetic compounds" 794 330 130 0.9 2.1 1.2 1.3 1.7 1.0 0.8 0

0.8 1.1 1.0 0.9 1.6 1. 0 1 .o 2.0 1.0 1.4 1.0

2.5 2.5 2.6

3.0 2 .o 2.0

0 0 0 0 0 0 0 0 1.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 1.5 0 0 0 0 0 0 0 0 0 I .O 0 0 0 0 0 0 0 0 0 0

0 0

0

0.08 ml. undiluted beer/disk.

* 80 .g./disk. a

See Table I1 for explanation of abbreviations. Numbers in table refer to radii of zones of inhibition (in cm. ) .

V. Correlative Observations in Microbial and Mammalian Systems The great question confronting the investigator considering the use of microbial systems as indicators for probable drug activity against human tumors is that of correlation between microbial and mammalian (including human) metabolic systems. That a complete positive correlation betwezn them does not exist is recognized by all, and this fact is taken by

244

FRANK M. SCHABEL, JR., AND ROBERT F. Pll’TILLO

some to indicate that attempts to obtain knowledge concerning mammalian cell metabolism by studying drug inhibition of microbial systems is unrealistic and rationally indefensible. Unquestionably the “unity of biochemistry” theory, holding that “the liver cell and E . coli, the meristematic plant cell, and the purple bacterium are sisters under the skin, their biochemical differences being principally ones of minor detail” (Stanier, 1954), is far too rigid to fit the facts. Among the bacteria themselves and even among different strains of the same species important exceptions to metabolic norms are commonly observed ( Cohen, 1955). The degree of positive correlation between the inhibitory activity of anticancer drugs against animal neoplasms and human tumors has seldom been established. Certainly one of the most critical deficiencies in our current effort to discover drugs effective against human neoplasms is our lack of objective data indicating positive or negative correlation between drug inhibition of animal neoplasms and human neoplasms. The laboratory worker should not be recriminatory to the clinicians who state that animal screening fails to correlate with observations in man. Reliable objective clinical data from humans are far more difficult to obtain than are reproducible experimental data from well-controlled studies in animals. Another factor often overlooked in this type of academic “in-fighting’’ is that the laboratory experimentalist and the human clinician are not even conducting the same general type of experiment. Usually the laboratory worker is studying tumor inhibition in an essentially normal animal with a small and recently established tumor while the human clinician is attempting to treat an advanced and massive tumor in a debilitated and often terminal patient. Under these circumstances one marvels that any positive correlations in the field of cancer chemotherapy between man and animals have been seen. Lacking objective evidence of extensive positive correlation between observations on drug inhibition of microbial systems and human tumors, how can we rationally defend further work with microbial systems in cancer chemotherapy studies? We must take our conviction that studies with anticancer drugs in microbial systems will yield useful information concerning the anticancer activity of the same drugs in mammalian tumor systems including human tumors from the examples of positive correlation available to us. Examples of useful anticancer drugs first suggested on the basis of inhibition of microbial systems were presented in Section I. The use of drugs in combination in cancer chemotherapy has great potential usefulness. When a cell grows in the presence of suboptimal amounts of an essential metabolite, several cell divisions may take place before cell division ceases. During this growth the cells are depleted of

MICROBIAL TOOLS FOR ANTlTUMOR RESEARCH

245

the metabolite in question and also all of its products in the metabolite sequence. Theoretically, this depletion renders the cell hypersensitive to antimetabolites acting at any subsequent stage along the metabolic pathway. The demonstration of such sequentially acting antimetabolite inhibitors suggests drug combinations with possible greater inhibitory activity than that possessed by either drug alone. Elion et al. (1954) have described a procedure which allows the in vitro assessment of compounds in combination for potentiation of inhibitory activity against bacteria, and we have utilized this procedure extensively in our own laboratories in searches for possible potentiating combinations of anticancer drugs using E . coli as the microbial test system. Wheeler et al. (1956) have published the results of some of these studies and Table VII lists examples of some of the results obtained. The usefulness of microbial studies in the instances of positive correlation is obvious, and the examples of negative correlation might be reduced if the potentiating combinations in bacteria were tested against other tumor systems-particularly tumor systems that are inhibited by one or the other drug in each combination. It is of particular interest to note that the potentiating activity of combinations of deoxypyridoxine and acid hydrazides was first detected in microbial systems, and they have been subsequently shown to be highly active against Sarcoma 180 in mice (Brockman et al., 1956). Active potentiating combinations of 6-mercaptopurine plus amethopterin (Skipper et al., 1954) and 8-azaguanine plus amethopterin (Law, 1952; Skipper, 1953) against Leukemia L1210 have been reported. The combination of azaserine plus 6-mercaptopurine is potentiating against Leukemia L1210 in mice (Skipper, 1954; Stock et al., 1955; Goldin and Mantel, 1957) as is the combination of azaserine plus 8-azaguanine (Skipper, 1954). Azaserine or DON plus 6-mercaptopurine, 6-methylmercaptopurine, 6-chloropurine, or thioguanine are potentiating combinations against the RC mammary carcinoma in mice (Tarnowski and Stock, 1957) and azaserine plus thioguanine is a potentiating combination against Ehrlich ascites carcinoma and TA3 ascites carcinoma in mice (Sartorelli and LePage, 1958). Probable potentiation of azaserine plus 6-mercaptopurine against Sarcoma 180 has been reported (Stock et al., 1955; Sartorelli and LePage, 1958). Unequivocal potentiation of azaserine plus 6-mercaptopurine in the treatment of leukemia or solid tumors in man has not yet been shown. In addition to these obvious positive correlations between potentiation of drugs in combination in microbial systems and mammalian tumor systems, data have been reported suggesting potentiation between amethopterin plus 2,4-diamino-5( 3',4'-dichlorophenyl ) -6-methyl-

246

FRANK M. SCHABEL, JR., AND ROBERT F. PITTILL0

TABLE VII EFFECTIVENESS OF COMBINATIONS OF DRUGS AGAINST E . coli AND SOMEEXPERIMENTAL TUMORS COMMONLY USEDIN SCREENING FOR ANTICANCER AGENTS

Comhination Sulfanilamide plus: Deox ypyridoxine Adenine sulfate Daruprimc 6-Mercaptopurine Hypoxanthined Dr,Ethionine 3-( 1,2,4-Triazolyl)alaninc Azaserine. plus: 6-Mercaptopurine 3-(1,2,4-Triazolyl)alanine 2,6-Diaminopurinc 1l)eoxypyridoxine

lhraprim plus: Canavanine sulfated 2,6-Diaminopurine Sulfanilamide Deoxypyridoxine plus : 3-(1,2,4-Triazolyl)alanine DbEthionine Sulfanilamide Bensoic acid hydrazide Isonicotinic acid hydraside p-Aminobenzoic acid hydraside 1,5-Diaminobiurct Azaserine

3-(1,2,4-Triazolyl)alanineplus: 6-Mercaptopurine Canavanine sulfated Dcoxypyridoxine Adenine sulfate 6-Chloropurine 2,6-]>iaminopurine

Potentiating against E. coli

+ + + + + + + f

+ + + + +

+

Potentiation of anticancer activitya Positive

Negative

L1210b

Not tested L1210b Sa 1 8 O b L121P Sa180'

L1210b

L1210b Sal80b Sa180b L1210b

L1210 Sa180 L1210 Not tested Ad756 L1210 Sa180 L1210 Not tested 1,1210 Sa 180 Sa 180 Sa180 L1210 Sal80 Sa 180 Sa 180 Sa180 Ad755 Sa180

L1210

L1210 L1210 Sa180 Not tested Not tested Not tested

247

MICROBIAL TOOLS FOR ANTITUMOR RESEARCH

TABLE VII (Continued)

Combination 3-(1,2,4Triazolyl)alanine plus (cont.): 5-Bromouracil Azaserine 2-Thiazolealanine Sulfanilamide Streptomycin sulfate plus: 6-Mercaptopurine Dithiouracil Azaserine

Potentiating against E . coli

+ + ++ + + +

Potentiation of mticancer activity Positive

Negative

Not tested L1210 Not tested Not tested Not tested Not tested Not tested

' The experimental neoplasms used were Leukemia L1210, Sarcoma 180, Adenocarcinoma 755. * In tests of anticancer activity, amethopterin was used instead of sulfanilamide. 2,4-Diamino-5-p-chlorophenyl-6-ethylpyrimidine, Calculations were based upon a subinhibitory level of this compound and, therefore, the combination is perhaps much more active than figure in table indicates. a 0-Diazoacetyl-L-serine,

pyrimidine against Leukemia L1210 ( Nadel and Greenburg, 1953). Since 2,4-diamino-S-( 3',4'-dichlorophenyl ) -6-methylpyrimidine is closely related to Daraprim, this example probably can be logically considered to be positive correlation with the potentiating combination of sulfanilamide plus Daraprim against E. coli (Table VII). Clarke et al. (1954) have reported probable potentiation of streptomycin plus thioguanine against Sarcoma 180 which could be considered as a positive correlation with the potentiating combination of streptomycin plus 6-mercaptopurine against E . coli (Table VII). Lowery and Foster (1959) have reported results of the screening of culture filtrates in combination with some known anticancer agents, and also combinations of culture filtrates, for potentiation of microbial inhibition. In both cases potentiating combinations were observed. No data were presented to establish positive or negative correlation between the potentiating activity of these combinations in microbial systems and mammalian tumor systems, but the growing interest in and potential importance of antibiotics as anticancer agents of possible usefulness suggests that this method of searching for potentiating combinations should be utilized much more extensively than it has been to date. It is in the area of drug resistance that the most striking and probably most significant positive correlation between observations on anticancer

248

FRANK M. SCHABEL, JR., A N D ROBERT F. PIITILL0

drug activity in microbial systems, animal tumor systems, and human tumors has been seen, An understanding of the underlying basic mechanisms involved in the development of drug resistance by cancer cells is probably the most important single problem facing the cancer chemotherapy investigator. In the treatment of lymphatic leukemia of man it is possible that cures would have been obtained by this time were it not for the fact that resistance to the inhibitory action of temporarily effective drugs appears in the leukemic cell population as treatment continues. That similar drug resistance may appear in solid tumors of man under treatment with effective drugs, while not nearly as well established as with the leukemias, is nonetheless a likely possibility. Studies in microbial systems have contributed greatly to our current understanding of the mechanisms involved in the development of drug resistance to antileukemic agents and the positive correlation between observations in drug-resistant bacteria, drug-resistant animal leukemias, and drug-resistant human neoplastic cells has been striking. Brockman et al., have shown that resistance to purine analogs in microorganisms [ S . faecalis (Brockman et al., 1959a)l and in mouse neoplasms [Leukemia L1210 (Brockman et al., 195913; Brockman, 1960) and P388 (Brockman et al., unpublished observations)] is accompanied by decreased purine ribonucleotide pyrophosphorylase activity. It was further observed by Brockman that resistance to 6-mercaptopurine and to 8-azaguanine in growing bacteria was accompanied by cross resistance to these analogs and by loss of capacity to metabolize hypoxanthine, guanine, and analogs of these purines to the corresponding ribonucleotides, these enzymatic conversions being readily demonstrable in the drug-sensitive lines from which the drug-resistant ones were derived. When these studies were extended to mouse neoplasms similar results were obtained (Brockman et al., 1959a, b; Brockman, 1960). The main pathway by which purines and purine analogs are converted to ribonucleotides is by reaction with 5-phosphoribosyl-l-pyrophosphate (PRPP) (Korn et al., 1955; Kornberg et al., 1955). A study of the purine ribonucleotide pyrophosphorylase capacity of enzyme preparations from bacteria and mouse neoplasms revealed that a specific decrease or loss of guanylic and inosinic acid pyrophosphorylase activity accompanied resistance to 8-azaguanine and 6-mercaptopurine while adenylic acid pyrophosphorylase activity was not significantly altered. Dr. George Kelley of these laboratories has isolated in cell culture a 6-mercaptopurine-resistant line of H.Ep-2 cells, a human epidermoid carcinoma originally isolated from man in cortisone-treated hamsters by Toolan (1954) and adapted to cell culture by Moore et al. (1955). When Brockman et al. (1961) compared the enzymatic activity of the line of

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6-mercaptopurine-resistant human cancer cells with the 6-mercaptopurine-sensitive parent line, he found that the drug-sensitive line reacted guanine, hypoxanthine, 8-azaguanine, and 6-mercaptopurine with PRPP to yield the corresponding nucleotides while identically prepared enzyme preparations from the 6-mercaptopurine-resistant line did not catalyze formation of ribonucleotides of these same purines and purine analogs. Adenylic acid pyrophosphorylase activity was high in the 6-mercaptopurine-sensitive and -resistant lines of H.Ep-2 cells ( Kelley, unpublished observations), Similar losses in enzyme activity have been seen in microorganisms resistant to pyrimidine analogs. A 6-azauracil-resistant S. faecalis apparently lost a nucleoside phosphorylase present in the drug-sensitive line ( Handschumacher, 1957). The 6-azauracil-resistant line of bacteria was still sensitive to 6-azauridine (Welch, 1959). As with resistance to 6-azauracil, resistance to 5-fluorouracil has been associated with inability to anabolize the base. Reichard et al. have reported that enzymatic preparations from 5-fluorouracil-resistant Ehrlich ascites carcinoma cells were unable to convert uracil to the ribonucleoside and hence were unable to form uridylic acid (Reichard et al., 1959). Pasternak et al. have observed that mouse Leukemia L5178, resistant to 6-azauridine, growing in cell culture had markedly reduced ability to convert uridine or 6-azauridine to uridylic acid (Pasternak et al., 1961). Reduced capacity for uracil and 5-fluorouracil anabolism has been seen in 5-fluorouracilresistant lines of mouse Leukemia L1210 (Reichard et al., 1959) and the mast cell neoplasm P815 (Brockman and Law, 1960) as well as in a 5-fluorouracil-resistant line of E . coli (Brockman et al., 1960). A great many additional data have been collected using microbial systems, mammalian tumor systems, and mammalian tumor cell culture systems in studies on mechanisms of action of anticancer drugs and mechanisms of resistance to anticancer drugs. While these data have not always shown complete positive correlation between microbial and mammalian systems, the degree of positive correlation observed strengthens our view that microbial systems can be very useful as models for an undetermined but probably significant number of mammalian tumor systems. A comprehensive review of recent studies on the enzymatic aspects of resistance to purine analogs, pyrimidine analogs, and other types of antimetabolite anticancer agents has recently been presented by Anderson and Law ( 1960). Table VIII illustrates the striking positive correlation between the enzymatic activity of purine-analog-sensitive and -resistant bacteria, mouse leukemic cells, and human cancer cells in cell culture. Decreased pyrophosphorylase activity accompanying resistance to

COMPARISON OF

THE

TABLE VIII RELATIVE CAPACITIESFOR ENZYMATIC CONVERSION OF BASETO RIBONUCLEOTIDE BY SENSITIVE AND PURINE ANTAGONIST-RESISTANT BACTERIA AND MAMMALIANNEOPLASTIC CELLS Base

Enzyme source A. Bacterial (I)* S. faecalis/Sensitiveh S. faecalis/6-Ptlercaptopurine resistant S. faecalis/&Azaguanine resistant B. Mouse Neoplasms (2, 3)a 3 LIZ10/Sensitiveb L1210/6-Mercaptopurine resistant LlZlO/GThioguanine resistant L1210/&Azaguanine resistant P388/Sensitiveb~ P388/&Azaguanine resistantc C. Human Neoplasm (4)H .Ep-2 /Sensitiveb," H.Ep2/6-Mercaptopurine resistantb

+ 5-phosphorihosyl-1-pyrophosphate

+ rihonucleotide

GMercaptopurine

Hypoxanthine

S-Azaguanine

Guanine

hdenine

100 0 0

100 0 0

100 0 0

100 2 2

100

100 4 4 4

100 8

100

100 94

14 6

6

100

100

0 100

2

0

0

100 25 33 7 100 4

100 0

100 0

100 0

100 0

100

0

100

138

128 114 100 93 100

(1) Brockman et al., 195%, ( 2 ) Brockman et al., 1959b, (3) Brockman, 1960, ( 4 ) Brockman et al., 1961. Conversion of base to nucleotide by the drug-sensitive bacterial or mammalian cells is expressed as 100; nucleotide formation by the drug-resistant sublines is expressed relative to that of the parent line. The observations on the enzymatic activity of these mammalian cells were made upon cells grown in cell culture.

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purine analogs has recently been extended to Salmonella typhinturium resistant to 6-mercaptopurine, 8-azaguanine, and 2,6-diaminopurine (Kalle et al., 1960), to Diplococcus pneumoniae resistant to 8-azaguanine (Brockman et al., unpublished observations ), and to E . coli resistant to 2,6-diaminopurine ( Remy and Smith, 1957).The results of Brockman with S. fmcalis have been confirmed by Salser et al. (1960). Decreased pyrophosphorylase activity in other purine antagonist-resistant mammalian cells has also been reported, e.g., mouse leukemic cells resistant to 2,6-diaminopurine, 8-azaguanine, and 6-mercaptopurine (Lieberman and Ove, 1960) and Earle's Strain L mouse fibroblasts resistant to 6-mercaptopurine (Tomizawa and Aronow, 1960).

VI. Discussion a n d Future Considerations '4s mentioned, cancer in both man and animals is not a single disease but a host of diseases. The common characteristic of all malignant neoplasms is uncontrolled growth of cells. From all other considerations cancer is a widely diverse biological phenomenon, be it site of origin, cell type or types involved, pathogenesis, natural history, or response to physical or chemical agents. Great advances have been and are being made ( a ) in our understanding of some causes of cancer with the promise of being able to reduce cancer incidence by removing or controlling these causative factors and ( b ) in treatment by surgery and ionizing radiation and supportive post-surgical treatment. The hard fact remains that cancer is currently the second leading cause of death in man in the United States, and its ranking in mortality statistics will probably remain static or rise as the mean age of the population advances due to further control of infectious and other diseases. Since metastasis has occurred in between one-half and two-thirds of human cancer cases at the time of first detection, these cases never come under the possibility of control by surgery or ionizing radiation. Research designed to shed knowledge on cancer induction is being diligently pursued, but at present it appears that successful prophylaxis is not imminent. To the partisans of chemotherapy, and we count ourselves among them, it appears that the development of drugs that will selectively destroy cancer cells, wherever they may be in the body, without unacceptable harm to vital normal cells offers the greatest promise for successful control of the cancer cases beyond treatment at time of first detection. Successful biological research efforts demand: ( a ) inductive reasoning to establish reasonable hypotheses and ( b ) usable experimental tools to put the hypotheses to objective test. Searching for anticancer agents in human cancer patients is a practical impossibility for a variety of reasons. Tumor-bearing experimental animals have been the test systems of choice

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in researches for and evaluation of anticancer agents. While direct positive correlation between observed anticancer activity in tumor-bearing experimental animals and human cancer patients has been demonstrated in only a few instances, we believe that it is likely that a chemotherapeutic agent that would cause regression and cure of an established tumor in experimental animals might also be effective in the treatment of one or more malignant neoplasms in man. Screening for effective new anticancer agents and evaluation of congeners of known anticancer agents against a variety of experimental animal tumors is demanding of time, laboratory space, technical labor, money, experimental animals, and the drugs being tested. As a result in vivo screening for new agents and extensive evaluation of known agents must, of necessity, await the availability of in Vzvo procedures. The possibility that in vitro procedures using either microbiological or mammalian cell culture systems might be useful in searches for and evaluation of anticancer drugs has been the point of departure for a number of different investigations. In this presentation we have considered microbial systems as tools for use in searches for and evaluation of anticancer agents. As primary screening tools microbial systems have not been extensively evaluated. Historically, the biological inhibitory activity of some of our most useful anticancer agents was first detected in microbial systems (see Section 11), and studies with representatives of the major classes of known anticancer agents indicate that a relatively limited spectrum of bacteria would select most of the known agents with a considerable (but still acceptable for primary screening) number of false-positive and falsenegative results (Foley et al., 1958). The unanswered question is whether or not microbial screening might select materials useful against human tumors that do not respond to currently known agents. Studies supported by the CCNSC to attempt to answer that question are currently in progress, Unquestionably microbial assay of new antibiotic antitumor agents has been very useful in the isolation and purification of those now known. Attempts to isolate anticancer antibiotics using in vivo animal tumor assay systems, while possible, are extremely difficult due to the physical requirements of the assay system, the quantitative demands of the assay samples required, and the lack of quantitative precision of most experimental in vivo tumor systems. The use of antitumor-agent-resistant microorganisms as tools for preliminary identification of new anticancer agents and of drug-sensitive or drug-resistant bacteria for gaining information concerning possible sensitive metabolic sites inhibited by new anticancer agents has great potential. In addition, the relative ease with which anticancer-agent-resistant bacteria can be isolated, as compared to anticancer-agent-resistant cancer

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cells in vitro or anticancer-agent-resistanttumors in uiuo, makes such bacteria very useful in searching for drugs selectively inhibitory for drugresistant tumors, The problem of correlation between the response of bacteria and of mammalian tumor cells to anticancer drugs is one of paramount importance that cannot be unequivocally resolved on the basis of current knowledge. That excellent positive correlation exists concerning the mechanism of resistance in purine-antagonist-resistantbacteria, mouse leukemic cells, and a line of human cancer cells in cell culture has been firmly established and similar correlation between bacteria and animaltumor cells resistant to pyrimidine-antagonists anticancer agents is indicated. In addition, the observed positive correlation between bacteria and animal tumors in vivo in response to possible potentiating combinations of anticancer drugs strongly suggests that bacterial systems may be useful in vitro tools with which to search for such drug combinations for use against tumors. In vitro studies in microbial systems, either in searching for new anticancer agents or characterizing known anticancer agents, are economical of time, space, labor, money, and drug; and, in addition, they are quantitatively reproducible as compared to in vivo studies. We have not considered virus systems in this discussion, since not enough data are available, especially in relation to chemical control of virus-induced tumors, to allow critical evaluation. A number of cancers of animals are known to be virus induced, and the possibility that virus association, if not indeed virus induction, may be important in the human disease seems a likely possibility as our knowledge of cancer grows. The possibility, therefore, of using viral systems in screening for anticancer agents may shortly be under consideration. A number of laboratories are currently searching for antiviral drugs and, whenever possible, any leads turned up in such studies should be evaluated in experimental tumor systems. Asheshov et al. tested some standard anticancer drugs as well as other inhibitors against a wide spectrum of bacteriophages (Gellhorn and Hirschberg, 1955), and their results showed that the individual bacteriophages responded quite differently to the standard agents, even among those having a common bacterial host. The utility of bacteriophages as test systems for searching for and evaluating anticancer agents has not been established, but such systems should be borne in mind if viral association with mammalian malignant neoplasms continues to grow in importance and interest.

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The Classification of Actinornycetes in Relation to Their

An tibiotic Activity ELIOBALDACCI Institute of Plant Pathology, University of Milan, Italy

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... 257 11. The “Formation of Antibiotics” as a Taxonomic Characteristic. . . . . . . . . . . . 258 111. Rules of Nomenclature ............................................ 265 IV. General Lines for Classifying.. ...................................... 270 A. Genus in the Streptomycetaceae Family ........................... 270 B. Subgenus and Series in Streptomyces.. ............................ 271 C. Species and Infrasubspecific Taxa in Streptomyces. . . . . . . . . . . . . . . . . . 271 V. Summary and Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

1. Introduction The increase in species, consequent to the study and the discovery of antibiotics produced by actinomycetes, has brought the microbiologist’s attention to the criteria and the methods of formation of species or speciation, whichever one may wish to call it. The question used as a starting point in many reviews of the present situation, as far as the classification of actinomycetes is concerned, is this: how can one determine a species, or, what is a species? It must be observed immediately that a question of this kind has never been codified for any category of microorganism and this is because the criteria for the formation of the species cannot be given a priori, but can only be worked out from one time to the next or almost “traced down” by the investigator on the basis of his knowledge of the microorganisms with which he is working. Thus the classifications are subject to change and to modification continually with the passage of time and with the changing of ideas which are had of the various categories of microorganisms. The rules of nomenclature, which is to say the language itself used for the classification, have been codified and are not subject to arbitrary variation by the investigator, These rules must be observed by everyone, independent of the category of microorganisms on which the investigator is working. It is for this reason that an International Code of Botanical Nomenclature and one for Zoological Nomenclature has been in existence for some time. The microorganisms, since they be257

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long to one of the two Kingdoms of nature, should have been classified according to one of the two codes. But microbiologists have found themselves faced, more than once in the past and also recently, with problems of classification in forms and conditions so different from those encountered by botanists and zoologists that it is not always easy to find a way to apply the rules of nomenclature established by these codes. These legitimate difficulties suggest the creation of an International Bacteriological Code of Nomenclature, following in the footsteps of the models which are already in existence but imposing some rules which have not been provided for or overlooking other rules not applicable to microorganisms. The first drawing up of the Bacteriological Code of Nomenclature has been approved by the 4th International Microbiological Congress in Copenhagen, Denmark in 1947 (Buchanan et al., 1948). After several revisions, an edition in book form was published in 1958 with the title “International Code of Nomenclature of Bacteria and Viruses.”

II. The “Formation of Antibiotics” as a Taxonomic Characteristic Consider the question we must ask ourselves if we wish to use the characteristic “formation of antibiotics” for classifying actinomycetes: “in which subdivision or category of taxonomic groups (taxa) would we place them?.” And, more specifically, can we use this characteristic to determine the species? The Round Table Conference on Streptomyces at Stockholm (Kiister, 1958) [not without some comments on the part of Krassilnikov and the writer (Baldacci, 1958)] rejected the formation of antibiotics for a characterization of species. In the records of the meetings, the reasons for this rejection are listed in summary form as follows (Kiister, 1958) : ( a ) This property is not considered constant, because a loss of antibiotic formation can be frequently established under laboratory conditions; ( b ) there are numerous species of Streptomyces which contain inactive strains as well as producers of antibiotics; ( c ) in several cases antibiotics were formed from only one strain; ( d ) the same antibiotic can be formed by different species. Similar arguments were also brought up at the Round Table Conference on Taxonomy of the Actinomycetes at Chicago in 1958, where, nonetheless, the opinions were also in disagreement. To summarize, these objections raised against the use of the characteristic “formation of antibiotics” for the characterization of species are the following: ( a ) the variability of the formation of the antibiotic in respect to different culture conditions of the producing strain; ( b ) the fact that one and the same antibiotic can be formed by “species” which had previously been established on the basis of different diagnostic characteristics.

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These objections do not seem to be either sufficient in themselves nor clearly enough formulated to be used as a basis for rejecting the use of the above characteristic. As regards the variability of antibiotic formation, the following remarks could be made, It is quite evident that a characteristic should be constant in order to be used for classification, that is, it should appear identical to all investigators under the same culture conditions. That is why taxonomists have preferred those characteristics which vary little or not at all under different conditions of observation when the characteristics to be used for classification are chosen. But it is well known that bacteria show considerable variability in their characteristics during their cultivation and, for that reason, a different, distinct code of nomenclature was set up. Then, as far as the actinomycetes are concerned, other characteristics have been used in their classification and are still being used, the variability of which could also be examined. For example, the so-called pigment or soluble color of the substrate has been widely used in various classifications, but it is very doubtful whether this characteristic is constant and whether its loss or decrease is not often observed to occur under laboratory conditions. It is for this reason that the characteristic was rejected at the Round Table Conference in Stockholm. Its use as an element in classification was limited to the appearance of a “melanoid” color in certain substrates. In the same way, the aerial mycelium show variability in their coloring, as do the vegetative mycelium, in relation to the substrates of the culture, the pH, and the temperature. Morphological characteristics, for example, relating to the structure of the sporophores, also vary under observation. In this respect, the results of a project which has up to now remained unpublished, performed, with colleagues including 36 investigators throughout the world, on strains distributed by the writer after the Stockholm conference, could prove this point. The author wishes now to make known some of the results. For example, the color of the aerial mycelium to be classified within pre-established color categories has been recognized, for the same strain, by 36 experimenters as being gray 48 times, white 39 times, yellow 2, orange 1, pink 9, red 2, brown 6, on a substrate common to all investigators, This observation is in no way an isolated instance1 As far as the structure of the sporophores is concerned, one strain has been evaluated 58 times as spira, 8 times as rectus, 14 as rectus-flexibilis, 10 as rectinaculum-apertum, 7 as monoverticillatus-spira, 2 as biverticillatus-spira. Of 25 strains, 14 were considered by all the experimenters to present all the possible forms of sporophore listed above. As a result, this characteristic, too, should be considered to be variable. Here we can bring out the point which we feel to be most pressing,

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namely, that no characteristic “alone” can characterize the species, in the case of actinomycetes, since no one characteristic has a perfectly equal constancy under all working conditions. From the work of the investigators given above it is to be concluded that only the shape of the spore under the electron microscope and the appearance of a melanoid color in a certain substrate (agar-peptone) are to be taken as absolutely constant characteristics for all investigators, This is really very little on which to base the formation of a species. Let us now come to the second objection, It is said that different “species” are able to produce the same antibiotic. More exactly, we should say that once the species have been classified according to other characteristics, namely, color of the mycelium, structure of the sporophore, etc., these species can then be considered producers of the same antibiotic, It would be sufficient to establish at the next convention that the diagnoses of species be made on the basis of the formation of a particular antibiotic, to have these objections removed. In other words, if we would place together all the producers of antibiotic A, then those producing antibiotic B, and so on, we would be able, within these “species,” to distinguish “subspecies” or infrasubspecific subdivisions for other characteristics (color of the aerial mycelium, structure of the sporophore). This is a purely conventionalist thesis, and it is surprising that a scholar such as Krassilnikov (1959) should consider it as an objective thesis, based on a supposed genic stability of the formation of the antibiotic. Krassilnikov (1960) has gone so far as to want to formulate rules for the classification of antibiotic-producing actinomycetes. But it is plainly evident that all the actinomycetes must be classified independent of the fact that some of them produce antibiotics. Formation of an antibiotic may constitute a characteristic to be taken into consideration when classifying “all” the actinomycetes. It is not understood why Krassilnikov should have made an appeal to undertake “linguistic” agreements among investigators in all countries; these linguistic agreements are already in existence and, in this respect, we shall discuss them in the third section of this work. The only rules to follow are those of the Code of Nomenclature and the reports of the experimental results of research need to agree with those rules. But, once again, it appears that the most important consideration in this respect is not yet clearly formulated. If we should wish to decide the “species” of the actinomycetes on the basis of the antibiotic formation, that is, if we wished to proceed in the above-mentioned manner, putting all the producers of antibiotic A in one species and in a second species, all those producing antibiotic B, etc., we would really run into serious difficulties, both of a formal and of a substantial nature. The first,

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more banal objection is that most of the presently known species turns out to produce more than one antibiotic: some of them produce three or four; sometimes even twelve or thirteen ( S . Zuvendzclue!). If, then, we were to want a classification on the basis of antibiotic formation, it would be necessary to nullify all that has been done up to now and, immediately afterward, make almost as many subdivisions for the “species” which produces antibiotic A as there are species known today and the same for the “species” producing antibiotic B, etc. It would be a labor of Sisyphus, without constructive results. Still another substantial objection against the use of “antibiotic formation” in the characterization of the species exists. Research for an antibiotic is research within the chemical domain and requires the special equipment of a chemical laboratory; it falls almost entirely within the competent authority of a chemist, It is not possible to make taxonomic science depend on chemical research and to make the classification of species depend on chemical analyses. Once more we could repeat here a phrase made by an illustrious taxonomist, Dr. C. Thom, who wrote in a monograph on Penin’llium, “A monographer is not necessarily a geneticist and not a cytologist.” More recently he clearly reaffirmed his concept, with which we entirely agree (Thom, 1954), “The taxonomist is a morphologist. If he is also a microbiologist, he must use a microscope. If his organisms are really small, the best apochromatic objectives should be regularly at hand and used. I know places where using any oil immersion objective is a unique circumstance. There is a limit, however. I read a paper the other day in which the worker reported that the only dependable character separating his two species appeared to be the length of the chromosomes. I am afraid that the ordinary working mycological laboratories will be compelled to dump those organisms together and perhaps hyphenate their species’ names.” In turn, this writer has read at length a work in which the investigators asserted that only three strains from among those collected all over the world could be referred to a certain species. In order to find this species, they had made extensive chemical analyses on the synthetic activity of the strains. It must be concluded that a monographer is not a chemist, and, returning to the actinomycetes, it must be stated that it would be impossible-for every strain which is traced down-to first study its formation of antibiotics and then to proceed with its classification. The contradiction to be found in this statement is so plain that it need not be emphasized. It is possible, then, to state that a classifkation which places the characteristic “antibiotic formation” at the species level contradicts our work as taxonomists, which aspires toward making an order in the

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nomenclature of the great variety of living organisms. It could more simply be considered that a classification for antibiotics would be no more than a disguised classification of chemical substances and not really a classification of organisms; moreover it would be entirely insufficient. It is not within the scope of our authority to criticize the present classification of the antibiotics, but it is permissible to point out its difficulties and its incompleteness. The classifications made constitute useful work toward clarifying the synthetic activities of microorganisms, but they demonstrate a one-sidedness so far as the purposes which we are here discussing. The classifications presented, for example, in the works of Goldberg (1959) or of Szemiakin and Koklov (1953),consist of groupings of the antibiotics within categories of chemical compounds, but these categories do not turn out to be related to one another by any particular characteristic. Instead, it becomes obvious that it would be very difficult to place antibiotics in the much greater and more extensive classification of organic substances. In the work of Korzybski and Kurytowicz (1959), in which the authors have tried to reunite perhaps the greater number of known antibiotics, the classification is rendered invalid at the beginning by a microbiological distinction, in that one starts by distinguishing first on the basis of the “taxonomic genus” of the producing organisms ( antibiotics produced by Penicillium, antibiotics produced by Actinomyces, and so on). A classification takes place within this framework which groups categories of antibiotics on the basis of their chemical composition along with categories created on the basis of their action on certain microbes, for example, polypeptide antibiotics and antibiotics acting on acid-resistant bacilli. That is to say, one proceeds with heterogeneous categories which have no related characteristics for classification, but which are only change groupings. These remarks have, as their purpose, simply an exposition of the difficulties in classifying antibiotics and do not constitute a criticism of the investigators who have gathered together the present knowledge of these substances in valuable and useful works, These difficulties appear even more evident if it is postulated that chemical ideas be applied to antibiotics for the purpose of biological classification. Actually, those who are working on research in the antibiotic field do not raise questions of classification, nor do they even generally have co-workers who are engaged in the taxonomic field. Rather, these investigators are particularly interested in another phase of the research; often the research is carried out on a single strain or on a few strains which are thus studied thoroughly and profoundly under specific, predetermined conditions, and it is difficult to obtain the same information for other strains. In that way, the researcher who has in-

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vestigated “his own” strain so thoroughly is prompted to distinguish it from others, perhaps for no other reason than for the particular study which he has made. In this way the vicious cycle is created about which there is so much complaint nowadays; thus we have the increase in the number of species of actinomycetes which is so extraordinarily bound together with chemical research on antibiotics. In a previous work (Baldacci, 1956), the author illustrated the results of a classification by Dr. A. Di Marco, carried out according to the type of metabolism from which the substances of each single group have seemingly taken their origin. This classification, which offers considerable advantage in that it refers back to the metabolic activity of the microorganism and that it agrees with the general criteria of biological research, has turned out to be insufficient, nevertheless, for the purpose of tracing down a useful relationship between the classification of the actinomycetes and that of the antibiotics. The conclusion which can be drawn, upon due consideration of the above, is quite precise, but is not exclusive. Formation of an antibiotic is not a characteristic which can be used for the classification of species of actinomycetes. But we shall show the existence of numerous taxonomic categories lower than that of species and we shall present evidence to the effect that these categories have originated just for the very reason that it is difficult to include in a species microorganisms which have numerous and very particular characteristics. It is, furthermore, a fact observed by many investigators that in the “species” which have been formulated using other criteria (morphological and cultural) inactive strains are found, that is, strains which do not produce antibiotics, or have even a single strain which produces an antibiotic; thus, it could be said that such a characteristic may be suitably used at the subspecific level, The characteristic, “antibiotic formation,” does not have to be completely rejected for use in classification, but, instead, it must be concluded that this characteristic may be used at a taxonomic rank below that of species, for the reasons examined above, In some previous works (Baldacci, 1956; Baldacci et al., 1955), the author gave several examples of this criterion. But this solution is difficult to accept for reasons which affect, but which are not directly involved with, scientific research. The existing regulations in various countries are bound by patenting of strains producing antibiotics only if the strain is recognized as a “new species.” In this way there is an inevitable push toward the formation of species in order to be able to protect a productive output for industrial use. But the legal defense or the protection of a strain could also be achieved at taxonomic ranks lower than that of species, since these are already recognized by microbiologists in their own specific

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Code. As we shall bring up later, it could also serve as a factor which could be used as an example to make it easier to change the existing legislation on the subject of patents for the microbial production of antibiotic and other substances. Some investigators, desiring to avoid or to get around the difficulties of chemical research pointed out above, have proposed, as has Kutzner ( 1958), that the spectrum of antibiotic activity on determined microbes tested be used instead of a knowledge of the chemical nature of the antibiotic, This examination can be performed readily without having to make long chemical analyses, but its effectiveness for diagnostic purposes is limited. Kutzner concluded that the results of such an investigation would have served for the classification of subspecific ranks. The spectrum can be brought about by the same antibiotic or, as the case may be, by the same mixture of antibiotics and, furthermore, this method also offers difficulties for purposes of standardization like other tests of a biochemical nature, for example, carbon compound utilization, peptonization of milk, etc. Nevertheless, this proposal represents a reasonable inclination to use some of the ideas regarding antibiotic activity on the part of the taxonomist. Finally, another factor must be taken into consideration in limiting the use of the characteristic “antibiotic formation’* to subspecific ranks; it is that of natural and induced variability of the actinomycetes. In agreement with Jones (1954), we can distinguish the variations in actinomycetes as temporary or as permanent variations. Temporary variations can be attributed to the direct effect produced by the environment, to the age of the spore, to the density of the inoculation, to the effect of the substrate of culture when it is the same for a long period of time, and so on. These can be referred to as natural variability, which depends on the conditions under which the microbiologist works. The permanent variations, which are also called mutations, are rare under normal culture conditions, but they can be more readily obtained when the cultures are subjected to special treatment, such as X-ray, ultraviolet radiation, chemical agents, etc. These are of particular interest in the case of the actinomycetes because they produce considerable and peculiar modifications in the formation of antibiotics, and treatments such as those mentioned above have been widely used for such purposes. It is therefore legitimate to suspect that many of the “new species” which have been brought to light in these last few years are “species” as the result of artificial inducement. There is a great deal to be learned scientifically in knowing the morphological characteristics of species which have given origin to highly active strains or which have produced new antibiotics, although apparently no correlations have been found

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between morphological type and antibiotic yield. But there is just as much interest involved in defending the “species” obtained by artificial induction, if it produces a considerable quantity of antibiotic or has a new type of antibiotic activity. Here the contrast between the requirements of industry and those of pure science are once more repeated. This contrast could be reduced or eliminated if the legitimate protection of the formation of antibiotics could be obtained at subspecific ranks. The “species” are established on the basis of what is prevalently or entirely their morphological characteristics and the variations or mutations obtained in the field of biochemical activity could be made known if they were adequately protected by patent laws by changing the existing laws as indicated. A different problem, though, is that of morphological variations produced by the above types of treatment, Few investigations have been carried out at the present time using systematic criteria. This subject requires attention and will be discussed. It should certainly lead to better knowledge of the genetics of the actinomycetes and their life cycle and this would also help to clear up the choice of characteristics which would be the most suitable for purposes of classification. But this is another matter, not to be dealt with at this time.

111. Rules of Nomenclature In relation to the problem under investigation, it will be well to bring up some of the general considerations used to introduce the Code. General consideration No. 1 states, “The progress of bacteriology can be furthered by a precise system of nomenclature which is properly integrated with the systems used by botanists and zoologists and accepted by the majority of bacteriologists in all countries. The bacteriological Code applies to bacteria, related organisms and the viruses. Botanical and Zoological Codes provide for the nomenclature of certain other microbial groups such as the yeasts and fungi, algae and protozoa. The special nomenclatural problems of these groups require cooperation with zoologists and botanists.” This consideration makes it evident that every advance in solving the problems of classification should be placed in a fundamental relationship to a precise system of nomenclature and that this system must be respected. As to the suitability of grouping together bacteria and virus in the same code of nomenclature, the author is not in agreement with this, but that is beside the point. I now refer to general consideration No. 3, “Provisions for emendation of rules, for special exceptions to rules, and for their interpretation in doubtful cases have been made through the establishment of an International Committee on Bacteriological Nomenclature for the Inter-

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national Association of Microbiological Societies and of its Judicial Commission.” This sanctioned the existence of the forum to which was referred every controversy arising from questions of the application of the rules of nomenclature, The Nomenclature Committee had been organized in 1930; the Judicial Commission was set up in 1939. The Nomenclature Committee has appointed several subcommittees to consider the problems of certain categories of taxa. One of these is the Subcommittee on Streptomyces, presided over by Dr. D. Gottlieb of which subcommittee the author is also a member (Anonymous, 1959). The existence of a Bacteriological Code of Nomenclature alongside that for Botanical Nomenclature raised a legitimate question after the Round Table Conference on Streptomyces in Stockholm in 1953, namely, which of the two codes should be used to classify the actinomycetes. It is evident that this question raises the further question of whether the actinomycetes belong to the order of bacteria or to fungi. My opinion in this respect is as follows: up to now the actinomycetes have been considered to belong to a category of taxa together with bacteria, even though it was recognized that there were aspects approaching those of fungi. Thus, now that a Bacteriological Code of Nomenclature has been established, it is legitimate to say that one should be obliged to follow the latter. In fact, at the Symposium of the Actinomycetales in Rome (Baldacci et d.,1953) the author called the attention of those present to these and other problems relating to the nomenclature of the various genera and species of actinomycetes, commenting on and comparing the rules which had been established and sanctioned for the various cases involved in the two codes. These observations did not lead to any action for the simple fact that microbiologists at that time still were not interested enough in problems of that kind. Recently such problems have been taken up again in an exchange of letters with Dr. R.E. Buchanan, Chairman of the Judicial Commission. In his turn, Dr. D. Gottlieb, Chairman of the Subcommittee on Streptomyces, has raised the above-mentioned question to Dr. R. E. Buchanan, and in turn to Dr. D. P. Rogers and Dr. C. W. Hesseltine. But the answers have not been unanimous and this has obliged Dr. Gottlieb to consult with all the members of the Subcommittee. The answers are now under study. THE CATEGORIES OF TAXONOMIC GROUPS(TAXA) The Bacteriological Nomenclature Code sanctions the existence of precise categories of taxa, “The terms which denote the rank of taxonomic groups (taxa) are defined as follows: ( a ) Every individual is treated as belonging to a number of categories of consecutive rank and consecutively subordinate; of these the species is the basic one” (Principle No. 7 ) . The

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categories of taxa that are recognized in the Bacteriological Code may be summarized as follows: 1. Division (Divisio) 2. Subdivision ( Subdivisio) 3. Class (Classis) 4. Subclass ( Subclassis) 5. Order (Ordo) 6. Suborder ( Subordo) 7. Family (Familia)

8. Subfamily (Subfamilia) 9. Tribe (Tribus) 10. Subtribe (Subtribus) 11. Genus (Genus) 12. Subgenus (Subgenus) 13. Species (Species)

The categories below the species level will be discussed later. It should immediately be pointed out that there have been considerable concessions made to the fact of the existence of taxonomic levels which are intermediate (sub . . . ), and this concession must be understood as a working concept in the classification. The purpose of the classification is to put a certain number of lower units within the ranks which have been so arranged and in these alone; in the genus category one would place a certain number of species, while a certain number of genera would be placed in the family category, etc. Principle No. 8 clearly affirms, “The primary purpose of giving a name to a taxonomic group is not to indicate the characters or the history of the group, but to supply a means of referring to it.” Thus the purpose of the classification is to give investigators the means for understanding one another; this is the reason why the Code does not go into the merits of the methods and criteria used for the formation of the ranks for the various microorganisms, but, instead, limits itself to giving the rules of nomenclature for all.

I. Inframbspecific Taxa As to the subdivisions of the species, one should refer to Rules Nos. 7 and 8 (Chapter 3 ) of the Code. Rule No. 7 states, “Names of subspecies (varieties) are ternary combinations consisting of the name of the genus followed by specific and subspecific epithets in order.” Rule No, 8 states, “Subdivision of species (other than subspecies or varieties) and subdivision of subspecies are given vernacular names or designated by numerals or letters or, in special cases, are given names in Latin form. These are termed infrasubspecific subdivisions or forms, The names given to an organism included in infrasubspecific subdivisions need not conform to the rules governing the naming of subspecies and higher taxa. These rules do not determine the naming of forms of infrasubspecific rank.” The infrasubspecific subdivisions are : strain, biotype, serotype, morphowe, phagotype, group, phase, form or forma specialis, variant, mutant, and stage (or state). As can be seen there is such an abundance as to leave no doubt as to the concept which inspired it; it is desired to

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leave ample freedom to the classifier to arrange the individuals in infrasubspecific subdivisions, keeping in mind the considerable difficulty involved for the microbiologist in this regard. For the purpose of investigations made on actinomycetes, it would be well at this point to call the reader’s attention to the term “group.” The Code has already used this word as having a general significance to designate the categories of taxa. Note 1 to Principle No. 5 states, “The use of word taxon (plural is taxa) is appropriate for the term taxonomic group or the word group wherever used in the sense of taxonomic group in this Code, The word group has as its preferred use in bacteriology that indicated in Recommendation 8A.” This recommendation states, “The term group in bacteriology should be used with great care and be well defined if ambiguity is to be avoided. It has been used in somewhat different senses by those working in various fields of bacteriology. ‘Group’ is used to designate congeries of organisms having common characteristics. In many cases the groups are based upon antigenic analyses, they are assemblages of related serotypes.” But those who have studied the classification of the actinomycetes have adopted the word “group” in another way, having still another meaning. They have, in fact, used it to mean the subgenera categories, that is, those between genus and species, in the sense of “group of species.” Waksman and Lechevalier (1953) write giving this meaning, gathering together the species under designations like Streptomyces antibioticus group, S . Zavendulae group, S . grism group, etc. The word “group” is used to have the same meaning by Flaig and Kutzner (1954, 1960). In the same way, investigators of the Zurich school (Ettlinger et al., 1956) use the term “group” to bring together various species of actinomycetes producing the same antibiotic or antibiotics. In the same way also Krassilnikov (1957, 1959) in his work dedicated to the study of the relationships between the antibiotic formation and classification, uses the word “group” as a combination of different species, although it does not appear clear just at what point he considers the group as infraspecific or infrageneric in rank. The same comment could be made with respect to the work of Kuchayeva ( 1958). In the cases above, one sees an example of an obvious lack of respect for the rules of the Code of Nomenclature, in that the correct terms in accordance with the Code have not been adopted. Let us now see what would have been the proper terminology to use in the above cases. 2. Subgeneric Tam In the categories of taxa listed between genus and species, there is only one intermediate category, the subgenus. As provided in the Botanical

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Code of Nomenclature, which, in its 1956 edition introduced five infrageneric ranks, named in descending sequence subgenus, section, subsection, series, and subseries, a similar proposal has been advanced for amendment of the Bacteriological Nomenclature Code to the Judicial Commission, When the author published (Baldacci et al., 1953) the first essay on the classification of the actinomycetes species, the terms sectio and series were adopted, according to the Botanical Nomenclature Code. Series was understood to be an intermediate subdivision which included species that could be grouped together for one characteristic or more. That term should have been used by the above-named investigators when, on reporting their findings, they put more than one species of actinomycetes together for diagnostic and taxonomic purposes. There are many reasons for accepting the above proposals for creating infrageneric taxa. Here we will put forth those which concern the actinomycetes, If, when diagnosing genera, a single determining characteristic is used, for example, the morphology under the optical microscope, separation of species becomes a complex matter because of their excessive number. Such is the case with the genus Streptomyces. There has been a large increase in knowledge concerning this genus because of antibiotic formation and other biochemical activities associated with it and, even more, so far as it concerns the ecology of this genus, its behavior under different culture conditions. Lastly, knowledge about the characteristics of the mycelium and the sporophore has been both improved and enlarged, along with that of the morphology of the spores through electron microscopy. To this rapid build up of knowledge can be added the fact that the knowledge acquired has been used in different ways for classificatory purposes, from one country to another and from one investigator to another, and this has inevitably created confusion. For example, knowledge about a given strain’s ability to form antibiotics was used to raise it to the category of species, while similar information has not been found for other “species” already existing and diagnosed using other factors, some of which were common to the “new species.” The extension of the diagnosis has not proceeded at the same rate and, quite to the contrary, there has been a tendency to cut it short. There are two printed pages for the diagnosis of Streptomyces albus Rossi Doria in 1891; there are ten or twenty printed lines for the diagnosis of the species in the work of Krainsky (1914) and, lastly, there are two printed lines for the diagnosis of the species S. rimosus (Finlay et al., 1950),the essence of which remains defined perhaps prevalently by the cultures which have been distributed, As a result, it becomes evident that we

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are being informed in an entirely heterogeneous manner about the “species” of the actinomycetes, but it should also be added that this situation is quite common in the history of many genera and many taxonomic matters in the botanical field, The problem today strongly affects those who study the actinomycetes because a criticism of the taxonomic characteristics of the object of their study has far-reaching effects among them. As a definitive statement, we would say that the need for infrageneric taxa of the kind proposed appears to be obvious. That is why we favor their use in accordance with the rules which have been codified.

IV. General lines for Classifying A. GENUSIN

THE

STREPTOMYCETACEAE FAMILY

In a recent work by Lessel (1960)) more than 200 names are given of genera which are connected with the nomenclature of the order of the Actinomycetales. Nevertheless, the names which have the closest relationship with the family of the Streptomycetaceae (Bergey’s Manual, 1948; “Actinomycetaceae,” Baldacci, 1941) are certainly smaller in number, even if more than a few. Their number has increased, and their characteristics have been made clearer and more precise during the last few years. This tendency should be encouraged, in my opinion, because the increase in genera will make it easier to classify the species which are too numerous and which crowd the Streptomyces genus. The characteristics used for the formation of genera refer to kinds of sporulation, and it will be better to keep to that single criterion in order to avoid an overlap of heterogeneous modes of classification. In one of the author’s previous papers (Baldacci, 1958), the key to the genera was thus presented: FAMILY: STREPTOMYCETACEAE

( A ) Spores produced on short sporophores 1. with a single spore Micromonospora Orskov 2. with two spores Microbispora Nonomura and Ohara = Waksmania Lechevalier et al. 3. with more than two spores ( a ) isolated or aggregated sporebearing hyphae Streptomyces Waksman = Actinomyces AA. p. p. ( b ) verticillate spore-bearing hyphae Streptoverticillium Baldacci ( B ) Spores produced in sporangia 1. nonmotile Streptosporangium Couch 2. motile Acthoplanes Couch

A brief comment should be added to the key. As I had personally written to Professor Waksman, and, as is written by Lessel, it is not pos-

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sible to give oneself the priority of publication which would give the right to a priority of use between the two names Microbispora and Waksmania. “The date of publication is customarily designated as the date on which the article wherein the name was proposed was placed in the mail or was otherwise made available to bacteriologists. Both Microbispora and Waksmania were published in August of 1957; however, the exact dates of publication of these names have not been definitely established, and which name has priority has not been determined.” Both publication dates, according to the writer’s investigation, are in August, but the American publication starts a new volume in that month. As far as the name Streptomyces is concerned, which replaced the name Adinomyces, for the events and interpretations dealing with the latter in Europe and in the United States, I would refer to one of my previous papers (Baldacci et al., 1953).

B. SUBGENUS AND SERIESIN Streptomyces Having pointed out that it is better to keep to the criterion of sporulation form, other characteristics remain available for the infragenerical names, subgenus, and also-since the acceptance of them can be considered certain-for section, subsection, series, subseries as proposed in Section 111. The author already has mentioned the reasons for his choice of the characteristic “color of the aerial and vegetative mycelia” for the section and series subdivisions. All workers have made use of this characteristic in the past and all of them presently do so, since every diagnosis, however brief or incomplete, never fails to give a description of the color of the vegetative mycelium during the first hours of growth, and of the aerial mycelium upon reaching maturity. The use of this characteristic therefore makes it possible to include a greater number inside the genus and perhaps even the whole totality of the species described and also makes it possible to subdivide them according to an easy characteristic which is both immediate and objective if a color atlas is used, as will be pointed out in more detail further on. As far as the suitability of using the characteristic “color of the vegetative mycelium” at the “section” rank is concerned, the reader is referred a published work (Baldacci, 1958) which specifies that vegetative mycelium must be taken to mean the mycelium which originated from the germination of spores. The color of this mycelium is observed on the very surface of the agar, and not on the reverse of the culture, 24 hours after incubation at 3OOC. (approximately). Research on the color of the vegetative mycelium similar to that done on aerial mycelium has been carried out in the Institute of Plant Pathology by Paulin (1960) using colorimeter reflection with a photoelectric Tristimulus colorimeter

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J 20 (Hilger, London) and also an Istophotometer, with two centrable diaphragms for studying absorption curves, for determination of transmission or absorption. The research confirmed the existence of colorless vegetative mycelium alongside colored mycelium; and, this confirms the suitability of using this characteristic to distinguish between two sections. If it is kept in mind that the investigators postulated a two-phase life cycle in Streptomyces, distinguishing in the vegetative mycelium and haploid mycelium (by supposition) the seat of the process of nuclear fusion, it appears that this mycelium is anything but negligible for the purposes of taxonomic classification. Let us now examine the aspects of morphological research on the two mycelia. It is true that the color of the mycelia can present itself differently according to the culture’s substrate, but it is also true that investigators indicate the substrate used each time. Thus when identifying a species previously described, a good method of working is to use the substrate used previously by whoever outlined the species in order to compare the strains being examined. Standardization of substrate is desirable, but the recommendation that synthetic substrate be used instead of those derived from vegetable or animal products (potato, oats, milk, meat, etc.) may only be partially justified by the availability of organic compounds which are uniform in all countries; standardization cannot constitute an exclusive criterion because the use of substrate of a special kind such as have been used in the past, as has already been pointed out, is necessary for the re-identification of the species, In addition, for the characterization of the species, it might be well to use “natural” substrate from time to time, In general, substrates that lack too many organic substances do not make examination easier because development of the actinomycetes turns out to be limited, vegetative mycelium is scarce, and sporulation is reduced. In place of potato agar which was criticized because it is of undefined chemical origin, we have proposed using a Bacto-casein Czapek agar made by the Difco Laboratories (Baldacci et al., 195413). Research published later (Kuster and Grein, 1955; Grein and Kiister, 1955) added another contribution to the characteristics of potato agar substrate which was largely used by the writer in research on the cultivation of the actinomycetes. By using potatoes taken from ten different cultivars, with 100 strains of actinomycetes, the characteristics of the culture (color and development) did not vary for those potatoes which were from the early-harvest cultivars. Upon using late-harvest cultivars, it was observed that the soluble color of the substrate (the so-called pigment) proved to be diminished in strength with respect to the same cultures using early-harvest potatoes. Other, less evident differences were noted in the color of the aerial

CLASSIFICATION OF ACTINOMYCETES

273

mycelium, One and the same potato cultivar does not give rise to differences in the behavior of the actinomycete cultures, whether they are harvested in Italy or in Germany. Finally, by comparing potato agar made by means of the same cultivar, by using healthy tubers or those attacked by virus (particularly, those attacked by leaf-roll virus) no differences were noted in the aerial mycelium and in the vegetative mycelium of the actinomycete colonies. The only difference was that one could observe more accentuated shades of soluble color in the substrate (pigment) when using potatoes attacked by virus. Therefore, the use of potato agar for the study of the actinomycetes does not present any difficulty even when used in different countries; one would only need to state the characteristics of the cultivar of potatoes used, for purposes of greater accuracy. Contrary to every expectation, Difco potato agar has no advantage in use for actinomycete culture, over that prepared in the laboratory (Baldacci et al., 1961). In this same paper, we have been able to show that another natural agar (oat agar) offers superior advantages in comparison with four other substrates, two of which were synthetic, for the conservation and growth of actinomycetes. The other substrates are yeastagar, asparagine-glycerinate agar (also known as Conn agar), and Difco potato agar. The results have been the same from four different laboratories, using the same strains, 50 in number. On the basis of our investigations, we believe that standardization of the substrate would be highly advisible, but standardization should not necessarily lead to the exclusive use of synthetic agars. On the contrary, it would be well to use more than one kind of substrate, among which would be included several “natural” ones, for which the means of preparation would be specified, as well as others which could be called “historical” ones because they make it possible to compare past and present diagnosis using those same substrates. The noting of the color of the mycelia should not be made on the basis of a pre-established standard, as has sometimes been done (Pridham d al., 1958) and as was postulated in the work established at the Stockholm Round Table Conference. It would be preferable to use a color atlas for the purpose. Research carried out by Paulin (1960) brought out the fact that the use of an atlas ensures complete objectivity in the reading of the color. In fact, numerical values of wavelengths of the colonies’ reflection data compared with those of the atlas cards picked out beforehand turned out to be very close. Thus, the designation of color takes on the status of an objective indication which can be checked and verified by other investigators; this would be lacking if one were to establish fixed color categories beforehand.

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In the work previously referred to (Baldacci et aZ., 1961) observations have been made as to the constancy of color during one year of culture preparation at ambient temperature and repeated transfers and at a temperature of 4OC. without transfer up to a year. The results from four laboratories which use the same strains (50 in number) confirm that the color constancy is excellent for oat-agar and for asparagine-glycerinate agar under both of the two above-mentioned working conditions. The variations have more to do with the tonality of the color than with its precise qualification. Our conclusion is that the use of mycelia color gives the best results for grouping some species, since such a method provides a characteristic which is easy to observe and which has the possibility of being standardized. As we have already seen on other occasions (Baldacci et al., 1954a), the observation and noting of this characteristic is sufficient for the identification of not only series, but also of species, because here we are dealing with typical entities having but little variability and limited diffusion [for example, S. sulphurew, S. cueruleus, S. madurae (Baldacci, 1937, 1944)l; in other cases this characteristic leads to more species being established, but nonetheless they are quite typical (S. viridis, S. albus, etc.) (Baldacci, 1939).

C. SPECIESAND INFRASUBSPECIFIC TAXAIN Streptomyces We now have the following characteristics for the species: ( a ) branching manners of the sporophores: irregular branches, tufts, cluster broomshape and clump types branches; long main stems which alternate branches (Mayama, 1959; Nomi, 1959); ( b ) form or structure of the sporophores (rectus, rectus-flexibilis, retinaculum-apertum, spira) in conformity to what has been proposed by Pridham et al. (1958); ( c ) shape of the spores for impression preparation for electron microscopy (Kriss et aZ., 1945; Flaig et al., 1952; Kiister, 1953; Baldacci and Grein, 1955; Baldacci et al., 1956). The use of these characteristics for speciation offers the advantage of establishing characteristics which are all related to a single morphological process, that of the sporeforming apparatus. In so doing, one also follows a tradition of the science of taxonomy, in which preference will be given to characteristics dealing with this apparatus for the classification of species, whenever possible, Finally, accepting this succession in the use of characteristics, the situation may then be outlined. In the series there is a collection of species which have been outlined in the past and, in some cases, now, on the basis of the Characteristics of “color of vegetative and aerial mycelium”; within each series, the species are defined by the micromorphological characteristics

CLASSIFICATION OF ACTINOMYCETES

275

given. These species will be considered according to the rules established for the purpose by the code (Principle No. 5 and Rules 12 and 14) for “valid” species. Those for which the morphological characteristics of the sporophore and of the spores are missing should not be qualified as “unknown” according to a different terminology sometimes used by microbiologists; they must be analyzed according to the rules of the code and their names can then be considered to be “legitimates,” “synonyms,” “homonyms,” and, furthermore, “nomen nudum” and “nomen conservandum” in accordance with the rules of the expressions used in the code. Numerous examples can be cited. Species which are described incompletely (incompletely according to present ideas, but completely or saciently according to the ideas existing at that time) can be recognized, re-identified, and amended, Much of author’s previous work has been directed toward the recognition of these species (Baldacci, 1937 et passim). We have the duty of conserving the cultural patrimony created and developed by researchers who have preceded us and we cannot work by simply making a clean slate of the past to start over from the beginning. Science is a written tradition and it requires a respect for and a study of those works which have been previously performed. The significance of the existence of a code of nomenclature is also this. A work of this kind being used by taxonomists, botanists, and zoologists has the advantage of limiting the number of new species, obliging investigators to analyze the diagnoses already in existence before going ahead with the creation of new nomenclature. A greater liberty of coining new terms is allowed for the subspecific and infrasubspecific ranks. For those ranks we now have many other characteristics available and, above all, those of the so-called biochemical type; in addition, there are those of the serological type, those pertaining to cultures in liquid substrates to antibiotic activity particularly. As we have seen, there are as many as twelve infrasubspecific categories allowed by the code, but perhaps none of them has been yet used for the actinomycetes. In conclusion, several remarks could be made on the failure to apply these taxonomic categories in the nomenclature to actinomycetes; it may be observed that this failure is due principally to : ( a ) the existence of relatively few species of Streptomyces before the discovery of their antibiotic activity; ( b ) the lack of knowledge of the rules of nomenclature on the part of those engaged in research on the actinomycetes; ( c ) legislation in different countries, requiring the creation of “new species” for protection rights of a patent for a compound having biological origin. In Table I which will bring this paper to a close, a model of the diagnostic procedure going from series to species to infrasubspecific rank is illustrated by the writer.

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A MODELOF

TABLE I DIAGNOSTIC PROCEDURE

White

Aerial mycelium

ALBUS

SERIES Sporophores Spore shape under E.M. Other morphological characteristics

SPECIES

Spiral Spiny

-

Straight Smooth Yes

Novus

QUALIS

TALIS

+ ++ -

Soluble pigment Carbon compounds utilization Antibiotic activity Phage Other biochemical activity

SUBSPECIES (or INFRASUBSPECIFIC RANK)

Straight Hairy Yes

Yes

NOBILIS

FACIENS

CHROMOQENUS

V. Summary and Conclusions This paper discusses whether the characteristic, “formation of antibiotic,” can be used as an element in the classification of actinomycetes, with particular attention being given the genus Streptomyces. Section I1 gives the various reasons which advise against using the characteristic, “formation of antibiotic,” at the species level. The chief difficulty lies in the fact that research on antibiotics is research of a chemical order and this cannot be asked of a taxonomist, who is, essentially, a morphologist. This same characteristic could be used to advantage at the infrasubspecific taxa provided for by the Code. The chief obstacle to this solution is to be found in the legislation of various countries. The protection of patents for biological production obliges the applicant to present “new species.” This legislative situation is not the subject being examined in this paper; comment is limited to the statement that these laws do not take into account the Bacteriological Code of Nomenclature. There does not appear to be any reason why patent protection could not be given at levels below that of the species, since these taxonomic ranks or taxa are explicitly recognized in the code. Section I11 comments on several rules of the International Code of Nomenclature of Bacteria and Viruses, which deals with the subject in hand. Section IV presents the general criteria of a classification of actinomycetes, according to ideas currently held and according to the rules of

CLASSIFICATION OF ACXINOMYCETES

277

the Code. Following an accepted tradition of taxonomic science, it is proposed that the formation of genera and species be limited to the known elements of the morphology of the sporeforming apparatus; data dealing with the culture, biochemical data, and data dealing with the formation of any antibiotic are to be limited to the intermediate categories (subgenus, series, subspecies, and infrasubspecific taxa) . The advantages of such criteria for classification are brought out.

REFERENCES Anonymous ( 1959). Intern. Bull. Bacteriol. Nomenclature and Taronomy 9, 173174. Baldacci, E. (1937).Atti bt. botan. Uniu. Pavb [IV] 9, 244-271, 299314; 10, 126-159. Baldacci, E. (1938).Atti ist. botan. Uniu. Puoia [IV] 10,321429. Baldacci, E. (1939).Atti ist. botan. Uniu. Pauia [IV] 11, 191-231. Baldacci, E. (1941).Atti Irt. botan. Untu. Pauia [IV] 13, 99-129. Baldacci, E. (1944).Ist. botan. Untu. Lob. crittogam. Pavfa Atti [V] 3 (3), 139192. Baldacci, E. ( 1956).Giorn. microbiol. 2, 50-62. Baldacci, E. ( 1958).Giorn. rnicrobiol. 6, 10-27. Baldacci, E. (1959).Intern. Bull. Bacteriol. Nomenclature and Taxonomy 9,81-88. Baldacci, E., and Grein, A. ( 1955).Giorn. microbiol. 1,28-34. Raldacci, E., Comaschi, G. F., Scotti, T., and Spalla, C. (1953).Intern. Congr. Microbiol. 6th Congr. Suppl. Repts. ist. Sanitd Romu pp. 2&34 Baldacci, E., Spalla, C., and Grein, A. ( 1954a).Arch. Mikrobiol. 20,347457. Baldacci, E., Grein, A., and Spalla, C. (1954b).Nuovi ann. tgiene e rnicrobiol. 5, 296-298. Baldacci, E., Grein, A., and Spalla, C. (1955).Giorn. microbbl. 1, 127-147. Baldacci, E., Gilardi, E., and Amici, A. (1956).Giorn. rnicrobiol. 1, 512-520. Baldacci, E., Giolitti, G., Kuster, E., and Scotti, T. (1961).Gbrn. microbbl. 9 (in press ) . Buchanan, R. E., St. John-Brooks, R., and Breed, R. S. (1948).J . BacterloZ. 55, 287406. Ettlinger, L., Corbaz, R., Keller-Schierlein, W., and Ziihner, H. (1956). Ciorn. rnicrobiol. 2, 91-97. Finlay, A. C., Hobby, G. L., P’an, S. Y., Regna, P. P., Routien, J. B., Seeley, D. B., Shull, G. M.,Sobin, B. A., Solomons, I. A., Vinson, J. W., and Kane, J. H. (1950). Science 111, 85. Flaig, W., and Kutzner, H. J. (1954).Naturwissetwchaften 12,287. Flaig, W.,and Kutzner, H. J. (1960).Arch. Mikrobbl. 35, 105-138. Flaig, W.,Beutelspacher, H., Kiister, E., and Segler-Holzweissig, G. (1952). Z. Pfkznzemmiihr. Dung. u. Bodenk. 57,42-51. Goldberg, H. S. (1959).“Antibiotics, Their Chemistry and Nonmedical Uses.” Van Nostrand, Princeton, New Jersey. Grein, A., and Kuster, E. ( 1955).Ann. microbiol. 6,269-272. “International Code of Nomenclature of Bacteria and Viruses” ( 1958). Iowa State Univ. Press, Ames, Iowa. Jones, K. L. (1954).Ann. N . Y. Acad. Sci. 60,124-135.

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Korzybski, T., and Kurylowicz, W. ( 1959 ) . “Antybiotyki.” Panstwoure Wydawnictivo Nankowe, Warsaw. Krainsky, A. ( 1914). Zentr. Bakteriol. Parasitenk. Abt. I1 41, 649-688. Krassilnikov, N. A. (1957). Ann. inst. Pasteur 92, 597-604 Krassilnikov, N. A. (1959). Ann. inst. Pasteur 96, 434-447. Krassilnikov, N. A. (1960). 1. Bacteriul. 79, 65-74; 75-80. Kriss, A. E., Rukina, E. A., and Issaiev, B. M. (1945). Mikrobiology ( U .S. S . R . ) 14, 172-176. Kuchayeva, A. G. (1958). Foliu Biol. 4,266-273. Kiister, E . (1953). Intern. Congr. Microbiol. 6th Congr. Romu 1, 114-116. Kiister, E. (1958). Intern. Bull. Bacteriol. Nomencluture and Taxonomy 9, 15-26. Kiister, E., and Grein, A. (1955). Aiaturwissenschaften 42, 52. Kutzner, H. J. (1958). Intern. Bull. Bncteriol. Nomenchtttre rind Taxonomy 9, 79-80. Lessel, E., Jr. (1960). Intern. Bull. Bacteriol. Norrienclnture und Tuxonomy 10, Suppl., 87-192. Mayama, M . (1959). Ann. Rept. Shionogi Research Lab. (Amagasaki, Japan) 9, 1185-1212. Nomi, R. (1960). 1. Gen. and Appl. Microbiol. (Tokyo) 5, 180-192. Paulin, N. ( 1960). Giorn. microbiol. 8, 91-144. Pridham, T. G., Hesseltine, G. W., and Benedict R. G . (1958). Appl. Microbiol. 6, 52-79. Szemiakin, M. M., and Koklov, A. C. (1953). “The Chemistry of Antibiotics.” Gozchimizdat, Moscow. Thom, C. (1954). Ann. N . Y. Acad. Sci. 60,24-34. Waksman, S. A,, and Lechevalier, H. A. (1953). “Guide to the Classification and Identification of Actinomycetes and their Antibiotics.” Williams & Wilkins, Baltimore, Maryland.

The Metabolism of Cardiac Lactones by Microorganisms ELWOOD TITUS Laboratory of Chemical Pharmacology, Nationul Heart Institute, Nationul Institutes of Health, Bethesda, Maryland

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hydroxylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Oxidation and Reduction ....................................... C. Dehydrogenation .............................................. D. Hydrolytic Reactions ........................................... 111. Conclusion ...................................................... References ......................................................

279 281 281 287 287 288 290 290

I. Introduction Since the first reports of the anti-arthritic properties of cortisone a decade ago there has been an intensive effort to produce modifications of steroids by microbiological means. Some of the most important adrenocortical hormones possess an oxygen function at carbon-11, and the initial work was largely prompted by the need to circumvent the cumbersome chemical methods for the introduction of oxygen at this position. Since then other structural changes produced by fermentation with microorganisms have been found to change the physiological activity of certain steroids. Introduction of an additional 1,2 double bond into ring A of cortisone, for example, greatly enhances glucocorticoid and mineralocorticoid activity (Runim, et al., 1955). The literature on the metabolism of steroids by microorganisms has been reviewed a number of times (Fried ct al., 1955; Wettstein, 1955; Eppstein et al., 1956; Vischer and Wettstein 1957, 1958). Some biochemical aspects of the microbiological alterations have been discussed by Talalay (1957). By far the greatest part of this work has been carried out with C,, and C,, steroids structurally related to testosterone and progesterone. Since these compounds include many of the reproductive as well as the adrenocortical hormones, this emphasis is not surprising. Until rather recently there appear to have been no attempts to produce new cardiac lactones by fermentative methods. This may be due to the large number of these steroids already available from plants and toad venom although by no means all the possible variants of the basic cardenolide and bufadienolide structures have been isolated, 279

280

ELWOOD TITUS

Enough is known of the relationship between structure and activity to suggest that new derivatives produced microbiologically may well have useful properties. The pharmacological action of the glycosides depends on two factors, the binding to cardiac tissue, which is largely a function of the sugar with which the steroid is conjugated, and the structure of the steroid nucleus itself. Digitoxigenin ( I ) the simplest of the cardiac aglycones, exhibits the structural features required for cardiotonic activity. These include 3p- and 14phydroxyl groups, the cis fusion of the A and B rings, the characteristic a$-unsaturated lactone ring, and the cis fusion of the C and D rings. Relatively simple structural differences markedly affect the activity. Replacement of the lactone ring, for example, by the six-membered ring of the toad poisons, e.g., bufalin, results in a fourfold enhancement of the activity. Strophanthin, with an aldehyde group on the 10 position, is twenty times more active than the corresponding C-10 methyl compound, periplogenin ( Hajdu, 1957). Epimerization at positions 3, 5, or 17, reduction of the double bond in the lactone ring, and certain hydroxylations can greatly reduce or eliminate the activity (Fieser and Fieser, 1959; Titus et al., 1960).

METHODS The methods used for studies of the metabolism of cardiac steroids by microorganisms are generally the same as those used with other steroids. The substrate, dissolved in a small amount of a nontoxic solvent miscible with water, is added to a culture of the organism (usually a fungus) and agitated under aerobic conditions for periods up to several days. The transformation products are recovered by extraction of the medium with solvents and are isolated by chromatography on paper or columns. A number of solvent systems have been found useful for paper chromatography of the aglycones. These include toluene saturated with propylene glycol ( Burton et al., 1951), chloroform saturated with formamide (Schindler and Reichstein, 1951), and mixtures of benzene, water, and ethyl acetate or methanol (Sasakawa, 1955; Bush and Taylor, 1952). Kaiser (1955) has successfully used mixtures of xylene, methyl ethyl ketone, and formamide as well as a chlorofonn-tetrahydrofuran-formamide system. Chromatography of the aglycones on silicic acid (Titus et al., 1 9 0 ) as well as the more commonly used alumina or Fluorisil has proved useful for large-scale separations. The cardenolides are readily located on paper chromatograms by spraying with 3,s-dinitrobenzoic acid in alkaline methanol (Bush and Taylor, 1952). The bufadienolides give no color with dinitrobenzoate. = 300 ma) Because of their strong absorption in the ultraviolet (,imax. they can be located by making contact prints of the chromatograms on

METABOLISM OF CARDIAC LACTONES

281

photographic paper in filtered ultraviolet light (Bernasconi et d.,1955). A few chemical reactions have been particularly useful in elucidating the structure of metabolites. Under appropriate conditions oxygen in the presence of platinum will convert only the 3-hydroxyl of a cardenolide to a ketone (Sneeden and Turner, 1955a, b; Katz, 1957). The ease of preparation of reference compounds simplifies the identification of transformation products oxidized at this position. Since reduction of steroid carbonyl groups by metal hydrides yields the equatorial hydroxyl (Sigg et aZ., 1953), the configuration of alcohols produced by microbiological

FIG.1. Comparison of the configurations of progesterone and digitoxigenin; L denotes the position of the lactone ring in the latter.

reduction of ketones is readily established by comparison with NaBH, reduction products. II. Reactions A. HYDROXYLATION Hydroxylation appears to be the most frequent type of reaction of cardiac aglycones. These compounds differ in several respects from most of the steroids that have been subjected to microbiological transformation and an interest in the effects of substrate structure on the mode of enzymatic attack prompted the hydroxylation experiments of Tamm and Gubler (1959a). Figure 1 illustrates some of the differences between digitoxigenin and progesterone, which may be considered a prototype of the classically used substrates. In this configurational representation it can be seen that the nearly planar ring A of progesterone is replaced by a

282

ELWOOD TITUS

chair-formed ring which is sharply out of line with the rest of the molecule because of the cis fusion of the A and B rings. The cardiac aglycones resemble the bile acids in having a cis A/B fusion but differ in the A ring hydroxyl which is 38 (axial) in the aglycones and 3a (equatorial) in the bile acids. It would be of interest to discover how the changed environment in this region would affect the action of hydroxylating microorganisms that normally attack the A ring. Such experiments do not appear to have been reported. The cardiac aglycones are unique among naturally occurring steroids in possessing a 148hydroxyl ancl a cis C/D ring junction. This hydroxyl and the lactone ring, represented for the sake of clarity by L in Fig. 1, can be seen in models to lie in a position to hinder access to the D ring. It may be that steric hindrance accounts for the observations of Tamm and Gubler that Filsarium lini ( Rolley ), which normally hydroxylates the 15a position (Gubler ancl Tamm, 19%b), does not attack this position on the cardiac lactones. When digitoxigenin ( I ) (see Fig. 2 ) , the simplest of the biologically active aglycones, wits incubated with F . lini in a synthetic medium the only product to appear was the 12p-hydroxy derivative, digoxigenin ( I1 ) . Since this compound has long been known, the fermentation product was readily identified by comparison with authentic samples. It is of interest that 128-hydroxylation is also the fate of cardiac lactones administered to animals (Brown et ul., 1957; Repke, 1958a, b ) . This organism could also convert 3-dehydrodigitoxigenin ( I11 ) to a compound identifiable as 3dehydrodigoxigenin ( IV) by oxidation of digoxigenin. Substitution of a six-member lactone ring for the five-membered ring at C-17 did not change the position of hydroxylation. Bufalin (VII) (see Fig. 3) was converted by incubation with F . lini for 11 days to a new compound that could be identified as 12p-hydroxybufalin (VIII ) [3p,128,14-trihydroxybufadien(20,22)-olide], by degradation to the known 3~,12~-diacetoxy-14-hydroxy5p,l4fi-etiocholanic acid (Tamm and Gubler, 1959b). On the basis of these results F . lini appeared to be a trustworthy general 12p-hydroxylator of cardiac aglycones. The conversion of gitoxigenin ( V ) (16p-hydroxydigitoxigenin ) to a product identical with a recently isolated new aglycone, diginatigenin (Tamm and Gubler, 1958), was therefore considered to be a confirmation of Murphy’s proposal that the latter was 12p,16p-dihydroxydigitoxigenin( VI ) ( Murphy, 1955). The 12p position of the lactones appears to be particularly susceptible to hydroxylation, since several laboratories have independently observed the microbiological conversion of digitoxigenin to digoxigenin. A group at Takeda Pharmaceutical Industries (Nawa et aZ., 1959) has reported chromatographic evidence for this hydroxylation by Helicostylum piri-

&'

METABOLISM OF CARDIAC LACTONES

f

Y

&& + & &'

no

."

un I

I

283

II

&o

OH

0

m

Ip

Y

PI

HO

, HO

FIG.2. 12p-Hydroxylation of cardenolides b y Fusoriunr h i .

PII

YIlI

FIG.3. 12@-Hydroxylationof bufalin by Fusurium h i .

forme and Gibberella fujikuroii ( S'iwada ) Wollenweber. Investigators of

the Shionogi Laboratories have found that Nigrospora sphaerica, ordinarily an Il~,l5p-hydroxylatorof C,, steroids, and Calonectria clecura, a 12p- and 15a-hydroxylator,produce small amounts of digtoxigenin from digitoxigenin ( Nozaki et al., 1960). The highest yields of 12P-hydroxycardenolides thus far reported have been obtained at the University of Tokyo, where Okada and associates (1960) have observed that Gibberella

284

ELWOOD TITUS

saubinetti ( Montagne ) Saccardo can effect the 128-hydroxylation of digitoxigenin and gitoxigenin in yields of over 70 and 6% respectively. A number of other hydroxylations have recently been reported. Both H. piriforme and Cunninghumella blakesleeana Lendner convert digitoxigenin to its 16p-hydroxy derivative, gitoxigenin, and produce in addition an unidentified new aglycone (Nawa et d., 1959). Ishii ( 1960) has observed the 5p-hydroxylation of digitoxigenin to periplogenin (IX) (see Fig. 4 ) by Mucor parasiticus. Since the newly introduced hydroxyl could not be acetylated and was therefore on a tertiary carbon, and since the fermentation product could be converted

Ix

x H FIG.4. Conversion of periplogenin to anhydroperiplogenone.

to 3-dehydro-5-anhydroperiplogenin (XI ) ( anhydroperiplogenone ) , by

oxidation and dehydration, the structure was established unequivocally. Two new cardiac aglycones have recently been produced by fermentation. The first of these, 7p-hydroxydigitoxigenin (XII) (see Fig. 5), m.p. 287-272OC. (Ishii et al., 1960; Nozaki et al., 1960), was obtained by incubation of digitoxigenin with Rhbopus arrhizus, an organism that hydroxylates progesterone in the 68 and l l a positions (Eppstein et nl., 1953). The new aglycone was identified by oxidation to a diketone (XV) and dehydration of the latter to a product (XVI) that could be identified spectrally as an a$-unsaturated ketone. The formation of a carbonate (XIV) with COCl, and the molecular rotation data were in agreement with the proposed structure. The hydroxylation at 78 rather than at 6 p or l l a may reflect the influence of the structure of the A ring. Rhixopus arrhizus also introduces a C,, steroid with the same a 7p-hydroxyl into allopregnane-3p-ol-2O-one, A/B junction and axial 3-hydroxyl as the cardenolides (Murray and Peterson, 1952). A second new monohydroxydigitoxigenin, m.p. 268-275OC., [aID*5 37O, that is inactive in the frog heart assay of Hajdu (1957) has been isolated from fermentations of digitoxigenin with Tricothecium roseum

+

285

METABOLISM OF CARDIAC LACTONES

(Titus et al., 1960). The molecular rotation data suggest that the newly introduced hydroxyl, which is readily benzoylated, may be in the 6p position (XIX) (Fig. 6). Both the molecular rotation data and the lack of biological activity would also be in accord with an lla-hydroxy compound in which the bond between the lactone and carbon-17 has been isomerized to the a-configuration. Tricotheciurn roseurn is an lla-hydroxylator of both progesterone (Meister et al., 1954) and digitoxigenin, which is converted to sarmentogenin (XVIII) by this organism (Titus et al., 1960). Since Al6-anhydrogitoxigenin (XVII) was also among the

HO

&y/yLpoy-yp OH

OH

O’CqJ

XTP:

x

xm: FIG. 5. Proof of structure of 7p-hydroxydigitoxigenin.

fermentation products and since the microbiological conversion of Alepregnen-20-one to lla-hydroxy-17-isoprogesterone( Meister et al., 1953) provided a precedent for the isomerization of the 17 position by reduction of a 16,17-unsaturated intermediate, it was not impossible that the new compound might be 17-isosarmentogenin.Isomerization of sarmentogenin by the procedure of Kuritzkes et al. (1959), however, yielded a product that could be distinguished chromatographically from the new aglycone. Paper chromatographic comparisons undertaken after these publications appeared indicate that the products from R . arrhizus and T. roseurn are probably identical. It has been reported that the yield of hydroxylated steroids from incubations with Cunninghamella blakesleeana are considerably improved if various organic substances are added to the nutrient solution (Mann et al., 1955; O’Connell et al., 1955). This may be true also of T . roseurn. Approximately 50% of the added digitoxigenin could be recovered unchanged from incubations in either corn steep liquor or a synthetic

286

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medium. Only with the former, however, were appreciable amounts of hydroxylated derivatives obtained. As has been noted with other organisms (Vischer and Wettstein, 1958), all the transformation products appeared in the medium, while the mycelium of T . roseurn contained only unchanged digitoxigenin. Although most fermentations of cardiac aglycones have been carried out with digitoxigenin, Tamm and Gubler (1959,) have used a number of substrates in an effort to define the effects of structural variations on

w= 'v

HO

m 268-75'

\r 'OH

[a]

25

t 37"

FIG. 6. Hydroxylation of digitoxigenin by Tricothecium roseum to 16-anhydrogitoxigenin (XVII), sarmentogenin (XVIII), and @( ?)-hydroxydigitoxigenin (XIX).

the hydroxylation of cardenolides by F. h i . The introduction of double bonds between positions 14 and 15 or 16 and 17 gave products no longer susceptible to hydroxylation. Dihydrodigitoxigenin, obtained by catalytic reduction of the 20,22 double bond in the lactone ring, was extensively degraded to smaller fragments. Hydroxylation was greatly diminished or eliminated as more oxygen atoms were introduced into the steroid nucleus. Although, as has been mentioned, gitoxigenin was hydroxylated in small yield, sarmentogenin was not. There was some hydroxylation of 5fl-hydroxydigitoxigenin(peri-

METABOLISM OF CARDIAC LACTONES

287

plogenin) to unidentified products, but none with strophanthidol, which has an additional hydroxyl on C-19. These effects may occur because passage of the hydroxylated steroids into the lipoidal surface of the cells is more difficult than that of the less polar digitoxigenin. Polarity cannot be the only factor, however, since 3-epidigitoxigenin is not metabolized by F . h i . Uzarigenin, another analog that differs from digitoxigenin only in the configuration at one carbon atom (C-5), has yielded only traces of unidentified metabolites, Anhydroperiplogenone ( X I ) is of interest since the A and B rings are identical to those of the commonly used substrate, progesterone, while the C and D rings remain those of a typical cardenolide. There is paper chromatographic evidence for the production of two metabolites of this substance by F . Zini but yields and identities have not been established.

B. OXIDATION AND REDUCTION The 3-hydroxyl groups of several cardenolides are oxidized to ketones by certain fungi (Nozaki et al., 1960). Yields as high as 12 and 16%of 3-dehydrodigoxigenin were obtained from digitoxigenin with Nigrospma sphaerica and Calonectriu decora. The same organisms oxidized gitoxigenin to the 3-dehydro derivative. The extent of oxidation, at least by Rhizopzcs urrhizus, appears to depend on the pH of the medium. This organism, which produces 7p-hydroxydigitoxigenin when cultured in glucose-peptone-corn steep liquor at acid pH, yielded the 3-dehydro-7phydroxy compound when the glucose content was lowered to the point where the culture maintained an alkaline pH. Reduction of 3-keto groups is also common among the fungi. Gubler and Tamm (1958a) observed that F . lini produced both 3-epidigitoxigenin and 3-epidigoxigenin from 3-dehydrodigitoxigenin. The same transformations were carried out by G . saubinetti (Okada et al., 1960). Microbiological reductions of the keto aglycones have thus far always yielded the equatorial 3wepimer. C. DEHYDROGENATION The conversion of digitoxigenin to A'"-anhydrogitoxigeninby T . roseurn (Titus et al., 1960) seems to be the only reported instance of the introduction of a new double bond into an aglycone. This may have occurred by spontaneous dehydration of the unknown intermediate, 17n-hydroxydigitoxigenin, since T . roseunt is known to hydroxylate at the 17a position. The intermediate would be particularly susceptible to dehydration since loss of the tertiary hydroxyl would be favored by the resonance stabilization of the resulting A18.2u("2) -cardadienolide.

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The work with T. roseurn was originally undertaken in the hope of preparing the 17a-hydroxy compound for pharmacological testing. It has been observed by Hajdu ( 1957) that several adrenocortical steroids have a digitalis-like effect on the frog heart. The corresponding l7a-hydroxy corticoids, however, act in the opposite sense, tending to enhance rather than diminish the Bowditch “staircase” phenomenon, which is used as a measure of activity, It was hoped that 17a-hydroxycardenolides might be antagonistic to the action of the conventional aglycones. In spite of the mildness of the conditions of incubation and isolation, only the anhydro compound was obtained. The product was readily identified by its characteristic ultraviolet absorption band at 272 mp and by dehydration in acid to digitaligenin (3p-hydroxy-14-anhydro-A1s*20(22)-cardadienolide) ( Windaus and Schwartes, 1925) which absorbs near 340 mp.

D. HYDROLYTIC REACTIONS Both esterases and glycosidases occur commonly in the fungi and as expected these enzymes have been found to attack steroidal lactone substrates. Tamm and Gubler (1959a) have investigated the effects of incubation with F. l i d on a variety of acetylated cardiac glycosides. Acetyl groups were efficiently removed from both the sugar hydroxyls and those on the steroid nucleus. The esterases showed no positional specificity since acetoxy groups on both carbons-3 and -16 were hydrolyzed. In general the yields of hydrolysis products were of the order of 30 to 50%of the original glycoside. The glycosidases of F. h i , like those of most fungi (Stoll et al., 1951), would attack only glucoside linkages. The experiments with k-strophanthoside may be regarded as typical. This glycoside, in which steroid and sugars are in the sequence, strophanthidin-D-cymarose-Dglucose-D-glucose, was acetylated chemically to the hepta-O-acety1-kstrophanthoside. Incubation of the product yielded 52% of cymarin, strophanthidin-D-cymarose. An interesting deacylase system has been found in G. saubinetti at the laboratory of Professor Ishidate (Okada et al., 1960). This organism efficiently removes the acetyl groups from 3-acetyldigitoxigenin and 3-acetylgitoxigeninYbut effects little if any hydrolysis of oleandrigenin ( 16-acetylgitoxigenin) . The studies of Tamm and Gubler ( 1959a) suggest that the increased polarity of hydroxylated derivatives of digitoxigenin may hinder their access to the hydroxylating enzymes of the fungi. Acetylation of the 16-hydroxyl of gitoxigenin should reduce the polarity to such an extent that the acetoxy compound would be efficiently hydroxylated, and this has indeed been observed by the Japanese

METABOLISM OF CARDIAC LACTONES

289

workers. Oleandrigenin was converted in 15%yield to 18acetyldiginatigenin. An acetylated hydroxylation product has not previously been obtained, presumably because of the ubiquitous and nonspecific esterases in most fungi. Although the natural glycosidases of fungi are exclusively glucosidases, other enzymes have been induced. An interesting application was the work of Stoll and his collaborators (1951) at the Sandoz Laboratories. Here the problem was the assignment of the position of the double bond in the A ring of the aglycone of Scillaren A (XX) (Fig. 7). All the

FIG.7. Hydrolysis of Scillaren A by acid and induced rhamnosidase of PentciUfum strain 889.

chemical methods of hydrolysis that were sufficiently rigorous to split the aglycone from the accompanying sugars invariably caused dehydration to the dianhydro compound (XXI) so that the position of the double bond in the steroid nucleus remained equivocal until the true aglycone could be obtained. Efforts to accomplish the required hydrolysis enzymatically were unsuccessful since both the glycosidase from mammalian tissue (Stoll and Renz, 1951) and those from microorganisms were specific for glucosides and did not attack the rhamnoside linkage at position 3 in the steroid nucleus. A strain of Penicillium was finally encountered in which an active rhamnosidase could be induced, and with the aid of this enzyme scillarenin (XXII) was liberated from scillaren A. The desired rhamnosidase was induced by growing the Penicillium

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in a peptone medikm containing 2%rhamnose. The enzyme appeared in the culture medium and attained its maximal concentration after about 18 generations. De-adaptation occurred rapidly. If the organism was returned to a rhamnose-free medium the rhamnosidase activity was usually completely gone after 2 generations. With free scillarenin now available assignment of the position of the double bond was quickly made. The ease of dehydration of the dianhydro compound was more in accord with a a4-3p-hydroxy compound than with the A5 analog which was the alternative possibility. The difference between the molecular rotations of scillarenin and the dianhydro derivative were also in accord with the formation, and with this evidence the last uncertainty about the structure of the squill glycosides was removed.

Ill. Conclusion It appears from the limited data available that the enzymatic systems in fungi that attack the Cl!j and C,, steroids will metabolize other steroids of similar polarity in spite of differences in structure. Although no work has been done on the biochemistry of the hydroxylation of cardenolides, there is no reason to believe that these reactions differ from those of other steroids, which require reduced triphosphopyridine nucleotide and accept the newly introduced oxygen atom from atmospheric oxygen (Talalay, 1957; Hayano et al., 1956). It is interesting that enzymatic introduction of the hydroxyl group is accomplished with retention of the original configuration of the carbon. Hydroxyl entering at the l l a position, for example, displaces only the lla-hydrogen (Corey et al., 1958; Hayano et al., 1958). The mechanism of steroid hydroxylations is still obscure and much remains to be done. Many of the hydroxylating systems, if not all, are inducible (Talalay, 1957; Vischer and Wettstein, 1958). Whether it is the hydroxylating system itself that is induced or a permease governing access of the steroid to the cell is not known.

HEFERENCES Bernasconi, R., Sigg, H. P., and Reichstein, T. (1955). HeZo. Chirn. Actu 38, 17671775. Bunim, J. J., Pechet, M. M., and Bollet, A. J. (1955). J . Am. Med. Assoc. 157, 311318. Burton, R. B., Zaffaroni, A., and Keutmann, E. H. (1951). J . B i d . Chem. 188, 763-768. Bush, I. E., and Taylor, D. A. H. (1952). Biochem. J . 52,643-648. Brown, B. T., Wright, S. E., and Okita, G. T. (1957). Nature 180, 607.

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Corey, E. J., Gregoriou, G. A,, and Peterson, D. H. (1958). 1. Am. Chem. 80, 2338. Eppstein, S. H., Meister, P. D., Peterson, D. H., Murray, H. C., Leigh, H. M., Lyttle, D. A., Reinecke, L. M., and Weintraub, A. (1953). J. Am. Chem. SOC. 75, 408-412. Eppstein, S. H., Meister, P. D., Murray, H. C., and Peterson, D. H. (1956). Vitamins and Hormones 14,359432. Fieser, L. F., and Fieser, M. (1959). “Steroids,” pp. 727-809. Rheinhold, New York. Fried, J., Thoma, R. W., Perlman, D., Herz, J. E., and Borman, A. (1955). Recent Progr. in Hormone Research 11, 149-181. Gubler, A., and T,amm, C. (1958a). Helo. Chim. Acta 41, 297400. Gubler, A., and Tamm, C . (1958b). Helv. Chim. Acta 41,301405. Hajdu, S . (1957). J. Pharmacol. Exptl. Therap. 120, 90-98. Hayano, M., Saito, A., Stone, D., and Dorfman, R. I. (1956). Biochim. et Biophp. Acta 21, 380-381. Hayano, M., Gut, M., Dorfman, R. I., Sebek, 0. K., and Peterson, D. H. (1958). J . Am. Chem. SOC. 80, 2336. Ishii, H. ( 1960). Personal communication. Ishii, H., Nozaki, Y., Okumura, T., and Satoh, D. (1960). Yakugakrc Zasshi 80, 1150-1151. Kaiser, F. (1955). Chem. Ber. 88, 556-563. Katz, A. (1957). Helo. Chim. Acta 40, 831-846. Kuritzkes, A,, von Euw, J., and Reichstein, T. (1959). Helo. Chim. Acta 42, 15021515. Mann, K. M., Hanson, F. R., O’Connell, P. W., Anderson, H. V., Brenner, M. P., and Kamemaat, J. N. (1955). Appl. Microbiol. 3, 14. Meister, P. D., Peterson, D. M., Murray, M. C., Reinicke, L. M., Weintraub, A., and Leigh Osborn, H. M. (1953). J. Am. Chem. SOC. 7 5 , 5 5 5 6 . Meister, P. D., Reinecke, L. M., Meeks, R. C., Murray, H. C., Eppstein, S. H., Leigh Osbome, H. M., Weintraub, A., and Peterson, D. H. (1954). J. Am. Chem. SOC. 76, 4050-1051. Murphy, J. I 3 (1955). J. Am. Phann. Assoc. Sci. Ed. 44,719-722. Murray, H. C., and Peterson, D. H. (1952). U. S. Patent 2,602,769. Nawa, H., Uchibayashi, M., Kamiya, T., Yamano, T., Arai, H., and Abe, M. (1959). Nature 184, 469-470. Nozaki, Y., Masuo, E., Ishii, H., Okumura, T., and Satoh, D. (1960). Alistr. Papers Symposium on the Chem. of Digitalis Cardiac Glycosides, Tokyo, 1960 p. 114. O’Connell, P. W., Mann, K. M., Nielson, E. D., and Hanson, F. R. (1955). Appl. Microbiol. 3, 16. Okada, M., Yamada, A., and Ishidate, M. (1960). Chem. G Pbarm. R i d . ( T o k ! / o ) 8, 530-534. Repke, K. (1958a). Naturwissenschaften 45, 94. Repke, K. (195813). Naturwissenschaften 45, 366. Sasakawa, Y. (1955). Yakugaku Zasshi 75, 946. Schindler, O., and Reichstein, T. (1951). Helo. Chim.Acta 34, 108-116. Sigg, H. P., Tamm, C., and Reichstein, T. (1953). Helo. Chim. Acta 36, 985-989. Sneeden, R. P. A., and Turner, R. B. (1955a). 1. Am. Chern. SOC. 77, 130-134. Sneeden, R. P. A., and Turner, R. B. (195513). J. Am. Chem. SOC. 77, 190-191. StoU, A., and Renz, J. ( 1951). Helo. Chim. Acta 34, 782-786.

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Stoll, A., Renz, J., and Brack, A. (1951).Helu. Chim. Acta 34,2301-2311. Talalay, P. ( 1957).Phydol. Revs. 37,362-389. Tamm, C . , and Gubler, A. (1958).Ilelv. Chim. Acta 41, 1762-1769. Tamm, C.,and Gubler, A. ( 1959a).Helv. Chim. Acta 42,239-259. Tamm, C., and Gubler, A, ( 1959b).Helu. Chim. Acta 42,473-481. Titus, E.,Murray, A. W., and Spiegel, H. E. (1960).J. B i d . Chem. 235, 3399-3403. Vischer, E.,and Wettstein, A. ( 1957).Angew. Chem. 69,456-463. Vischer, E.,and Wettstein, A. (1958).Advances in Enzymol. 20,237-282. Wettstein, A. (1955).Experimentfa 11,465-479. Windaus, A.,and Schwartes, G . (1925).Ber. dart. chem. Ges. 58.1515.

Intermediary Metabolism and Antibiotic Synthesis J. D.Bw’Loc~ Department of Chemistry, The University, Manchester, England I. Antibiotics as Secondary Metabolites ................................ 293 11. A Classification of Secondary Metabolites ............................ 295 A. General Observations .......................................... 299 B. Some Structural Categories ..................................... 296 C. The Relation to Taxonomic Classification .......................... 308 111. Connections between General and Secondary Metabolism . . . . . . . . . . . . . . . 311 A. The Selection of Precursors ..................................... 311 B. The Use of Parallel Mechanisms ................................. 313 C. Unit Processes ................................................ 313 D. Some Special Synthetic Mechanisms ............................. 316 IV. The Interaction of General and Secondary Metabolism . . . . . . . . . . . . . . . . . 326 A. Relation of Secondary Metabolites to Growth ...................... 326 B. Secondary Metabolites as “Shunt” Products ....................... 330 C. Effects of Mutations ........................................... 331 D. Enzymic Induction Effects ..................................... 332 E. Efficiency of Secondary Biosynthesis ............................. 334 V. The Functions of Secondary Metabolism ............................ 335 VI. A Postscript ..................................................... 339 References 339

......................................................

1. Antibiotics as Secondary Metabolites In the twenty-two years since the clinical utility of penicillin was established, the study of antibiotics has tended to dominate the approach to certain aspects of microbiology. Not only has the “screening” process involved superficial examinations of an enormous range of microorganisms, but, more fundamentally, great efforts have been made toward understanding the mode of action of antibiotic substances and toward the improvement and control of industrial fermentations. The direct and indirect stimuli from all this research have affected fundamental aspects of microbiology, organic chemistry, and even engineering. Yet meanwhile, just because the antibiotics are primarily defined, detected, examined, and ultimately marketed as agents acting upon other organisms, the fact that they are themselves organic products has become relatively overshadowed. It is with an attempt to consider this aspect, the antibiotics as functions of the producing organisms, that this chapter will be concerned. 293

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J. D. BU’LOCK

We begin with something of a pgradox, namely that in order to consider antibiotics in the way proposed we have first to ignore the antibiotic effects which define them, In general we must admit that the distinction between antibiotically active and inactive metabolites is an artifact of humanly contrived situations, and has less significance to the producing organism. Thus, considered as metabolic products the two polyacetylenes diatretyne I and diatretyne I1 (Fig. 1) clearly belong HO&.CH=CH.C=C.C=C.

CONHI.,. . . . . . inactive C E N . . . . . . . . . . antibiotic

FIG. 1. The diatretynes.

together, being produced by one organism simultaneously and largely by the same mechanism, yet I1 is an antibiotic and I is not (Anchel, 1955). Again, to a test bacterium there is a world of differeke between gentisic acid and patulin, while to the producing organism it is merely a matter of a few micrograms of iron (Brack, 1947). But if, for the next few thousand words, we are to forget about antibiotic activity, what becomes of our subject? What category of substances can we discuss which will embrace both the antibiotics and their inactive poor relations? Now ever since Perkin, failing to make quinine, founded the dyestuffs industry, the organic chemists have found the study of “natural products” an inexhaustible source of exercises, which can be performed out of pure curiosity even when paid for in the hope of a more commercial reward. As a result, the organic chemist’s view of nature is unbalanced, even lunatic, but still in some ways more exciting than that of the biochemist. While the enzymologist’s garden is a dream of uniformity, a green meadow where the cycles of Calvin and Krebs tick round in disciplined order, the organic chemist walks in an untidy jungle of uncouthly named extractives, rainbow displays of pigments, where in every bush there lurks the mangled shape of some alkaloid, the exotic perfume of some new terpene, or some shocking and explosive polyacetylene. To such a visionary, both the diatretynes are equal prizes, to be set together as “natural products.” We shall do the same, but since to a more sober eye both are in a sense rather less “natural” than, say, glycine or adenosine triphosphate ( ATP), we may prefer the term “secondary metabolites.” Given the generally acceptable view that there are basic patterns of general metabolism, on which the variety of organic systems imposes relatively minor modifications, we can define secondary metabolites as having, by contrast, a restricted distribution (which is almost species specific) and no obvious function in general metabolism. Such sub-

INTERMEDIARY METABOLISM

295

stances are already known in great number and variety though we know only a fraction of the full range. For present purposes we shall restrict ourselves to products formed by microorganisms, but among these we shall include, for reasons hereafter apparent, some special cases of substances which are extraordinary not in their structure but in the amount in which they are sometimes formed, such as the riboflavin accumulated by Ereniothecium nshbyii. The study of these substances began before penicillin had been noticed-as a systematic program, perhaps with Birkinshaw and Raistrick’s classic papers of 1931-and has continued steadily even when disguised as a study of antibiosis. Today it has reached a critical and interesting stage, and perhaps when we have surveyed it we may return briefly to questions of biological activity.

It. A Classification of Secondary Metabolites A. GENERAL OBSERVATIONS Since the most obvious characteristic of secondary metabolites is their extreme variety, from oxalic acid to erythromycin, any useful classification will simplify their consideration. As already noted, a manifestation of antibiotic activity is not a significant criterion at this stage, and by similar arguments most other incidental properties, such as color, redox potential, metal-binding action, optical activity (to name a few which at one time or another have been given prominence), can be overlooked. The most helpful classifications seem to be by chemical structure and by species of origin, and these should ultimately converge when the structural classification is understood in terms of biochemical processes and when the taxonomic classification specifies biochemical as well as morphological relationships. Since this convergence is not yet in sight, and since the structural classification has had a more successful history and underlies many of the recent developments, it will be considered first and at greater length. It should first however be noted that the structural types encountered in a study of secondary metabolites are likely to reflect the methods and interests of the investigator, who in the first place decides which organism to study. It is easy to come to attach undue importance to naturally occurring quinones, for example, if pigmented organisms are persistently chosen for a series of investigations. Only by using relatively nonspecific criteria for further study, such as screening for deficits in carbon balance or (what is easier) for antibiotic activity, is a more representative selection of structures likely to be encountered, and even here the emphasis will be, respectively, on compounds produced in large amounts,

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or having antimetabolite structures, both of which will still cause a bias. Again, most publications describe only the easily isolated metabolites, or the ones most easily detected because of some spectacular property, though their understanding may depend on a knowledge of minor substances formed simultaneously. Having tried hard to compensate for such distortions, perhaps the author will be forgiven if the choice of illustrative examples betrays his own personal interests.

B. SOME STRUCTURALCATEGORIES Every chemical structure epitomizes, though perhaps ambiguously, the processes of its formation, and in this light very diverse structures can be seen to be comparable, The list which follows is intended to be illustrative rather than final or exhaustive, and since it is set out in quasibiochemical terms some indication of the historical development of the categories should first be given. As an example, take the p-carotene of Phycomyces blakesleeunus, which might initially have been classified, rather simply, as a hydrocarbon ( C4,,Hae)along with, say, ethylene and heptadecane. The subsequent recognition by organic chemists of the “isoprene unit” as a component of a wide variety of plant products would allow the carotene to be transferred to a group of “isoprenoids,” defined at first in purely structural terms and later in terms of a hypothetical common origin. Eventually a common pathway with defined intermediates ( e.g., mevalonate) is discovered in biochemical studies of general metabolic processes, and in the light of these discoveries the carotene is seen to be classifiable alongside such diverse fungal constituents as ergosterol, mycelianamide, and gibberellic acid, a grouping which is justified by experimental studies of precursor incorporation. Most of the categories to be considered here have developed in some such way, but the next stage of their definition, in terms of detailed biochemical mechanisms, has for the most part still to be attained.

1. Simple Acid Derivatives A number of simple aliphatic acids, not directly related to lipids, are known as secondary metabolites (Fig. 2). Some (citric, fumaric, and CHz-CHOH-CHI LOZH ~ O Z H bOzH Citric

CHz=bCHa LOnH h O n H Itaconic

HOaC*CH=CH.COzH HOzC.CO,H Fumaric Oxalic 7-Methyltetronio FIG.2. Simple carboxylic acid derivatives.

INTERMEDIARY METABOLISM

297

perhaps oxalic) are only secondary in the sense that though normal intermediates of general metabolism they sometimes accumulate to an extraordinary extent, Others like itaconic acid or the various tetronic acids (below) are secondary in the more usual sense. All, however, seem to be rather simply related to general metabolism, the case of itaconic acid is a good example, since the producing strains of Aspergillus niger contain a special decarboxylase which withdraws cis-aconitate from the Krebs cycle ( Bentley and Thiessen, 1957). 2. Fatty Acid Derivatives In addition to the well-known examples of unusually vigorous fat synthesis by certain microorganisms, recently reviewed by Woodbine (1959), there are many metabolites that seem to be related to the fatty acids in that they too contain straight chains of aliphatic carbon atoms assembled by head-to-tail linkage of C,units related to acetate (Fig. 3 ) . The polyacetylenes of Basidiomycetes are good examples of this (Bu’Lock and Gregory, 1959) and perhaps also the polyene chain in such substances as fumagillin. The category also includes some substances related equally to category 1, e.g., the fungal tetronic acids and citrate derivatives such as agaricic acid. Since propionate-derived C, units, and longer acyl residues, can be incorporated into fatty acids (Horning et al., 1960), the category also includes such branched-chain analogs as D-Smethyloctanoic acid (in the polymixin antibiotics) and probably the curious branched-chain acids from mycobacteria. When thus extended the category will also include the main “aglycone” moiety of the typical macrolides, such as erythromycin ( propionate-derived as shown by Corcoran et al., 1960) as well as such polyene antibiotics as lagosin. There is also a connection between this group and that of the acetate-derived aromatic compounds (below, 7 ) , with substances such as curvularin, which appear to be only partly aromatized, as a possible link (Birch et al., 1959). 3. Isoprenoid Substances This category (Fig. 4 ) has already been mentioned, being defined as substances wholly or partly derived from mevalonic acid and its transformation products and containing C,isoprene units which may be variously modified. It includes carotenoids, the rather ubiquitous ergosterol, the triterpenoids of Basidiomycetes, and more modified isoprenoids such as rosenonolactone (Birch et al., 1958f; Britt and Arigoni, 1958), trichothecin (Jones and Lowe, 1960), and gibberellic acid (Birch et al., 1958a). In such substances as mycelianamide (Birch et al., 1958c) or auroglaucin (Birch et al., 1958g), only part of the molecule is isoprenoid.

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J. D. BU'LOCK

H C E C . C E C . CH=C=CH. CHOH. CH2CH?C02H

Neniotinic acid

Fumagillin

6-Methyloctanoic acid

Agaricic acid

Corynomycolic acid

Curvularin

co 0

OR

OR'

Erythromycin (R=desosamine, R'=cladinose)

Lagosin

FIG.3. Substances related to fatty acids.

299

INTERivlEDIARY METABOLISM

p- Carotene

Eburicoic acid

Rosenonolactone

M0

0

H

O

d. CO. CH=CH. CH,

W

co CO,H Gibberellic acid

Trichothecin

r-------

I I

Mycelianamide

O

Auroglaucin

FIG. 4. Isoprenoid substances.

H

300

J. D. BU’LOCK

Though large and clearly defined, this category is less important in microorganisms than in plants, at any rate if “importance” is measured by variety of structures. 4. Amino Acid and Peptide Derivatives Here are included (Fig. 5 ) a number of antibiotics such as D-cycloserine and L-azaserine, and such inactive substances as 0-carbamylserine, which are clearly classifiable as amino acid derivatives. Equally clearly OC-CHNH, NH,CO. 0. CH, D -Cycloser ine

L-Azaser h e

Carbamylserine

RCO. NH. CH-P’,

Fumarylalanine

?Me,

Ao-N-CH-

HO,C. CH=CH. CO. NH. CHMe. C02H

CO,H

Penicillins

HO,C. CH. CH,. CONHl

HO

I I CO..CH,. NH

CO,H

I I OH

NH. CMe

NH Lycomarasmin Phalloidin

QCO hpergillic acid

O

C

Cliotoxin

t

301

INTERMEDIARY METABOLISM

L-lactyl-

NMe

I D-valyl

NMe

I

I

I L-Pro

~-valyl

I

D-a-HOiSOValeryl

I I L- lacty I

D-a-HOisovaleryl D-Valyl

I

L -valyl-

Valinomycin

H

Actinomycin C,

Ergot alkaloids V-CO

Violacein

FIG.5. Amino acid and peptide derivatives.

the bacterial polypeptide antibiotics and such simpler substances as the fumarylalanine of Penicilliurn reticulosurn, or the penicillins, can be classed as peptide derivatives. Somewhat more variety is seen in substances such as the actinomycins, which have a very varied selection of peptide side chains (Brockmann, 1960), or such fungal metabolites as lycomarasmin, the various diketopiperazines, gliotoxin ( Winstead and Suhadolnik, 1960), and the Arnunitu toxins; provisionally one can also include here the group of “peptolide” antibiotics, such as valinomycin, with their mixed ester-peptide chains of amino and hydroxyl acids. In most of these substances the amino acids are the common ones, sometimes with the unusual D-configuration, but “special” amino acids are found in addition. The constituent amino acids appear generally to arise by “normal” routes ( MacDonald, 1960), and at least in certain cases it has been suggested that the abnormal optical configurations

302

J. D. RU’LOCK

arise after assembly of the peptides (Amstein and Margreiter, 1958; Katz, 1960), though this may not be general. The production of peptide derivatives of unusual nature seems to be especially characteristic of bacteria, an observation which may be significantly related to the question of their cell-wall structures (cf. Section V ) . One might, of course, regard this category as including any species-specific peptide or protein, though such an extension seems rather unprofitable. There are a small number of alkaloids from fungi (e.g., ergot) to illustrate amino acid derivatives which are not primarily of peptide nature; in higher plants such substances are of course so numerous as to warrant the creation of a special category. The pigment violacein, derived from tryptophan ( D e Moss and Evans, 1960), is another example of an amino acid derivative which is not a peptide.

5. Sugar Derivatives Many antibiotics contain sugar derivatives, at least as part of the molecule. Sometimes these are “common” sugars, as in the neomycins ( D-ribose ) or D-xylosylnemotinic acid, but more frequently they are “unusual” (Fig. 6) ranging from digitalose and fucose (in chartreusin) to amino sugars like desosamine (in several macrolides), nonnitrogenoiis substances like oleandrose ( oleandomycin ) , or branched-chain sugars like cordycepose ( cordycepin ) , While some antibiotics such as the streptomycins are wholly made up of such units, most contain characteristic “aglycones” as well, Thus the D-xylose of xylosylnemotinic acid arises by ‘‘normal” routes ( Bu’Lock and Gregory, 1959) and even the N-methyl-L-glucosamine of streptomycin ( Silverman and Rieder, 1960) of hygromycin A ( Elbein et ( i l . , and the 5-keto-6-deoxy-~-arabohexose 1960) are formed fairly directly from D-glucose; evidence that other more exotic “sugar derivatives” are really derived from sugars is implicit in several less complete studies of precursor incorporation into the aglycones. Some simpler examples of sugar derivatives as secondary metabolites are the various sugar acids and alcohols, accumulated by many microorganisms, and the glucose-derived substances like kojic acid (Arnstein and Bentley, 1953). There are also many unusual polysaccharides, such as luteic acid ( polymalonyl-p-glucose ) , about which relatively little is known. In the light of newer knowledge about the cellwall materials of microorganisms, some relationship between these and the sugarlike secondary metabolites may be anticipated (Section V ) . As with the previous category an extension of the classification to include all species-specific polysaccharides is at least conceivable, if not particularly helpful.

303

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jHO CHZ

I H- C- OMe

I HO-C-H I I

HO-C-H

CHO

I

CHOH

I

CH HOH,C’ ‘CH~OH

I

CH,

L-Oleandrose

Desosamine

Cordycepose

0

HyTNj CH,OH

OH

OH Streptomycin

COWR I

OH Hygromycin

FIG.6. Sugar derivatives.

6. The C,;C:,Substances

The C,,C:,,or phenylpropane, skeleton has been recognized as a structural and biogenetical unit in a very wide range of plant products; like the isoprenoids, this group is somewhat less conspicuous in microorganisms (Fig. 7 ) though the corresponding general metabolites (phenylalanine, tyrosine, etc.) are of course universal. Some examples of this

304

J. D. BU'IACK

P-MeO. C,H,. CH-CH. C0,Me

(from Lentinus sp.)

P-NO,.Coho CH. CH. CHZOH

Chloramphenicol

I

I

OH NH.CO.CHCI,

H o r l ' = C H . CO. CH,. i!O Hispidin

HO \

TYoZH H \

HQ HO

HO

Protocatechuic acid

Gallic acid

\

Pyrogallol

Echinulin

Novobiocin

FIG.7. Phenylpropane derivatives.

group, such as the ergot alkaloids, gliotoxin, or the phenylalaninecontaining peptides, are as easily classed as amino acid derivatives; intermediate cases are compounds such as chloramphenicol and mycelianamide. Closer analogs of the nitrogen-free series in plants are relatively few, but include hispidin and other cinnamic acid derivatives in Basidiomycetes. As the C,C, series is defined biochemically as deriv-

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305

ing from the shikimic acid pathway, this category can also include related CeC1 compounds such as protocatechuic and gallic acids, and indole derivatives like echinulin. Chambers et al. (1980) showed that both the Ceca and CeC1 portions of novobiocin may be formed from tyrosine. In some cases, e.g., the pyrogallol from some penicillia, only experiment will show whether a substance belongs to this or to the following category, but in general the structural indications are fairly clear.

7. Acetate-Derived Aromatic Substances A very large number of aromatic secondary metabolites, especially of fungi, have a common biogenesis in that most or all of the carbon skeleton, and some other structural features such as the pattern of oxygen substituents, can be shown to have arisen from the head-to-tail linkage of C-CO units derivable from acetic acid. There is here a clear analogy with the over-all process of fatty acid synthesis, which in at least one typical case (Bassett and Tanenbaum, 1960) extends as far as the participation of acetate as acetyl-CoA.’ Again, as with category 2 (above), other acyl groups may be substituted, such as propionate in pyrromycin (Ollis et al., 1980). A study of 0l8incorporation has shown that this process of aromatic biosynthesis diverges from that of fatty acid synthesis in lacking the systematic dehydration and reduction reactions of the more general process (Gatenbeck and Mosbach, 1959). The recognition and experimental demonstration of this category, primarily by Birch and his co-workers (Birch and Donovan, 1953, and later papers), is only just beginning to be linked to more strictly biochemical studies; within this limit, the category has been adequately reviewed (most recently by Johnson, 1980), so that little special comment is needed here. A group of mechanisms for assembling and cyclizing the C, residues in various ways seems to be common to the whole category, while the individual features of particular metabolites seem to result from the further operation of various “unit processes” such as oxidation, reduction, alkylation, substitution, ring-opening, ring-coupling, and rearrangement reactions. The structures illustrated as examples (Fig. 8) are set out in such a way as to show some of the scope and variety of these processes.

8. Purine and Pyrimidine Derivatives Cordycepin and amicetin (Fig. 9 ) exemplify a characteristic group of substances which are clearly related to normal nucleotide components. ‘Added in proof: It is now confirmed that the “acetate chain” in aromatic (Bu’Lock and Smalley, 1961) and derived (Bentley and Keil, 1961) metabolites is built up, as in fatty acid synthesis, from one acetyl-CoA terminal group and a series of malonyl-CoA units.

306

J. D. BU’LOCK

Aur oglaucin

Mycophenolic acid

OMe -(C,) I

* =ringclosur e (Mosbach, 1960) ‘(fission) Penicillic acid

E

-Pyrromycinone

FIG.8. Acetate-derived aromatic substances.

307

INTERMEDIARY METABOLISM

Geomycin IV

Cordycepin

NMe,

ia

CHzOH

CH,OH

I

NH. CO. C,H,. NH. COCMe I

Amic etin

FIG.9. Some nitrogenous substances.

Some other nitrogenous substances, e.g., geomycin, may well be more obscure examples, related to intermediates in the synthesis or degradation of the heterocyclic bases. Very little is known about the role of nitrogen compounds in secondary metabolism.

9. Metabolites of Mixed Origin It will have been sufficiently clear from the examples already cited that many secondary metabolites are built up from units belonging to more than one of the above categories. It seems to be generally accepted that in the biosynthesis of such substances the supply of one such unit can be rate-limiting, but little is known about the fate of the remainder of the molecule under such circumstances. For example, it would be interesting to know the nature of the product formed (if any) from “acetate” if the synthesis of tetracyclines could be limited by a deficiency of glutamic acid, which Snell et al. (1960) suggest is the probable precursor of ring “A.” A study of situations of this kind might

308

J. D. BU’LOCK

throw much light on questions concerning the regulation of secondary biosynthetic processes.

C. THE RELATIONTO TAXONOMIC CLASSIFICATION Whereas in higher plants the occurrence of secondary metabolites, both individually and in groups, correlates rather well in most instances with the botanical classification of the producing species, and the way is open towards a degree of biochemical specification of the taxonomic groups, the situation with microorganisms is far less satisfactory. Since the microbiologists themselves are so often in disagreement over taxonomic questions this is perhaps not surprising, so that in spite of serious gaps and some very bad discrepancies we might be allowed a little satisfaction to find any significant correlation at all. One objection must first of all be considered. It has been argued that antibiotics and other secondary metabolites are the products of “sick” and incorrectly nourished laboratory freaks and therefore can have no relevance to the classification of a species, which is to be made in terms of “healthy” and “normal” individuals. It is true that there is a gap between most laboratory studies of cultural characteristics and the wild organisms, though there are several cases where such relationships have been explored without encountering inexplicable discrepancies. In any case, the objection is really rather a trivial one, for even if the actual production of some substance in a culture is strictly a laboratory artifact, the potential capacity to produce that substance under laboratory conditions is nevertheless a characteristic of the wild type. Moreover the morphological features of a culture are often themselves as much a product of laboratory conditions as the biochemical features. It is also curious that some of the taxonomists who refuse to consider antibiotic production as a criterion for classification are nevertheless quite satisfied to use their subjective impressions of color, which are after all dependent upon similar biochemical features. If particular secondary metabolites are to be regarded as characteristic of particular species, are the structural relationships and classification of those metabolites at all parallel to the relationships between those species in the taxonomic classification? The same question is of course posed in relation to more general metabolic features. If first we consider large categories there are several cases where such a relationship exists. Thus the polypeptide antibiotics are as characteristic of certain groups of Bacillus as are the rnacrolides of certain Streptomycetes; equally the higher fungi, especially the polypores and agarics, can be characterized by the frequent occurrence of a variety of triterpenoids and of polyacetylenes. It also appears that the greatest variety of acetate-

INTERMEDIARY METABOLISM

309

derived aromatic substances is produced by Ascomycetes and the related aspergilli and penicillia. However, when a closer analysis is attempted, serious difficulties arise, These are best illustrated with respect to the two groups from which the greatest number of secondary metabolites have been isolated, the Actinomycetes and the Fungi Imperfecti. In classifications of the Actinomycetes, the criterion of antibiotic production has been applied in taxonomy with varying success, and with different degrees of enthusiasm by different schools. Several such studies, in which the type of antibiotic produced has been correlated with the types of carbon source utilized by a species, have been summarized by Waksman (1957, 1959); the grouping of species thus attained does not correlate particularly well with morphological classifications. Waksman points out that the production or nonproduction of an antibiotic, and even the type of antibiotic produced, may be considered as strain rather than as species characteristics-in other words the biochemical and morphological features are not necessarily linked. For Krasil'nikov (1960), on the other hand, the production of a particular antibiotic ranks as a unique and characteristic feature of a particular species. Waksman and Lechevalier (1953), and others also, have set up groupings of Actinomyces based on cultural and morphological features, which sometimes correlate with groupings of antibiotics, so that the producers of, e.g., streptomycin, neomycin, or tetracyclines each fall together. So far as the structures of the relevant antibiotics are known, however, the applicability of the biochemical classification still appears limited; for example even in Waksman and Lechevalier's system both polyene producers and macrolide producers appear in more than one of the groups. The situation with regard to the Fungi Imperfecti is somewhat similar, particularly since the taxonomic status of the whole group is intrinsically dubious. To a limited extent relationships between metabolites are paralleled in the accepted classifications but beyond this limit there are serious incompatibilities, For example, of all the aspergilli, only species of the A. g2aucus group produce auroglaucin and flavoglaucin (Gould and Raistrick, 1934), but palitantin and frequentin, which biochemically are closely related, are metabolites of Penicillium pulituns (or P . yclopium) and P. frequentuns, respectively, species in two different sections of a different genus, while other quite closely related compounds (of the sclerotiorin type) occur in equally remote species of Penicillium and Monarcus. (The structures and relationships of these compounds are discussed in Section 111, D.) Again, the production of 6-methylsalicylic acid derivatives is fairly characteristic of species in the P . expansum, urticue, and claviforme groups of the section Asymmetrica-Fasciculata, but the same species, or other species of the same groups, also

310

J. D. BU’LOCK

produce metabolites such as griseofulvin, citrinin, or fulvic acid, which are equally characteristic of quite “remote” species. Similarly the P . gladiolii, palitans, and cyclopium groups of the section Fasciculata can be characterized by the production of penicillic acid, phthalaldehyde, and tropolone derivatives ( biochemically related) and also phenylquinoline derivatives, but penicillic acid and some phthalaldehydes are also produced by species of Aspergillus. The production of penicilPh I

CHO

Cyclopaldic acid

Cliotoxin

Viridicatin

Spinulosin

FIG. 10. Concurrent biosyntheses in dissimilar species.

lins characterizes not only the P. chrysogenum group but also species of Aspergillus and Cephdosporium. Sometimes quite distinct biosynthetic activities are apparently associated in more than one species (Fig. lo), for example those leading to the phthalaldehydes, etc., and to phenylquinolines in the above-mentioned Fasciculata; a striking example of linkage between quite dissimilar metabolites in widely separated species is the production of both gliotoxin and spinulosin by both Aspergillus fumigatw and Penicillium cinerascens. Faced with such a situation various extreme views can be taken. It can be held that the morphological classifications are uniquely “true,” the occurrence of particular metabolites wholly random, and the

INTERMEDIARY METABOLISM

311

coincidences and disparities exemplified above entirely accidental. To the writer this seems only somewhat more plausible than the less orthodox extreme, which would hold that the biochemical relationships, as for example epitomized in the metabolites, are the “true” ones and the morphological features incidental. It is in fact possible to make a fairly self-consistent classification of the Imperfecti using only the structural relationships between the secondary metabolites, though the utility of such an exercise is rather doubtful. The “truth,” if any, clearly lies between such extremes, and should be recognizable by its relevance to the phylogenetic origins of the different organisms. Seen in this light we must conclude that in going from ancestor to descendant the morphological and biochemical features have sometimes been transferred together and sometimes separately, and in this way both the limited coincidences and the far-flung inconsistencies have arisen. As an everyday illustration of this, it is easy to see how the surnames of a human population, which are derived from fathers and husbands, will afford a classification which is significantly, but not wholly or consistently, related to the blood kinships in that population; now if a second set of surnames, derived alternatively from mothers and wives, were available, this would afford an entirely analogous but quite different classification of the same individuals. The analogy at least suggests that any attempt to reconstruct the ancestry of microorganisms should consider both primary and secondary metabolic features as well as morphological taxonomy. The argument as to which serves as the “best” basis for classification is then partly a matter of convenience and partly a question as to whether the morphological or the biochemical features epitomize the larger and more fundamental portion of the hereditary determinants. To summarize, we cannot do better than to recall the conclusions of Kluyver and Van Niel (1936) concerning the characteristic metabolic features of bacterial species: , . . a morphological evolution has been primary and . . . an independent, though sometimes parallel, physiological evolution has occurred afterwards. Yet it does not seem excluded at all that in special cases the order of events has been the reverse and that in reality parallel morphological evolutions have taken place in two physiologically different groups.” “

111. Connections between General and Secondary Metabolism A. THESELECTIONOF PRECURSORS The secondary metabolites are of necessity formed from intermediates in general metabolism, and the two kinds of process can be regarded as diverging at particular points. These are the “prime” precursors of the

312

J. D. BU’LOCK

secondary metabolites, in the useful sense that they are the last identifiable substances common to both the general process and the specific synthesis being considered, For example, in the case of patulin (cf. 111, D ) one would today pick out acetyl-CoA as such a prime precursor (Bassett and Tanenbaum, 1980), but not glucose or 6-methylsalicylate, though both can be converted into patulin. Now it is remarkable that so far as these prime precursors have been identified they appear to form rather a restricted selection from the very wide range of general metabolic intermediates which might be thought to be available; conversely the great majority of general metabolites are not often involved as precursors in this way. Moreover, those intermediates which do function as prime precursors can independently be regarded as “key” intermediates of general metabolism, each being common to a number of important processes of breakdown, synthesis, and interconversion, and also subject to fundamental (if little-understood) control mechanisms. The case of “acetate” (acetyl-CoA) is a good example, since its central importance in catabolic and anabolic processes is as well known as its function as a prime precursor of the largest and most varied group of secondary metabolites. Other substances or groups of substances occupy a similar position, for example the precursors of isoprenoid and phenylpropane compounds, methionine, the common amino acids, etc. In fact it appears that the greater the variety of general synthetic processes involving a particular substance, the greater the uadety of secondary metabolites derivable, in one organism or another, from that same substame. In many cases the prime precursors have only been approximately identified, usually as substances “readily formed” from some exogenous and simple material such as sodium acetate or phenylalanine. It is conceivable that the special nature of secondary metabolism might extend to the mechanisms by which such substances were activated, but there is an increasing body of evidence that this is not the case, and that the actual reacting entities are the same as in general metabolism. Thus for many aromatic compounds the ultimate precursor is only known as “acetate,” but Bassett and Tanenbaum (1980)have now shown that for the 6-methylsalicylate series acetyl-CoA is an obligatory intermediate, and doubtless this will soon be more generally demonstrated. A similar role for malonyl-CoA seems very probable. In the acetate-derived polyacetylenes there is at least circumstantial evidence that malonate as well as acetate derivatives are implicated (Bu’Lock et al., 1981). The source of the methyl groups which, sometimes in oxidized states, occur very variously in secondary metabolites as ester, ether, N-methyl, or C-methyl groups, has been identified in several typical cases as the well-

INTERMEDIARY METABOLISM

313

known general metabolic sequence involving fonnyltetrahydrofolate and methionine derivatives, and almost certainly S-adenosylmethionine is the reactive substance here, The activation of amino acids for the synthesis of peptide antibiotics appears to follow a standard route, and the mannose of streptomycin B is at least likely to be transferred from its appropriate derivative, thymidine diphosphomannose ( Baddiley and Blumson, 1960). The incorporation of acetate into secondary terpenoids certainly proceeds by way of mevalonate, and prime precursors here are probably the same reactive phosphorylated derivatives as in general biosynthesis.

B. THE USEOF PARALLEL MECHANISMS If, as described above, important substrates are common to both general and secondary metabolism, is the same true of any important enzymes or enzyme systems? At first sight the answer would seem to be, of necessity, “no,” since the defining characteristic of secondary metabolism is the nongeneral nature of the end products, and this implies that the total mechanism of synthesis is peculiar to each case. However the formation of a metabolite is not necessarily a one-step process. Equally, not all enzymes of general metabolism are absolutely specific for their “usual” substrates. It is therefore conceivable that at least some of the reaction steps in the biosynthesis of various secondary metabolites might be characterized by the same enzymes, and moreover that some of these enzymes might also be involved in general metabolism. Although our knowledge of the reaction steps in secondary metabolism, let alone of the enzymes involved, is very limited, the available information does in fact lend some support to both of these possibilities.

c. UNIT PROCESSES The idea that some steps in secondary biosynthesis are effected by rather nonspecific means, common to a range of organisms and a range of metabolites, is one which can be supported by a number of examples of what may be called “unit processes.’’ Some of these appear to be wholly peculiar to secondary biosynthesis and are discussed later, but others are at least superficially parallel to known processes of general metabolism, and a few examples of these will be given here. The hydroxylation of aromatic rings appears to be a “unit process” common to the biosynthesis of numerous metabolites in both the acetatederived and C,C, series; usually it occurs at electronically activated positions in the ring. As an example of an analogous general process the formation of tyrosine from phenylalanine is the best known. An equally general process is that of methylation, and methyl groups of demon-

314

J. D. BU’IBCK

strably similar origins are found in secondary metabolites attached to phenolic and carboxylic oxygen, amine nitrogen, and even aromatic carbon. Moreover, in all the cases suggesting C-methylation as a step in the synthesis of acetate-derived aromatic substances, the methyl group is attached to a carbon atom derived from C-2 of acetate; it is not known at what stage in the synthesis such alkylation steps occur, but this element of specificity seems further to characterize a common process. An

‘--------I

__.-I

1

, NMe-CO L-..--.l

Echinulin (?)

FIG.11. Nonspecific isopentenylation in Aspergillus gluucus.

example of apparently indiscriminate esterification is provided by Polyp m s anthracophilus (Bu’Lock et al., 1957), in which various waterinsoluble acetylenic acids are synthesized and then esterified, either with acetylenic alcohols or with methyl groups, the latter almost certainly from the same source as the “extra” carbon atom which the fungus is simultaneously attaching to an acetate-derived triterpene (Dauben et al., 1957). A process of C-alkylation which is perhaps analogous to certain steps in the general biosynthesis of isoprenoids also seems to be rather widespread, and the case of the mould Aspergillus glaucus offers a striking example of such an apparently nonspecific isopentenylation mechanism (Fig. ll), common to the biosynthesis of two quite different

INTERMEDIARY METABOLISM

315

metabolites, auroglaucin (Birch et al., 19588) and echinulin (Birch, 1961). Similarly there appears to be a general sequence of oxidation which can modify both acetylenic and aromatic methyl groups through successive conversion to the alcohol, aldehyde, and acid, followed by decarboxylation (Jones, 1961). It is hardly necessary to add further examples of the possible “unit processes” that can be deduced from structural comparisons and a knowledge of the prime precursors, especially since these form the usual repertory of most “iogenetic schemes.” The rationale of these hypothetical constructions is that the steps are said to be “biochemically feasible”; this is in fact equivalent to the assumption that the reactions involved in secondary biosynthesis are not, after all, essentially different from those which biochemists have demonstrated in general metabolism. We are therefore entitled to inquire into the closeness of this connection. All the processes already exemplified can be paralleled in general metabolism so far as the functional transformations of the substrates are concerned, but we have as yet very little to show whether they are effected by the same enzymes, by modified forms of the same enzymes, or by enzymes which are merely functionally analogous and otherwise quite different. In at least one type of case, that of the aromatic hydroxylations and certain related processes such as quinone formation and oxidative coupling (Barton and Cohen, 1957), it does appear that the execution of such reactions is almost the only function that can be plausibly assigned to some well-known enzymes, the phenol oxidases of fungi; these enzymes are somewhat broad in their specificity and seem to be mainly involved in secondary metabolic changes, a matter which is referred to again in Section V. Another close connection with well-known reactions is seen in the ring-opening steps in the biosynthesis of patulin and penicillic acid; unfortunately in this case relatively little is known about any of the enzymes involved (see Section 111, D ) . In those cases where it is the quantity of a metabolite rather than its structure which is peculiar to some organism, cases which for functional reasons we have also classed as examples of secondary metabolism, it is of course very probable that all the mechanisms of synthesis are the same as in other organisms, the specific differences being in the control mechanisms that regulate the synthesis. Finally we should note that there is general and over-all parallelism between secondary and general biosynthesis, best exemplified by the fact that most of the categories set out earlier in our classification of secondary metabolites will serve equally well to classify the products of general metabolism. Thus there are common lipids and special lipid-like substances, common peptides and special peptide or peptide-like anti-

316

J. D. BU’LOCK

biotics, common structural and reserve polysaccharides and the unusual extracellular polysaccharides, and so on, and in each such group there are close similarities not only between the precursor units but also in their mode of assembly, For example, the difference between an unusual fatty acid like lactobacillic (Hofmann and Liu, 1980) and a “normal” CH~.(CH~)T.CH=CH.(CH~)~*CO~H Oleic

CH,.(CHZ),*CH-CH’(CHz)o*COzH

\

/

CHa Lactobacillic FIG.12. Oleic and lactobacillic acids.

one like oleic (Fig. 12), clearly results from a relatively minor addendum to the standard machinery of fatty acid synthesis. D. SOME SPECIALSYNTHETICMECHANISMS So far our deductions about the mechanisms of secondary biosynthesis have been reached indirectly, either from comparisons of structures or from demonstrations of over-all precursor transformations which give little information about intermediate stages. For the same reasons it has only been possible to consider those aspects of synthesis which can be closely paralleled in general metabolism. In this section we shall perforce use more evidence of the same kind, but in discussing some mechanisms peculiar to secondary biosynthesis we shall also be able to consider a few examples for which more detailed evidence is available. 1. Pmlcillins The biosynthesis of the penicillins has of course attracted more attention than that of any other secondary metabolite, and a considerable body of information was reviewed in the first volume of this series (Demain, 1959). The dynamics of the process will be considered later, and here we need only summarize what is at present known about the mechanism (Fig. 13). As a result of painstaking work by Arnstein and others (Amstein and Morris, 1980),it now appears probable that in the first step a tripeptide, (a-aminoadipyl)cysteinylvaline,is built up, from the a-aminoadipyl end. This is an open-chain form of the penicillin, cephalosporin N, in the subsequent formation of which the D-configuration of the valine moiety is apparently introduced during cyclization. Loss of the side chain from the cyclized tripeptide appears to be the source of 6-aminopenicillanic acid, while transpeptidation reactions may afford the other penicillins. For the biosynthesis, L-cysteine and L-valine

317

INTERMEDIARY METABOLISM

are required and are formed in the usual way; the origin of the a-aminoadipate is not known but it seems related to methiodine. The peptideforming reactions somewhat resemble those involved in the genera1 biosynthesis of glutathione (Fodor et al., 1953). One peculiarity of penicillin biosynthesis appears to lie in the cyclization and inversion process, which must be common to, and characteristic of, the whole 0- aminoadipate

cysteine

va ine

L____1

+

6-Aad-Cys

~ - C y s - ~ - V a;-l---------? 6-Aad-Cys-Val I

6-[6-Aad-amino]penicillanate

-

6-aminopenicillanate

(Cephalosporin N)

R . CO,H

transpeptidation'

I R . CO. NH. CH-CkS\FMe, &O--r[r--CHCOJi (Penicillins)

FIG. 13. Possible mechanisms of penicillin biosynthesis. After Arnstein and Morris (1960).

range of penicillin-producing organisms. The over-all process requires activating mechanisms which are blocked by, e.g. 2,4-dinitrophenol, and it has so far proved impossible to prepare cell-free preparations which will synthesize penicillins; as in some other instances, the synthetic apparatus appears to be rather easily destroyed. 2. Patulin and 6-Methylsalicyhte in the Penicillium urticae Series In the P . urticae series of the peniciIIia, a variety of aromatic metabolites has been found, at various times, in association with the heterocyclic antibiotic patulin. Largely through the work of Bassett and

318

J. D. BU’LKXX

Tanenbaum (1959, 1980) a plausible sequential arrangement of some of these substances can now be made (Fig. 14). In these species the first characteristic step in secondary biosynthesis is the transformation of acetyl-CoA (formed by glucose catabolism or by activation of exogenous acetate) into 6-methylsalicylic acid. This process is common Glucose ‘AC-COA-

Acetate/

E

0

2

H

heavy-metal enzyme

0-co

HOHaC

CHO

FIG. 14. Patulin and phenol formation in PeniciUIurn urticae, etc., after Bassett and Tanenbaum (1959); the compounds in parentheses have not been isolated and the detailed oxidation sequence is uncertain.

to the whole group of microorganisms and has been effected in a cellfree preparation. The 8-methylsalicylate sometimes accumulates as such, but frequently it may undergo a variety of oxidative changes (typical of those implicated in the synthesis of other acetate-derived substances) in which 3-hydroxyphthalic acid, gentisyl alcohol, or gentisic acid may all

INTERMEDIARY METABOLISM

319

be formed, as shown. Some species then carry out a further oxidative ring-opening of which the product is the open-chain form of patulin, which readily cyclizes. It is uncertain whether gentisyl alcohol or gentisic acid is the immediate substrate for this step ( Bu'Lock and Ryan, 1958), but with the alcohol as substrate and allowing for some internal hydrogen transfer processes the reaction can be written in the manner shown, which appears closely analogous (Fig. 15) to two known reactions of general metabolism, viz., the oxidation of 3-hydroxyanthranilate in nicotinamide formation ( Wiss and Bettendorf, 1957), and of homogentisic acid in tyrosine breakdown (Knox and Edwards, 1955). Data

OH2"."

C k/'H , C 0 2 H

H

OH

(Knox and Edwards, 1955)

( W i s s and Bettendorf, 1957)

FIG. 15. Analogs for patulin and penicillic acid formation.

for a detailed comparison of the special and general processes are not available; however, the mode of oxidation is also precisely analogous to one recently established for protocatechuic acid and catechol oxidation processes in certain microorganisms (Dagley et al., 1960). In all cases, heavy-metal enzymes seem to be involved, and this may explain the exacting trace metal requirements for patulin formation (Brack, 1947).

3. Penicillic Acid in the Penicillium cyclopiuni Series In the P. cyclopium and A. terreus series and in some other Imperfecti (Fig. 16) the formation of penicillic acid appears as the counterpart of patulin formation in P . urticae. Birch and co-workers (1958e) deduced from the mode of incorporation of acetate into this acid that an aromatic

320

J, D. BU’LOCK

compound was intermediate, and Mosbach (1960) then showed that it could be formed from exogenous orsellinic acid by P. baarnense (or by a cell-free preparation therefrom). This acid has not been isolated from this particular fungus, but on the other hand a whole series of aromatic Glucose

FHO

I

1-22

Cyclopaldic acid

‘ F O H OMe Penicillic acid

/ /

(- co*7)

P.puberulum

/

0

HO

OF Puberulonic acid

OH Stipitatonic acid

FIG. 16. Penicillic acid and other metabolites of Penicillium cyclopium, etc.; sequential details hypothetical.

metabolites, including many which are very characteristic of the P. cycloplurn group, such as the phthalaldehyde (Birch and Kocor, 1960) and tropolone (Richards and Feretti, 1960; Bentley, 1960) derivatives, would also seem, from their structures and from over-all precursor-incorporation

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321

studies, to be derived from acetate by the same pathway. The cyclization to orsellinate, as demonstrated by Gatenbeck and Mosbach (1959) in a species of Chaetmium, is distinctively different from that leading to 8-methylsalicylate, as the latter includes a reduction step which removes one of the acetate-derived oxygen atoms. As in patulin formation, the conversion of orsellinic into penicillic acid must involve various “unit process” transformations including decarboxylation and O-methylation, the order of which is not known, but the over-all course of the ringopening step is fixed by isotopic data and again appears to be rather closely analogous to 3-hydroxyanthranilate and homogentisate oxidations.

4. Resorcinols and Naphthalenes in Daldinia concentrica An ascomycete, Daldinia concentrica, has been studied in the wild state (Allport and Bu’Lock, 1960) as well as in laboratory cultures, and an interesting sequence of processes partly elucidated (Fig. 17). Most cultural strains of this species can kffect the aromatization of acetate units, in yet another pattern (for which other parallels are known); in this the terminal carboxyl of the acetate “chain” is incorporated into the aromatic ring. In Daldinia concentrica the process may involve either four or five acetate units, giving 2-acetylresorcinol in the first case and a group of 2-acetoacetyl .derivatives in the latter. However the wild D. concentrica and some laboratory strains can also effect a complete cyclization of the five C, units, giving 1,8-dihydroxynaphthalene.Some strains deficient in oxidase activity accumulate this phenol as its methyl ethers, but otherwise it undergoes oxidase-catalyzed coupling to give mixtures of a tetrahydroxydinaphthyl, a dihydroxyperylenequinone, and a quinonoid polymer, the last being a black pigment which invests the reproductive structures, conidia, or wild fruit bodies. The complex of processes is shown in the figure. The strain differences between all but the most defective cultures were found to be quantitative rather than absolute, and activities originally absent were found to appear on prolonged incubation or with fresh substrate.

5. Anthraquinones in Penicillium islundicum Several penicillia of the Biverticillata-Symmetrica produce anthraquinones and dianthraquinones structurally related to emodin, and one species, P . islandicum, which produces several of these compounds, has been especially studied by Gatenbeck (1960a, b ) . These quinones incorporate both 0 l 8 and C1*from added acetate in a way consistent with their derivation from acetate units, and the activation and assembly of these units seem to be relatively fast processes, which constitute yet another characteristic cyclization pattern. There are four main products,

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J. D. BU’LOCK

named in Fig. 18, which it is simpler to refer to as A, B, C, and D. Despite their structural similarities, by studying the time course of C1* incorporation it was found that each one arises by an independent, though parallel route, Now it can be seen that structures A and B differ

some strains

+

HO

polymers

OH 0

FIG. 17. Resorcinol and naphthalene derivatives in DaZdinM concentrica (Allport and Bu’Lock, lQ60).

from structures C and D in lacking one of the acetate-derived oxygen atoms (the difference being similar to that between 6-methylsalicylate and orsellinate, above), and we may reasonably surmise that this differentiation would take place rather early in the biosynthesis, while the differences between A and B, and between C and D, could arise later, still without any one of the four being a precursor of the others.

323

INTERMEDIARY METABOLISM

0

Emodin

Islandicin A

Rubroskyrin and luteoskyrin C

Iridoskyrin

Skyrin

B

D

FIG. 18. Anthraquinones from Penicillium islandicum ( Gatenbeck, 1960a, possible order of biosynthetic steps might be:

+cA

b). A

Ac-COA

(i)

&I-.:

where ( i ) includes a process directly affected in mutants, and ( i i ) includes a reduction step which is absent in (iii).

This itself indicates that the biosyntheses occur in a number of separable steps. [This situation is similar to that found with D.concentricu, above, where one of the 2-acetoacetylresorcinols was shown to be an alternative product to the naphthalenes and not a precursor, and to that in Neurospmu crQssu (Krzeminski and Quackenbush, 1960) where the various

324

J. D. BU’LOCK

carotenoids appear to be formed by partly parallel rather than wholly sequential reactions; it is in contrast to that in P . urticae (above) where isolable aromatic compounds can act as substrates for further transformations.] With a range of P . islandicum mutants, however, Gatenbeck and Barbesgard (1960) found that either all aromatization processes were blocked, or, all the metabolites of the parent strain were formed, albeit in different amounts and proportions; such observations are fairly common (see Section IV, C). It is apparent that some early reaction step is common to the biosynthesis of all the compounds and controlled directly by a single genetic unit, while later steps are distinct at various points, as indicated in the figure. 6. Some Other Speclal Mechanisns The few preceding examples virtually exhaust (at the time of writing) our more detailed information on particular secondary metabolic pathways, though there is reason to think that they do at least represent fairly typical cases. We can supplement the picture with examples of other special and characteristic mechanisms which seem to be implied by structural features and which are common to, or parallel in, the biosynthesis of more than one metabolite. Two typical cases may be instanced here. The structures shown in Fig. 19 exemplify how a common pattern of assembly of acetate units can be found in different compounds from different organisms, viz. Aspergillus glaucus (auroglaucin) (Birch et al., 1958g), Penicillium frequentam (frequentin), P. palitans (palitantin) (Birch and Kocor, 1960), P. sclerotiorum and P. multicolor (sclerotiorin and rotiorin), and Monas~csrubropundatus (rubropunctatin). The taxonomic diversity has already been discussed (see Section 11, C ) . The last three examples also suggest certain further mechanistic features (Haws et al., 1959; Birch et aZ., 1958d, g). Sclerotiorin contains chlorine, the introduction of which, as in the tetracycline series, is clearly an inessential step. Where in sclerotiorin a hydroxyl group is esterified with acetic acid, the same esterification in rotiorin, effected with acetoacetic acid, has been followed by an aldol-type condensation. In rubropunctatin a different organism has carried out the same basic synthetic process but with the omission of two acetate units and two (introduced) methyl groups from the side chain, and followed this by the same esterification and condensatioq as in rotiorin but with 3-ketooctanoic acid in place of acetoacetic. The whole picture thus implies a considerable sharing and grouping of synthetic mechanisms which are not wholly specific. A somewhat analogous but simpler case is that of the tetronic acid derivatives (Fig. 20), which in the somewhat diverse species (Birkinshaw and Samant, 1980) Penictllium terrestre, P. drldicatum, P. Charledi,

325

INTERMEDIARY METABOLISM

Auroglaucin

(C,) OH

O

W

O

H Palitantin

Rubropunctatin I

---_-___

I

Rotiorin

(Cl)

FIG.19. Variations on a cyclization pattern.

P. cinerascens, and Alternaria tenuis, are apparently formed by the same basic process, the condensation of an a-amino or a-hydroxy acid with an acetate-derived 8-ketoacid (Lybing and Reio, 1958; Sticking and Townsend, 1980). The reaction does not seem to be highly specific, since the known products (see Fig. 20) are derivable from isoleucine, malate, or lactate on the one hand and acetoacetate, 3-ketohexanoate, 3-keto-6-

J. D. BU’LOCK

326

CH,. CH. C0-I-CH,

Lactic

I

I

0

I

acetic

co

I

y-methyltetronic acid HO,C. CH,. CH. CO-I-CH.

Malic

I 0

;

I

I. co

CO.C,H, 3 - ketohexanoic

carlosic acid HO,C. CH,. CH. CO-4-CH.

Malic

I

0I

I

CO. CH,CH,CH,OH

3-keto-6-hydroxyhexanoic

do

carlic acid CH,. CH. C O A C H . CO. CH,CH,CH,OH

Lactic

‘; co I

I

0

3-keto-6-hydroxyhexanoic

carolic acid CH,. CH. CO-I-CH.

Lactic

I 0-co

‘ I

CO. CH,CH,CO,H

3-ketoadipic

I

carolinic acid H0,C. CH,. CH. C0-I-CH.

Malic

I 0

; I

CO. C,H,, I

co

3-ketooctanoic

virdicatic acid CH,. CH. CO-I-CH.

Lactic

I 0

‘; co I

CO. CH,CH,CHEt I

OH

3-keto-6-hydroxyoctanoic

terrestric acid

Isoleucine

Et.CH.CH.CO+CH.COCH,

I 1 ‘ I Me NH-CO

acetoacetic

FIG. 20. Tetronic acid derivatives.

hydroxyhexanoate, 3-ketoadipate, 3-ketooctanoate, or 3-keto-6-hydroxyoctanoate on the other.

IV. The Interaction of General and Secondary Metabolism A. RELATIONOF SECONDARY METABOLITES TO GROWTH 1. General So far we have considered the processes of secondary metabolism as though they were rather isolated activities, and while we have inquired

INTERMEDIARY METABOLISM

327

into their mechanistic resemblances to general processes the dynamic interactions between the two have not been considered. In particular, we have noted that the prime precursors of secondary metabolites are important intermediates in the general metabolic processes of the producing organisms, so that the two kinds of activity must be closely interconnected. Further interactions will result if, as has been suggested, the two sets of processes also share particular enzymes and coenzymes. Another mode of interaction will result from the fact that both general and secondary biosynthesis may be dependent upon the provision of reactive intermediates, usually from an oxidative phosphorylation process ( cf. several cases of the inhibition of antibiotic biosynthesis by 2,4-dinitrophenol, etc.). The most obvious manifestations of such dynamic interactions is the appearance of cell growth and secondary biosynthesis as competing or incompatible processes, an observation which is almost a commonplace of fermentation practice. Though there are a few cases in which the appearance of a secondary metabolite runs parallel to the growth of the producing organism, such cases are rather exceptional and for the most part they do not seem to involve any far-reaching synthetic activity; several are, in fact, rather clear examples of the partial utilization of substrates. More typically, the synthesis of secondary metabolites is wholly or partly suppressed while the cells in a culture are actively multiplying, and is most rapid when the culture reaches a stationary or resting stage. (With a heterogeneous cell population, as in surface cultures, the differences may be less marked since some cells reach this “resting” condition earlier than others, but the phenomenon usually remains quite apparent.) The same effect is also apparent if the rate of substrate utilization is measured instead of the cell weight or number, for this rate usually falls markedly when the multiplication phase ends; since the rate of the special synthesis then increases, the actual fraction of substrate converted-i.e., the efficiency of the synthesis-shows a marked increase. Essentially the same phenomenon is also frequently observed when a pregrown cell population is supplied with a replacement medium which is in some way inadequate for further growth but will still sustain the secondary metabolic process; the resting cells now convert an increased fraction of substrate into the metabolite and it is synthesized more rapidly. The general superiority of such ‘
328

J. D. BU’UKX

Observations of this kind are readily understood if, as is the usual case, the prime precursors of the metabolites are also essential for the integrated general synthesis which constitutes cell multiplication. When through a deficiency in some essential material, or for some other reason, this integrated activity is no longer possible, major readjustments must be made in the economy of the cells, and it is at this time that the secondary processes are activated. In a few cases, rather more detailed knowledge of these adjustments is available, chiefly from studies of commercially important antibiotic fermentations, and we shall therefore consider some of these,

2. Penicillin It is well known that the conditions for rapid penicillin production (reviewed by Hockenhull, 1959) are not the same as those for rapid growth of P. chysogenum, etc. In typical industrial fermentations the mycelium first multiplies at the expense of the most readily available nutrients (e.g. lactic acid), and when these are exhausted growth slows down and penicillin production begins; for this phase a slowly utilized carbohydrate such as lactose is commonly provided, though a slow addition of more easily available sugar has the same effect. Once the penicillinproducing stage is reached, though not before, the mycelium will in fact liberate considerable amounts of penicillin even when suspended in water, especially if a side-chain precursor is also added, but antibiotic synthesis is more prolonged on a minimal medium which also provides an energy source. The change from the growth phase to the production phase involves both cytological and biochemical alternations. Bekker and co-workers ( 1956) have described the morphological changes in some detail, and observed simultaneously with the onset of penicillin production the appearance of volutin granules, an increase in lipolytic activity, and a decrease in the fat content. All these changes seem to be part of a general adaptation to the changes in nutritive conditions. 3. Streptomycin

During growth of S. grkeus little streptomycin is produced, and maximum production is reached in the subsequent “mature” phase, Recently Hockenhull ( 1960) has reviewed the connections between antibiotic synthesis and “normal” substrate utilization by this organism, particularly the pathways of sugar catabolism and phosphorylation. On assuming the biosynthesis of streptomycin and mannisidostreptomycin to be comparable to that of more common oligosaccharides, and to depend on the provision of reactive phosphorylated sugars as precursors, the observed effects on the fermentation of such important variables as

INTERMEDIARY METABOLISM

329

oxygen, phosphate, glucose, and nitrogen supplies appear to be fairly reasonably explained. 4. Actinomycin In actinomycin formation by Streptomyces antibioticus (Katz et al., 1958), most antibiotic is produced after the growth rate has fallen off. If the main carbon and energy source provided is one which is readily utilized (such as glucose) then it is all exhausted by the time that antibiotic production sets in, so that the synthesis must be at the expense of accumulated reserves or autolysis products; hence the advantage of providing a less readily utilized nutrient (galactose), some of which will remain available at the end of the growth phase, so that a higher level of antibiotic production can be sustained. 5. Tetracyclines Changes in the Streptomyces sp. during the production of tetracycline antibiotics have been characterized frequently. Thus with S. aurofuciens Di Marco and others (Di Marco and Pennella, 1959) have described a first phase of rapid growth and synthesis of all cell constituents including nucleic acids, which is followed by a slower mycelial growth with falling protein and RNA content; this slower growth is accompanied by chlortetracycline production. With extra phosphate in the medium the features of the first phase are more marked whereas in the second phase the slow growth is inhibited and antibiotic production reduced. Prokofieva-Belgorskaya and Popova (1959) made similar observations for this and other Streptomyces; in low-phosphate media the antibiotic-producing phase was characterized by the presence of thin hyphae with low DNA content, which grow mainly by accumulation of cytoplasmic material, with falling RNA content and a high rate of antibiotic production, while in high-phosphate media thick hyphae, with much DNA, increasing nuclear elements, and inhibited cytoplasmic growth, were associated with low antibiotic production. These authors comment upon the strong interdependence between nucleic acid synthesis, pathways of carbohydrate metabolism, and antibiotic-producing capacity. Doskocil and co-workers ( 1958) described similar observations with oxytetracycline-producing S. rimosus. The first phase was a logarithmic growth of thick gram-positive mycelia, rapidly synthesizing nucleic acids and respiratory enzymes and using up the glucose, free amino acids, and phosphate of the medium, with an accumulation of pyruvate but no antibiotic formation. With the exhaustion of these readily available nutrients antibiotic production set in and the pyruvate disappeared (the tetracyclines are largely acetate derived; Snell et al., 1960); long gram-

330

J. D. BU’LOCK

negative mycelium was formed, growing at the expense of starch in the medium, with little DNA synthesis and a fall in RNA, and antibiotic production continued after the exhaustion of the medium until autolysis set in.

6. Miscellaneous Less well-characterized instances of similar phenomena are very numerous, and merely to enumerate them would add little. It should however be noted that the general situation is not peculiar to antibiotic production, and is observed as frequently in the formation of quite different metabolites including “normal” substances, e.g., the extracellular lipids of Rhodotorula sp. ( Deinema and Landheer, 1960), the carotenoids of Phyconiyces sp. ( Goodwin, 1959), and 5-ketogluconate in Acetobacter suboxydans strains ( Stubbs et al., 1940). In the production of the nemotin group of acetate-derived polyacetylenes there is a similar antagonism between growth and secondary biosynthesis, which is maximal in “resting” cells of the basidiomycete. This maximal condition can be realized either with glucose or with ethanol as the replacement substrate, and in either case it is found that the proportion of precursor acetate converted into the polyacetylenes is the same, the rest being oxidized or assimilated, However, the observed rate of synthesis from ethanol is considerably greater, i.e., the turnover of the precursor acetate is greater, It is believed (Bu’Lock et al., 1961) that this increased turnover is required to maintain a predetermined rate of mycelial assimilation, which with glucose as substrate can be effected without involving precursor acetate, the assimilated material probably being of carbohydrate nature. In this case, at least a part of a mechanism of interaction of general and secondary processes can be discerned.

B. SECONDARY METABOLITESAS “SHUNT”PRODUCTS It is at once apparent from the foregoing that the secondary metabolites cannot, as has sometimes been suggested, be lumped together as “reserve materials.” In any case the fundamental error of this view, as pointed out by Foster (1949), is that it can in no way account for the extracellular accumulation of substances which in many cases differ but little from other substances retained in the cells. It is also apparent that it is an oversimplification to state, in considering the ecological implications of secondary metabolism, that antibiotic production is characteristic of microorganisms in a rich nutritional environment; it would seem rather to be associated particularly with the exhaustion of such an environment, On the other hand the description of antibiotic and other

INTFXMEDIARY MmABOLISM

331

substances as “shunt metabolites,” as very cogently argued by Foster, seems to be more illuminating. The case of the nemotins mentioned above constitutes a good example of a “shunt” process. In the simplest sense, a shunt metabolite is one which is formed from a “normal” intermediate when other pathways for that intermediate are wholly or partly closed. The shunt pathway may involve one or many steps; indeed in the special case where the “normal” intermediate accumulates it involves no steps at all. Thus in citrate formation by Aspergillus niger the accumulation of this intermediate results from a fall in isocitric dehydrogenase activity, reinforced by a fall in aconitase, which is not accompanied by a fall in condensing enzyme activity or the overall intake of substrate (Ramakrishnan et al., 1955). If in this system an alternative pathway for the citrate were opened, perhaps as a result of enzymic induction, some other substance would then accumulate in its place. Such a situation appears to exist in the related species A. teweus, in which an enzyme acting upon cis-aconitate causes an accumulation of itaconic acid ( Bentley and Thiessen, 1957). In Acetobacter suboxydans the production of 5-ketogluconate appears to be a similar phenomenon; during the bacterial growth phase all the glucose is converted into gluconate, and the further oxidation of this begins sharply when the stationary phase of the fermentation is attained (Stubbs et al., 1940).

C. EFFECTSOF MUTATIONS It is not necessary that the blocked “normal” process and the newly opened shunt process should have the same immediate substrate, and indeed such a simple situation may be relatively uncommon. Only in this light, for example, can some of the effects of mutations on secondary metabolism be understood. It is true that the best-known examples of the effect of mutations on metabolic pathways are those in which the substrate of a process directly affected by the mutation accumulates, but this is because such mutations have been the ones selected by the investigators, since their study yields the most valuable information, For the most part, studies of this kind have been concerned with mutations which affect general, not secondary, metabolic reactions. The usual effect of mutations on secondary biosynthetic processes is either to block the whole series of reactions completely or else to accentuate it to varying degrees. In the first type of situation some step essential to the whole process has been eliminated, and the usual failure to detect accumulating intermediates of the secondary process suggests that the missing step is one in general metabolism, while the quantitative nature of the effect in the second type of situation suggests that the increased synthesis is a response to some all-or-nothing event relatively remote in the metabolic

332

J. D. BU'U)CK

network. The ultimate effect, the apparent intensification of a character as a result of mutation, is mechanistically the same as that attained by blocking general processes in other ways, as by the action of inhibitors or by limiting the availability of some essential substrate. Such effects are of course widely applied to increase fermentation yields; a similar study with P. kslandicum has already been cited. In some recent studies, however, rather closer connections between the genetic changes and a secondary biosynthesis have been observed. Thus in Streptmyces uureofuciens strains, the ultimate step in the biosynthesis of 7-chlorotetracycline (Fig. 21) appears to be reduction

enzyme absent in "W5"'

///

+

cofactor

'\'

'absent in "S-1308"

7-chlorotetracycline FIG. 21. Defect mutations in 7-chlorotetracycline formation ( McCormick et al.,

1960).

of the 5a(lla)-dehydro derivative. One mutant is known which is unable to carry out this reduction rapidly enough to convert all the dehydro derivative (which therefore accumulates); it appears to be blocked because it lacks a cofactor which other strains can synthesize. Another mutant contains the cofactor but still cannot effect reduction; in this case the reducing enzyme may be absent ( McCormick et al., 1960). It is possible, however, that these mutations are not solely, or even primarily, connected with the secondary biosynthesis, since the cofactor and the reductase may also have more general functions. Some mutants of Serratia mrcescens (Wasserman et al., 1960) are defective in their ability to synthesize the pyrrole pigment prodigiosin. This arises by a direct reaction (quite possibly nonenzymic) between two moieties, a dipyrrylaldehyde and 2-methyl-3-pentyl pyrrole, and mutants are known (Fig. 22) which are defective in the synthesis of either moiety and which accumulate the other. The actual sites of the genetic defects are not known.

D. ENZYMICINDUCTION EFFEC~S Given a novel accumulation of some precursor, there may well be cases where its transformation into some secondary metabolite will occur

INTERMEDIARY METABOLISM

333

spontaneously or can be effected by enzymes already in existence, but in other cases the acquisition or accentuation of a secondary pathway will require the creation of new enzymes or the increased synthesis of old ones. Where such synthesis of new enzymes is involved we might well suppose it to be induced by the accumulating substrate, and experiments to test this supposition would provide valuable information. Some evidence in support of this is already available. absent in "W-1"

absent in

"9-3-3"

w::Tc H

H

H

H

H Prodigiosin

FIG.22. Defect mutations in prodigiosin formation (Wasserman et al., 1960).

In the first place is the observation, already noted, that genetic changes are seldom observed to lead to an accumulation of intermediates in a secondary biosynthetic process, which in itself suggests that the enzymes responsible for that process are not always under the direct control of the genetic determinants. In such a situation induction seems to be the most probable controlling mechanism. The production of the enzyme tyrosinase, which is rather closely associated with secondary processes (see Section V ) in Neurosporu is apparently controlled primarily by an inductive mechanism and only less directly by the gene structure (Horowitz et al., 1960); this enzyme only appears at a particular developmental stage. It is commonly known that simple changes in the substrate are often quite sufficient to cause marked changes in secondary metabolism. Sometimes the effect is simply that new possibilities are opened to the biosynthetic system, for example the production of modified side chains in penicillins ( Hockenhull, 1959), or actinomycins ( Schmidt-Kastner, 1956; Katz, 1960), when new side-chain precursors are supplied. In other cases the effects are so bewildering-as when a mold produces one sub-

334

*

J. D. BU’LOCK

stance on Czapek-Dox medium and something totally different on Raulin-Thom-that any interpretation seems impossible. But some cases of intermediate complexity may usefully be considered. As shown by Brack (1947), certain strains of P. urticue can be made to produce either gentisyl alcohol or patulin by a small change in the iron content of the medium. It is now known (Section 111, D ) that the main additional reaction involved in this change is the ring-opening step, and though the relevant enzyme has not been isolated, various analogous reactions of general metabolism are catalyzed by iron-requiring enzymes. It therefore seems likely in this case that the effect of the substrate change is primarily upon the enzyme constitution, though the mechanism is not, of course, one of induction. A large number of Basidiomycetes have been tested by Jones and coworkers and found to produce polyacetylenes when grown on a supplemented glucose medium (Jones, 1961); it is known that the prime precursor of these polyacetylenes is “acetate” ( Bu’Lock and Gregory, 1959)When pregrown mycelium is supplied with glucose-acetate mixtures or with acetate alone, increased yields are frequently obtained, and there may be minor changes in the relative proportions of the products. Such effects have already been considered. However, in certain cases, the effect of added acetate is to stimulate the production of some quite new polyacetylene-for example a C, compound may be replaced by a C,, compound of quite different structure (Jones, 1961). Some specific enzyme changes seem to be involved here and to have been induced by an increase in the availability of substrate; such a case would not seem to differ superficially from the postulated one where similar changes follow internally determined substrate accumulations, In the related case of the diatretynes (Section I ) from Clitocybe diatreta, the situation appears somewhat more complicated; Silverman and Anchel (1959) showed that the two C, compounds on the one hand, and a related C,, compound on the other, are sometimes produced together, and sometimes singly-even when cultures from single-cell isolates are used. Another interesting case is that of Streptomyces griseus, in which a mannosidase which breaks down streptomycin B is apparently adaptive and inducible (Hockenhull, 1960); this induction can only be effected during growth, while the substrate does not appear until growth has ended!

E. EFFICIENCY OF SECONDARY BIOSYNTHESIS The efficiency of a fermentation can be related to the total substrate consumed, or to the relevant substrate consumed during a relevant period; as seen from the foregoing, the two are not necessarily the same, It is also apparent that while the yield of a particular metabolite can

INTERMEDIARY METABOLISM

335

be increased by suitable changes in the substrate, a limit to this increase will be imposed by the genetically determined enzymic constitution of the cells. Beyond this limit the yield may be further increased by selection of suitable mutants. But, as observed in the selection of high-yielding P . chrysogenurn strains, this effect is achieved by progressively blocking general pathways in the organism, and the limit to this process is set not only by the minimal requirements for the mutant to survive and to be propagated but also by the need for coupled catabolic mechanisms to “drive” the synthetic process. Moreover if the same substrate is to be used for the biosynthetic and the catabolic processes the efficiency of biosynthesis is correspondingly reduced; this effect explains the beneficial “sparing” effect of added fats on the synthesis of antibiotics such as penicillin, which is not a direct substrate effect.

V. The Functions of Secondary Metabolism No one can spend long in consideration of the so-called natural products before asking, or being asked, the question “What are they supposed to do?,” and to evade that question here would be unfair since we have already raised it, in defining secondary metabolites as having “no obvious function!” If the formation of these curious substances were only a freakish occurrence, or if it only involved a negligible fraction of total metabolic activity, we might be satisfied to dismiss the query on grounds of teleology, with its implication of a necessary purpose in biological events, but this is not the case. The formation of some kind of secondary metabolite is a feature of most microorganisms (and plants too), and under appropriate conditions an organism can survive quite effectively though devoting a very high proportion of its vital synthetic capacity to the production of some extraordinary substance. The capacity for secondary biosynthesis is a major feature of the organisms, and we must suppose it to have conferred some selective benefit in the course of their evolution. Now since the best-known secondary metabolites of microorganisms are antibiotics, it is very tempting to explain this selective advantage as one which operates by a toxicity mechanism in competitive situations. We shall return later to the question of whether antibiotic production actually has a significant effect in such situations, but at this point it is sufficient to reiterate what has already been observed, namely that no hypothesis based upon any one kind of intrinsic property or activity in the metabolites will account for the production of secondary metabolites in general, because these substances are known in such variety that no such single property can be found that is common to all of them. For every microorganism producing some extracellular substance antibiotic to its natural

336

J. D. BU’LOCK

competitors, there is one which produces by related mechanisms an intracellular substance which can have no such activity. Since the intrinsic properties are too restricted to afford a general explanation of secondary metabolism we are driven to a search for some common features, and in the preceding pages some such features have indeed emerged. They can be summarized thus: The formation of a secondary metabolite involves ( a ) the conversion of a normal substrate into important intermediates of general metabolism by standard mechanisms, and ( b ) the assembly of those intermediates in an unusual manner, by means of a combination of standard general mechanisms with a selection from a relatively small number of special mechanisms: ( c ) the special mechanisms are peculiar to secondary metabolism though not unrelated to general mechanisms; ( d ) the secondary metabolic activity appears, or is intensified, in adaptive response to nutritive conditions unfavorable for cell multiplication. An hypothesis consistent with these generalizations is to conclude that: the selective advantage of secondary metabolism is that it serves to maintain mechanisms essential lo cell multiplication in operative order when that cell multiplication is no longer possible. Though there is very little direct evidence as to the fate of cellular enzymes when deprived of their substrates, there is a general probability that they will tend to disappear; even in uitro, the substrate stabilization of enzymes is a somewhat general phenomenon. In default of special protective means, this would imply that when the integrated biosynthesis of new cell material is prevented, even by the exhaustion of one quite minor but essential substrate, a general breakdown of all these synthetic mechanisms would ensue, so that normal functioning at a later time would only be restored after extensive renewal of enzymes and coenzymes. Moreover the uptake of other nutrients would cease, leaving them available for the growth of competing orgqnisms which might not require the particular nutrient which is exhausted. The organism would thus be placed at a double disadvantage, both internal and external, A capacity for secondary biosynthesis will afford some defense against this situation; a relatively limited de m o synthesis of special enzymes will be enough to permit a number of the general synthetic mechanisms to continue operating despite the exhaustion of a particular substrate. The secondary synthetic process need not be very critically integrated since its end product is of no special significance; meanwhile the uptake of the remaining nutrients can continue freely, thus denying their use to any competing organisms. Since parts of the mechanisms are those which normally would help in the replication of a particular and characteristic type of cell, the characteristically specific nature of the end products is

INTERMEDIARY MGTABOLISM

337

understandable, and their lack of significant general intrinsic properties reflects the fact that it is the activity of syntliesis, rather than the nature of the end product, that is properly of value to the organism. Within the framework of this hypothesis, the possible significance of antibiotic activity can be re-examined, and the question restated: Given that secondary metabolism can confer a selective advantage irrespective of the nature of the end product, does antibiotic activity in the end product confer an additional advantage? The mere existence of antibiotics does not necessarily show that this is so, for simple reasoning indicates that some kind of biological activity is inherently rather probable in a secondary metabolite. The mechanisms of synthesis are in part those which normally lead to important cell constituents, which the secondary metabolites are therefore somewhat likely to resemble, and such limited resemblances, we know, are the basis for many kinds of antimetabolite activity. However, when antibiotic activity is no longer put forward as the primary advantage conferred by special metabolism, the arguments that it can confer some advantage remain. Such arguments, which are really far too lengthy to be reproduced here, are fortunately available in a very thoughtful review by Brian (1957),with the general conclusion that under certain circumstances antibiotic production may well confer a definite ecological advantage to certain organisms, but that the phenomenon is not necessarily general. In the same way the idea that secondary metabolites accumulated within the cells function as reserves can also be reconsidered. As pointed out by Foster (1949),most microorganisms will make some show of attacking quite extraordinary substances if sufficiently hard pressed, and this is no ground for describing all accumulated substances as reserves. However, it does seem plausible that under certain circumstances an organism might derive some additional advantage from having produced, and accumulated, a secondary metabolite which could if necessary be used as a nutrient. These and other possibilities, however, should be viewed as incidental advantages of secondary metabolism, not essential to its main function. It is not easy to think of direct experiments to test our hypothesis, but some published observations do seem to lend some support. We have argued that secondary biosynthesis is a response to conditions not permitting further cell multiplication; now in most microorganisms the long-term response to such conditions is the formation of specialized structures with the function of prolonging survival under adverse conditions and permitting a resumption of growth at a later time or in a different place-is., the formation of spores, conidia, sclerotia, etc. It is therefore interesting to note the growing number of instances of connections between such

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processes and typical examples of secondary metabolite formation. Thus in DaZdinia concentrica the various secondary metabolites from the mycelia are in a way imperfect representations of a functional process of which the complete expression is seen only in conidia and sporophores; this central pathway (Section 111, D ) leads to the formation of a polymeric pigment which invests the cell walls of these special structures making them hard, opaque, and generally resistant ( Allport and Bu’Lock, 1960). The phenolic pigments of a number of wood-rotting polypores e.g. P. hkpidzrs, are similarly related to structural elements in the developing fruit body ( Bu’Lock and Smith, 1981).In these cases, and perhaps in others, enzymes of phenol oxidase type are also implicated; we have already noted the general importance of this type of enzyme in secondary biosynthesis (Section 111, C ) and also that such enzymes are sometimes under the control of endogenous inducers (Section IV, D ) ; Hirsch ( 1954) has demonstrated their specific association with the formation of perithecia in Neurosporu. A different expression of the same kind of relationship may be seen in the case of some bacterial polypeptide antibiotics. It has frequently been remarked that these peptides and also the bacterial cell walls both contain the curious series of D-amino acids, which are rare elsewhere. Bernlohr and Novelli (1960a, b ) have recently shown that there is a close relationship between spore formation and bacitracin production in B . lichenifomis; both are responses to similar conditions, so that antibiotic is only formed under the conditions which eventually lead to sporulation, but there is a developmental stage beyond which the two processes appear to diverge and are no longer compatible. During the actual formation of the sporangia, part of the bacitracin which has been synthesized earlier is taken up by the cells and apparently incorporated into the spore coat. The processes of spore formation in microorganisms are exceedingly complex and not well understood, but it is at least clear that they involve drastic changes in cell walls (Salton and Marshall, 1959), requiring both breakdown and synthesis. In this general connection it is therefore interesting to note that rather a wide variety of secondary metabolites seems to be related in some way to structural elements in cell walls, Several actinomycete antibiotics, such as streptomycin, are somewhat firmly bound to the cell walls of the producing organisms (Waksman, 1959), while others, such as penicillin, have a strong (and inhibitory) affinity for enzymes concerned with cell-wall synthesis. It has been suggested that substances such as the “peptolide” antibiotics are involved in fungal spore-wall structures (Russell and Brown, 1960) as is the peptide fungisporin (Sumiki and Miyao, 1959). The association is not confined to

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antibiotics, for such various materials as prodigiosin in Serratia marcescens and the carotenoid pigments of Sarcinu lutea are also associated with the cell envelope. Many secondary metabolites do not appear in the medium until the autolysis phase of the culture is reached. We need not anticipate that all secondary metabolites will be found to be related to cell- or spore-wall structures; it is simply that changes in these structures are the most conspicuous, and at present the best known, of the deepseated changes produced in microorganisms as a response to a deteriorating environment; perhaps ultimately some other aspects of these changes will also be found to have similar reflections in the complex group of secondary metabolites.

VI. A Postscript A recognizable economic role has been suggested for the secondary metabolic activities of microorganisms. Can a related function be ascribed to the secondary metabolites of plants, in which every cell is continually adapting itself to changing functions in a multicellular complex? Equally, can we explain the rarity of such products in animals in terms of the greater specialization, and control of the internal environment, which in animals go along with a loss of cellular adaptability and a capacity to go somewhere else? There are arguments for such extensions, but they would be out of place here. Another parallel may perhaps be mentioned, however, since it will at least serve to show the real reason why the author finds this particular hypothesis attractive. In our anxious and overworked society it is pleasing to find some support for the idea that the eccentric and apparently useless activities of idle moments may after all serve to increase biological efficiency, And it is comforting to observe that microorganisms at least may be able to compete in other ways than by poisoning one another.

REFERENCES AUport, D. C., and Bu’Lock, J. D. (1960). J . Chem. Soc. pp. 654-662. Anchel, M. (1955). Science 121,607-608. Amstein, H. R. V., and Bentley, R. (1953). Biochem. ].54,493-522. Amstein, H. R. V., and Margreiter, H. (1958). Biochem. J . 68, 339-348. Amstein, H. R. V., and Morris, D. (1960). Biochem. I. 76, 357361. Baddiley, J., and Blumson, N. L. (1960). Biochim. et Biophys. Actu 39, 37K377. Barton, D. H. R., and Cohen, T. (1957). In “Festschrift Arthur Stoll” (C. M. Jacottet, ed. ), pp. 117-143. Birkhauser Verlag, Basel. Bassett, E. W., and Tanenbaum, S. W. (1959). I. B i d . Chem. 234,1861-1866. Bassett, E. W., and Tanenbaum, S. W. (1960). Biochim. et Biophys. Actu 40, 535537. Bekker, Z. E., Ostroikhov, A. A,, Smimova, A. D., Koscheleva, N. A., and Fadeevn, N. P. ( 1956). Antibiotiki 2, 40. Bentley, R. ( 1960). Biochem. Biophys. Reseurch Communs. 3,215-219.

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Bentley, R.,and Keil, J. ( 1981). Proc. C h m . SOC. (London) pp. 111-112. Bentley, R., and Thiessen, C. P. (1957). J. Biol. Chem. 226,873-701. Bemlohr, R. W., and Novelli, G. D. (1980a). Arch. Blochem. Biophys. 87,232-238. Bemlohr, R. W., and Novelli, G. D. (1980b). Bioch4m. et Biophys. Acts 41, 541544* Birch, A. J. (1981). Private communication. Birch, A, J., and Donovan, F. W. (1953). Australian J . Chern. 6,380470. Birch, A. J., and Kocor, M. (1980). J . Chem. Soc. pp. 888-871. Birch, A. J., Rickards, R. W., and Smith, H. (1958a). Proc. Chem. SOC. (London) pp. 192-193. Birch, A. J,, English, R. J,, Massy-Westropp, R. A., Slaytor, M., and Smith, H. ( 1958b). J. Chem. SOC.pp. 365389. Birch, A. J., English, R. J., Massy-Westropp, R. A., and Smith, H. ( 1 9 5 8 ~ ) .J. Chem. SOC. pp, 389375. Birch, A. J., Fitton, P., Pride, E., Ryan, A. J., Smith, H., and Whalley, W. B. (1958d). 1. C h m . SOC. pp. 45764581. Birch, A. J,, Blance, G. E., and Smith, H. (195%). J . Chem. SOC. pp. 45824587. Birch, A, J., Rickards, R. W., Smith, H., Harris, A,, and Whalley, W. B. ( 1958f). Proc. Chem. SOC.(London) pp. 223-224. Birch, A. J., Schofield, P., and Smith, H. (l958g). Chem. G Id.(London) p. 1321. Birch, A. J., Musgrave, 0. C., Rickards, R. W., and Smith, H. (1959). J . Chem. SOC. pp. 3148-3152. Birkinshaw, J. H., and Samant, H. S . (1980). Blochem. J. 74,389373. Birkinshaw, J. H., Raistrick, H., and others (1931). Phil. Trans. Roy. SOC. London Ser. B220. Brack, A. (1947). Helu. Chlm. Acta 30, 1-8. Brian, P. W. (1957). In “Microbial Ecology.” Symposia Soc. Gen. Mtcroblol. 7, 188-188. Britt, J. J., and Arigoni, D. ( 1958). Proc. Chem. SOC. (London) pp. 224-225. Brockmann, H . ( 1980). Fortschr. Chem. org. Naturstoffe 18, 1-54. Bu’Lock, J. D., and Gregory, H. (1959). Biochem. J. 72,322425. Bu’Lock, J. D., and Ryan, A. J. (1958). Proc. Chem. Soc. (London) pp. 222-223. Bu’Lock, J. D., and Smalley, H. M. (1961). Proc. Chem. SOC. (London) pp. 209-211. Bu’Lock, J. D., and Smith, H. G. (1981). Unpublished results, Bu’Lock, J. D., Jones, E. R. H., and Turner, W. B. (1957).J. Chem. SOC. pp. 1807-1817. Bu’Lock, J D., Gregory, H., and Hey, M. ( 1981). J . Chem. Soc. in press. Chambers, K., Kenner, G. W., Temple Robinson, M. J., and Webster, B. R. (1980). Proc. Chem. SOC.(London) pp. 291-292. Corcoran, J. W., Kaneda, T., and Butte, J. C. (1960). J. BtoZ. Chem. 235, PC29PC30. Dagley, S., Evans, W. C., and Ribbons, D. W. (1980). Nature 188, 580-588. Dauben, W. G., Ban, Y., and Richards, J. H. (1957). J. Am. Chem. SOC.79, 988970. Deinma, M. H., and Landheer, C. A. (1980). Blochim. et Biophys. Actu 37, 178179. Demain, A. L. ( 1959). Aduances tn Appl. Microbtol. 1,2347. De Moss, R., and Evans, N. R. (1980). J . Bacterial. 79, 729-733. Di Marco, A., and Pennella, P. (1959). Progr. in Id.Microbbl. 1,45-92.

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Doskocil, J., Sikyta, B., Kasparova, J., Doskocilova, D., and Zejicek, J. ( 1958). I. Cen. Microbiol. 18, 302304. Elbein, A. D., Mann, R. L., Renis, H. E., Stark, W. M., Koffler, H., and Gamer, H. R. (1960).Federation Proc. 19,141. Fodor, P. J., Miller, A., Neidle, A,, and Waelsch, H. (1953).J. B i d . Chem. 203,

991-1002.

Foster, J. W. (1949). “Chemical Activities of Fungi.” Academic Press, New York. Gatenbeck, S. (1960a).Acta Chem. Scand. 14,102-106. Gatenbeck, S. ( 1960b).Acta Chem. S c a d . 14,298402. Gatenbeck, S., and Barbesgard, P. (1960).Acta Chem. Scand. 14,230-231. Gatenbeck, S., and Mosbach, K. (1959).Acta Chem. Scand. 13, 1561-1584. Goodwin, T.W. (1959).Advances in Enzymol. 21,295368. Gould, B. S., and Raistrick, H. (1934).Biochem. J. 28, 1640-1656. Haws; E. J., Holker, J. S. E., Kelly, A., Powell, A. D. G., and Robertson, A. (1959). J . Chem. Soc. pp. 359t2-3610. Hirsch, H. M. (1954).Physiol. Pbntanrm 7, 72-78. Hockenhull, D. J. D. ( 1959).Progr. in Ind. Microbiol. 1,3-27. Hockenhull, D. J. D. (1960).Progr. In Znd. Mtcrobiol. 2, 133-165. Hofmann, K., and Liu, T. Y. (1960).Biochim. et Biophys. Acta 37, 364-365. Homing, M. G., Aartiu, D. B., Kamen, A,, and Vagelos, P. R. (1960). Blochem. Biophys. Research Communs. 3, 101-106. Horowitz, N. H., Fling, M., Macleod, H. L., and Sueoka, N. (1960).J. Mol. B b l .

2, 98-104.

Johnson, A. W. (1960).Scl. Progr. 48, 88-97. Jones, E. R. H. ( 1961).Private communication. Jones, E. R. H., and Lowe, G. (1960).J. Chem. Soc. pp. 3959-3962. Katz, E. (1960).I . Biol. Chem. 235, 1090-1094. Katz, E.,Pienta, P., and Sivak, A. (1958).Appl. Mtcrobiol. 6,236-241. Kluyver, A. J., and Van Niel, C. B. (1936).Zentr. Bakteriol. Pararltenk. Abt. ZI

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Lybing, S., and Reio, L. (1958).Acta C h m . Scand. 12, 1575-1584. McCormick, J. R. D., Hirsch, U., Sjolander, N. O., and Doerschuk, A. P. (1960). I . Am. Chem. Soc. 82,5008-5007. MacDonald, J. C. (1960).Can. J. Microbiol. 6,2742. Mosbach, K. ( 1960). Acta Chem. Scand. 14,457-484. Ollis, W. D., Sutherland, I. D., Codner, R. C., Gordon, J. J., and Miller, G. A. ( 1960). Proc. Chem. SOC. (London) pp. 347448. Prokofieva-Belgorskaya, A., and Popova, L. ( 1959).J. Gen. Microbiol. 20, 462. Ramakrishnan, C. V., Steel, R., and Lentz, C. P. (1955).Arch. Biochem. Biophys.

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2, 107-110.

Russell, D. W., and Brown, M. E. (1960).Biochim. et Biophys. Acta 38, 382383. Salton, M. R. J., and Marshall, B. (1959).J. Gen. Microbbl. 21, 415-420. Schmidt-Kastner, G. ( 1956).Naturwksenrchaften 43, 131-132. Silverman, M., and Rieder, S. V. (1960).J. B b l . Chem. 235,1251-1254.

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Silverman, W. B., and Anchel, M. (1959). Federatfon Proc. 18, 324. Snell, J. F., Birch, A. J,, and Thomson, P. L. (1960). J . Am. Chem. SOC. 82,2402. Stickings, C. E., and Townsend, R. J. (1960). Biochem. J . 74,36P. Stubbs, J. J., Lockwood, L. B., Roe, E. T., Tabenkin, B., and Ward, G. E. (1940). I d . Eng. Chem. 32, 1628-1631. Sumiki, U., and Miyao, K. (1959). Bull. Agr. Chem. SOC. Japan 24,23-30. Waksman, S . A. (1957). Bacterial. Revs. 21, 1-29. Waksman, S. A. ( 1959). “The Actinomycetes,” Vol. I. Williams & Wilkins, Baltimore, Maryland. Waksman, S. A,, and Lechevalier, H. A. (1953). “The Actinomycetes and Their Antibiotics.” Williams & Wilkins, Baltimore, Maryland, Wasserman, H. H., McKeon, J. E., and Santer, U. V. (1960). Biochem. Biophys. Research Communs. 3, 146-149. Winstead, J. A., and Suhadolnik, R. J. (1960). J . Am. Chem. SOC. 82,1644. Wiss, O., and Bettendorf, G. (1957). Z. phystol. Chem. 306,145-153. Woodbine, M . (1959). Progr. in Id.Microbiol. 1, 181.

Methods for the Determination of Organic Acids A. C. HULME Ditton Laboratory, Agricultural Research Council, Larkjield, Maidstone,

Kent, England

I. Introduction

....................................................

343

11. List of Acids to Be Considered.. .................................. 345 111. Preparation of Material for the Analysis of Acids. General Principles. . . . . 346

IV.

V. VI.

VII.

VIII.

A. In Organisms ........................................ B. In Media ............................................ Acids of the Citric Acid Cycle and Other Organic Acids (Groups 1 and 2 ) 349 A. In Organisms ............ B. In Media ................ C. Qualitative Analysis . . . . . . . D. Quantitative Determination Sugar Acids (Group 3) . . . . . . . A. Qualitative Analysis . . . . . . . . . . . . . . . . B. Quantitative Determination. . Keto Acids (Group 4) ........ A. Determination in Microorgani B. Determination in Media. .......................... Fatty Acids (Group 5 ) ....... A. Methods of Extraction from Organisms . . . . . . . . . . . . . . . . . . B. Extraction from Media ..... C. Qualitative and Quantitative Determination . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

1. Introduction It should be made clear from the start that the author is not a microbiologist and his sole qualification for writing this chapter lies in his experience in the determination of aliphatic and alicyclic acids in fruits. There are, however, certain general principles involved in the new techniques for the separation and determination of these acids which are of general application. A perusal of the volumes of The Annual Review of Microbiology, and, for example, Cochrane’s (1958) book on “The Physiology of the Fungi,” will produce a long list of organic acids which can be involved in the metabolism of bacteria and fungi-some of them very exotic and very specific to a particular organism. No attempt whatever will be made to cover such a vast field. Nevertheless the general methods described may 343

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often be adapted to the determination of many “odd” acids when once their structure, or partial structure, is known. An attempt will be made to cover, in addition to the acids of the citric acid cycle and modifications of the cycle, a representative selection of the sugar acids, the short-chain fatty acids, and some of the long-chain fatty acids, In regard to the last two groups, great strides have been made in the last two or three years by the rapid developments which have taken place in gas-liquid chromatography. The organically combined phosphoric acids lie outside the scope of this chapter, although the methods of ion exchange and paper chromatography to be discussed have been successfully applied to the separation of such acids (see Isherwood, 1954; Khym et al., 1957). The methods to be discussed have been used, in various forms, by several workers in different fields. The ones described have been selected somewhat arbitrarily because, in general, the author has used them himself or has had contact with the workers using them. No attempt will be made to list all the papers describing the use of these methods or modifications of them since this chapter is in no sense a review. Its object is to draw the attention of workers in the field of microbiology to the general methods which are now available for the study of the acids listed. In any case, it is very often necessary to modify a method to suit the particular problem on hand. It appears from the literature that microbiologists have been most often concerned with changes in one or only a few acids involved in some particular metabolic pathway under investigation. For this type of work specific tests or specific methods of estimation (e.g., the pentabromacetone method for citric acid, of Pucher et al., 1934) are often quite satisfactory. Care must, however, always be taken to be sure that other acids which may be present do not cause serious interference. The object of the present chapter is to provide the basis for the systematic separation and determination of all the acids which may be present in a microbial culture. Foster (1957) has drawn attention to the fact that evidence from microorganisms suggests that, apart from its role as an energy provider in the oxidation of carbohydrates, the tricarboxylic acid (Krebs) cycle is also an important mechanism for furnishing intermediates for many biosynthetic processes. He has also suggested that if modem methods for the identification, separation, and quantitative determination of organic acids had been available earlier, studies on the physiology of microorganisms might have advanced by many years our understanding of many metabolic processes fundamental to life in all its forms. When the literature concerned with the determination of acids in biological material is surveyed, chromatography in its various forms is

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taking the place more and more of the classic methods of, for example, the school of Vickery and his associates at Connecticut. In fact Palmer, himself one of Vickery’s school, has been a pioneer of the chromatographic examination of organic acids in plant material. As with any rapidly expanding technique, there are many variations on the theme of chromatography. Different workers use different methods for the extraction and preparation of acids for separation on ion exchange and silica gel columns; a variety of solvents have also been used for silica gel chromatography and paper chromatography of the acids. Comprehensive surveys of these different methods are given in detail by Wolf (1955) and by Ranson (1955). In this article, most of the procedures described are those which the author has found to be satisfactory in his own laboratory. For the separation of fatty acids-and probably this method will before long be successfully applied to all the organic acids-gas-liquid chromatography (“gas chromatography” or “GLC) with its elegant, rapid separation of the acids has made great strides in the last few years and many publications are appearing entirely devoted to this type of analysis. Two such publications have been particularly helpful in preparing the present article, namely “Gas Chromatography,’’ edited by D. H. Desty ( 1958) and the chapter on fatty acids by James (1960) in Methods of Biochemical Analysis, edited by D. Glick. The books on general chromatography by Lederer and Lederer (1957) and by Smith (1960) will serve as useful sources of reference to points not mentioned or only briefly touched on here. The quantitative determination of individual acids by means of specific organisms or isolated enzymes will not be described since microbiologists must be well aware of these methods, and the author prefers chemical methods.

II. List of Acids to Be Considered’ Group I . Krebs Cycle Acids Aconitic Citric Fumaric Isocitric Group 2. Other Organic Acids Acrylic Citraconic Citramalic

a-Ketoglutaric2 Malic Oxalacetic2 Succinic Lactic Malonic Mesaconic

In alphabetical order within groups.

* See also under keto acids.

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Group 9. (Continued) Crotonic Mesotartaric Dihydroshikimic Methylsuccinic Glutaric Oxalic Glyceric Oxalosuccinic* Glycolic Pyroglutamic a-Hydroxy-n-butyric Quinic 8-Hydroxy-n-butyric Shikimic or-Hydroxyglutaric Tartaric a-Hydroxypropionic Tartronic Itaconic Tricarballylic Ketomalonic* Various methyldihydroxybutyric acids Group 3. Sugar Acids Arabonic (lactone) 2-Ketogluconic Galacturonic Mucic Gluconic Saccharic Gulonic (7-lactone) Group 4. Keto Acids Mesoxalic (keto malonic) Acetoacetic Acetone-dicarboxylic Oxalacetic Glyoxylic Oxalosuccinic a-Ketoglutaric Pyruvic Group 6. Fatty Acids Subgroup (a) Acetic Crotonic Acrylic Formic n-Butyric Propionic Isobutyric Valeric Caproic Isovaleric Subgroup (b) All the saturated fatty acids Ce-Cze The following unsaturated fatty acids: Arachidonic Linolenic Decenoic Oleic Linoleic Undecenoic Also certain hydroxylated and branched-chain fatty acids.

111. Preparation of Material for the Analysis of Acids. General Principles

The first stage in the determination of organic acids (including fatty acids) in the material under investigation is a preliminary extraction of the raw material to reject as much unwanted material, cell debris, proteins, polysaccharides, etc., as possible. In this chapter only acids orig-

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inally in the free stage or combined with inorganic cations will be considered; esters, except for the glycerides, will not be dealt with (for esters see Meigh, 1955). The ideal extraction must achieve, without alteration due to artifact, the complete extraction of all the acids present in the material at the moment selected for analysis, with a minimum loss of the volatile lower fatty acids. It would be an advantage if the extraction of volatile acids could be combined with that for the determination of the organic acids but such “combined methods have, so far, been given little critical study and the author cannot recommend such a procedure for quantitative work. The literature on the mechanical breakdown and subsequent extraction of microorganisms for biochemical analysis is scanty. Especially is this so for the lipids of microorganisms. Magasanik (1957) in an article on the nutrition of bacteria and fungi, says, “The chemistry and metabolism of the lipides has remained one of the least-explored areas of biology.” Another difficulty in so far as microorganisms are concerned is that not only have the organisms themselves to be analysed but, generally, an analysis of the medium for acids “liberated” into it by the organisms is of equal importance. This introduces the added complication of separating the acids from the diverse substances originally present in the various media that may be used. Again this problem appears to have received little critical study and the author does not feel competent to suggest a completely satisfactory solution to this problem, although an attempt has been made to indicate the lines along which, at least a partial solution of the problem, might proceed. It would seem that in future work specifically designed as a biochemical investigation of the detailed metabolism of organic acids, it would be a considerable advantage if much simpler media could be devised than the traditional “blanket” media. These media no doubt contain all the essential compounds for the vigorous growth of a whole range of microorganisms; they also contain many compounds (generally unspecified as definite molecular types) not necessarily essential to the particular organism under study, but which render tedious and dif6cult the precise separation of individual compounds injected into the medium by the organism growing therein. But perhaps this suggestion is a case of “a fool rushing in where angels fear to treadf” Since each individual organism may differ and, since the media in which the organism is grown may vary in their composition, only general principles for the disintegration and extraction of the organism and of the medium can be given here. With the labile keto acids, the disintegration and extraction of organisms and of media will be dealt with separately, since here special

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methods of extraction must be used right from the commencement (see section on keto acids). A. IN ORGANISMS

1. Bacteria There appear to be three general methods for the disintegration of bacterial cells for extraction and analysis. ( a ) Where large quantities are involved, the bacteria must be collected by centrifugation in a continuous centrifuge of the ShaLples type. The “mush” of bacteria may then be ground in a mortar with sand, powdered glass, alumina, etc. ( b ) Smaller quantities may be disintegrated by vibrating with “ballotini” beads in a machine such as the “Mickle” vibratory tissue disintegrator. (See Salton, 1953.) ( c ) Sonic disintegration has been used very successfully for the breaking up of bacterial cells and there are several machines on the market especially designed for this purpose. The disintegrated cells may be extracted by one of the methods described in the sections dealing with the various groups of acids to be studied. 2. Fungi

After separation of mycelia, spores, etc., by centrifugation or filtration, the washed mycelia or spores should be ground with sand, etc., and the appropriate extracting liquid, depending on which of the various groups of acids are to be studied. If the organisms cannot be analyzed immediately they should be dried in a “vac-ice” drier and stored in a desiccator; volatile organic acids will, of course, be lost in this procedure. Homogenization in a Waring blendor with some of the abrasives already mentioned might be satisfactory although small particulate, leathery material such as fungal hyphae are not readily broken down completely by such methods. The Potter-Elvejhem type of homogenizer should prove useful for small quantities of material. B. IN MEDIA Because the preliminary treatment of media will be different for each group of acids, the treatment and extraction of media will be considered in each section dealing with the individual type of acid,

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IV. Acids of the Citric Acid Cycle and Other Organic Acids’ (Groups 1 and 2) A. IN ORGANISMS The material should be ground, as previously described, using cold 80%ethanol as the extractant. The residue is removed by centrifugation or filtration and re-extracted, etc., until the last extract is free from acid. Three or four extractions are usually sufficient. The ethanol extracts are combined and the ethanol removed by evaporation in UUCUO. The resulting aqueous extract is made up to volume and filtered through a pad of asbestos. If much pigment (or other phenolic material) is present the next stage is to shake the filtrate with a few grams of deactivated charcoal. [This charcoal (blood charcoal or other prosphate-free charcoal) is prepared by treatment with 5% acetic acid followed by washing free from acid (Partridge, 1949) .] The charcoal is filtered off on a Biichner funnel and thoroughly washed, The filtrate, or, if only a small amount of coloring matter, etc., was present in the original extract, the diluted filtrate from the asbestos pad (above), is passed down a column of Dowex 50 (or Zeokarb 215) to remove amino acids. The column is then washed until all the organic acids and sugars have passed through. The filtrate and washings from the column are then run onto a column of Dowex 1 (acetate form). The column is then washed free of sugars and any other nonacid material and the acids are eluted with acetic acid followed by formic acid by the method of gradient elution. Fractions (usually 2-3 ml.) are collected as they leave the column on a mechanical fraction collector. Details of the preparation of the columns and the apparatus and procedure used in gradient elution are given by Palmer (1955) and by Hulme and Wooltorton (1958). In gradient elution, acids generally leave the column in the order of their p K values but there are exceptions, unsaturated acids being more tightly bound to the resin than saturated acids of similar basicity and molecular weight. With this proviso, the acids leave the column in the order monobasic, dibasic, and tribasic, This is of considerable advantage in giving an initial clue to the nature of “unknown” acids appearing in the eluate from the column. As will be seen later, the acids in each fraction are finally checked by paper chromatography. * For the volatile fatty acids see the section on fatty acids and also Ranson ( 1955). Lactic acid is best determined by means of silica gel or Celite chromatography (see Phares et al., 1952). Ion exchange chromatography is not suitable for the quantitatioe determination of this acid.

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A. C. HULME

Although Hulme and Wooltorton (1958) give a definite sequence of increasing strengths of eluting acids (acetic and formic) suitable for the gradient elution of the acids of the apple, the strength of the eluting acid may be increased more slowly or more rapidly to suit a particular mixture of acids. For example, if the sample contains a high proportion of monobasic acids it may improve the separation of the acids if the strength of the eluting acid is raised more slowly. The detailed operation of gradient elution will be seen later from the flowing chromatograms obtained with various mixtures of acids ( Figs. 3-8). Before proceeding further with the details of the separation and qualitative and quantitative determination of the acids, the method of treating media for the extraction of the acids will be described

B. IN MEDIA The media, if solid, should be diluted to make a liquid and then sufficient sulfuric acid is added to make to pH 2.0. This will stop any enzyme action and precipitate some of the high-molecular-weight compounds, After stirring and allowing the precipitate to settle, the liquid is centrifuged. The precipitate is washed and supernatant and washings made to pH 9.4 with 2 N NaOH. The liquid is concentrated in zlucuo at 40°-450C. (preferably on a rotary evaporator) to a small volume; if frothing occurs in the early stages of evaporation, loss of material can be prevented by applying the vacuum gradually. Ethanol is then added to bring the ethanol concentration to 75-8M.After allowing the solution to stand for a short time, the precipitate formed is centrifuged off and the alcohol removed from the supernatant by evaporation in oucuo as before. The clear, colored, aqueous solution is now ready for passage down the Dowex 50 column, etc., as described in Section IV, A above. [It is important, when deciding the size of the column to h e , to make sure that the amount of resin is three times the amount necessary for all the amino acids present plus any other cations (e.g.,sodium and potassium) which may be present in the extract. The same argument applies as regards total anions when using Dowex 1.1 If the solution is very dark in color, it may be treated with the deactivated charcoal, etc. (as described in Section IV, A ) before passage down the Dowex 50 column.

C. QUALITATIVE ANALYSIS 1. Ion Exchange Chromatography

For both qualitative and quantitative analysis, the eluting acid (acetic or formic) in the acid-containing fractions from the Dowex 1 (above)

DETERMINATION OF ORGANIC ACIDS

351

must be removed by evaporation. This may be done rapidly, e.g., 24 fraction tubes at a time, in a hot air bath (45OC.) fitted with racks to hold the tubes. Capillary tubes attached to a manifold fit into each tube and air from the capillaries expedites the evaporation of the liquid in each fraction tube. The apparatus is described in detail by Hulme and Wooltorton (1958). Evaporation of each batch of 24 tubes takes less than an hour. For quantitative work the eluting acid must be evaporated off completely; for qualitative identification only of the acids present in the fractions, complete evaporation is not essential, (In practice, qualitative and quantitative determination may often be combined but the quantitative determination of the individual acids is only possible if their identity has been established.) With complex mixtures of acids it is necessary to make a preliminary survey of the acids present to establish their identity. The contents of the tubes (above) are dissolved in a small quantity of acetic acid and run on paper chromatograms in one or several of the solvent systems listed under the section on paper chromatography ( p. 354). When it is clear that all the acids (if there is more than one) in a fraction have been separated on the chromatograms, an attempt at identification should be made by means of the tests described in the section on paper chromatography, Confirmation of identity may be obtained by running the acids in several solvents with marker spots of pure samples of the “suspected” acid. In cases where an unknown acid is present (i.e., an acid unidentified alongside known and obtainable acids) larger samples must be obtained and purified either by running as a streak on thick (Whatman No. 3) paper and eluting, or by silica gel chromatography ( p. 352),and examining chemically, A good example of such a procedure is provided by Whiting and Coggins (1960) and by Hulme (1951, 1954a). Whiting and Coggins, in an examination of the acids formed during cider fermentations, separated a-hydroxyglutaric and its lactone and several methyl and ethyl dihydroxybutyric acids as groups on an ion exchange column and then as individuals on a silica gel column. If it is established during this qualitative survey that several acids are present in a fraction or fractions eluted from the Dowex 1 column, separation may often be achieved by means of silica gel chromatography (an example of partition chromatography). Acids having similar p K values, for example citramalic ( 1-methyl malic) and malic acids (Hulme, 1954a), may, because of the presence of a nonpolar group in one acid (the methyl group in citramalic acid), be readily separated by partition chromatography, i.e., .partition between water and an organic solvent (see Fig. 1 ) .

352

A. C. HULME

FIG.1. Chromatogram on buffered silica gel of citramalic (“C”)and malic acids run in methyl isobutyl ketone ( Hulme, 1954a).

2. Silica Gel Chromatography One of the earliest methods for the systematic separation of organic acids on columns of silica gel is that of Isherwood (1946) and later methods (Bulen d al., 1952) are, essentially, modifications of Isherwood’s technique, A simpler method of preparation of the silica gel than that of Isherwood, which the present author has found very satisfactory, is that described by Bradfield et al. (1947) and Bradfield and Penney (1948). The commercial preparation marketed in America by the Mallinckrodt Company is equally satisfactory. The preparation of the columns and the introduction of the sample onto the column has been described by Bradfield et al. ( 1947) (see also Synge, 1946). If it is assumed that an organic acid does not ionize in a organic solvent and that it is the undissociated acid which distributes between the solvent and water, i.e., the distribution coefficient is given by concentration of undissociated acid in solvent concentration of undissociated acid in water

then this “true” distribution coefficient can only be in operation if the acid is in the completely undissociated form. This is why partition chromatography (e.g., silica gel and filter paper chromatography) is

DETERMINATION OF ORGANIC ACIDS

353

normally operated at a low pH, i.e., in the presence of “swamping“ amounts of another acid or a buffer. As Bradfield has pointed out, it may still be possible, however, to separate acids having closely similar partition coefficients in a given solvent system by the employment of “apparent” distribution coefficients, Since Bradfield’s analysis of the situation has not been published it may prove useful to summarize it here: Let us suppose that the partition (distribution) coefficient for a particular acid this refers to partition of the undissociated acid molecule. If only is a fraction, n, of the acid is undissociated then the apparent distribution coefficient will be na. As n changes so will the apparent distribution coefficient. It can be shown that n varies in buffer solutions of differing pH values. In the present treatment the second ionization constant of a dibasic acid, which is relatively small, will be neglected. Consider a molecules of an acid dissolved in a liter of an aqueous buffer solution in which the hydrogen ion concentration is maintained constant at C , molecules per liter. Let x be the amount of the anion present at equilibrium. Then the concentrations at equilibrium are: (Y:

RCOOH a--z

H+

and

CE

+ RCOOX

Then the fraction of the total acid which remains undissociated under these conditions = ( a- x ) /a. If K is the dissociation constant of the acid, then:

K = -C H

xx

a--2

From which it can be shown that:

In the particular case when C H = K, i.e., pH = p K , then ( a - x)/a = If, for a series of acids for which the values of K are known, the values of ( a - x ) / a are plotted against pH, a series of curves are obtained such as the t w o shown diagrammatically in Fig. 2. Each curve will pass through a point where ( a - x)/a = 0.5 when pH = pK for the acid. On comparing the two acids A1 and A2 in the figure it will be seen that at the point where acid A1 is 50% dissociated, acid A2 is 80%undissociated. By bearing in mind what has been said earlier it will be realized that if the true distribution coefficients of A1 and A2 are both equal, then at this particular pH (3.5),the apparent distribution coefficients (my)of A1 = 0.5 and of A2 = 0.8 and a separation of A1 and A2 becomes possible by partition chromatography.

3.54

A. C. HULME

Most workers, following Isherwood’s lead, have used sulfuric acid as the “buffering” agent and mixtures of chloroform and tert-butanol (saturated with sulfuric acid) as the eluting solvent. The present author has found phosphate buffers at pH 2.2-2.9 for the column, and methyl isobutyl ketone (saturated with phosphate buffer) as the eluting solvent, to be more generally useful (Hulme 1951, 1954a). The methyl isobutyl ketone must be freshly distilled before use. For quantitative work the solution of the acids is mixed with a little silica gel and this gel placed on the top of the column (Hulme and Wooltorton, 1957). The acids leaving the silica gel column are collected in fractions on a fraction collector, the excess solvent evaporated off (without heat), taken up in water, titrated, and checked by filter paper chromatography.

0

2

4 PH.

6

FIG.2. Dissociation curves of two hypothetical acids (A1 and A2).

Examples of quantitative separations of organic acids by the use of ion exchange and silica gel chromatography will be given later, but further details will first be given of methods for the identification of the organic acids present in a mixture by means of filter paper chromatography and of specific spray reagents used in conjunction with the paper chromatograms. As already stated this is a necessary preliminary to the quantitative determination of the acids. 3. Filter Paper Chromatography

The number of papers published on this subject is now very large indeed; new solvent systems appear almost daily. The present author has, however, found by experience that most separations can be achieved by the use of two or three solvent systems. A new system has not been used unless existing ones have proved inadequate. The great advantage of remaining faithful to old and well-tried systems is that the worker

DETERMINATION OF ORGANIC ACIDS

355

becomes familiar with the patterns of the acids on the developed chromatograms, the appearance of a “new” acid becomes immediately apparent; often the group to which it belongs and even its individual identity may be quickly guessed by its position on the chromatogram in relation to “known” acids. For this reason, only the systems used by the author will be dealt with. It is not claimed that they are the best ones and in special cases the reader is advised to try different systems, many of the more recent of which appear in the Journal of C h r m t o g r a p h y (e.g. Howe, 1960). The author has found the descending solvent method generally most satisfactory although for small “check” chromatograms the ascending method is convenient (see Brimley and Barrett, 1954). Two-dimensional chromatography has been used (see Ranson, 1955) but the author has not found this particularly advantageous. Solvent systems for paper chromatography. The three solvent systems found to be generally useful are (1) butanol-formic acid-water (4:1:5, v:v), due to Lugg and Overell (1947). This is a two-phase system and the lower, aqueous layer must be run off before use and part of it run into the bottom of the chromatography tank to saturate the paper before the upper layer is added to the trough in which the chromatogram is placed for running. (2) Benzyl alcohol-tert-butanol-isopropanol-formic acid-water (24:8:8.8: 1.5, v:v), due to Stark and associates (1951). This is a misciblk solvent and is used in both the bottom of the tank and in the chromatogram trough. (3) n-Propanol-NH,OH-water (6:3: 1, v:v), due to Hanes and Isherwood (1949); this again is a miscible solvent. In this alkaline solvent system the acids travel down the paper as the ammonium salts. An example of the use of these three solvent systems to check the identity of an “unknown” acid (in this case citramalic acid) is given by Hulme (1954a). The solvent system (1) should be prepared 24 hours before it is required, to allow esterification of the formic acid by the butanol to reach equilibrium. For the accurate determination of Rr values, the spotted-up chromatogram should be allowed to equilibrate for 24 hours before it is run, i.e., before the solvent is added to the trough. The R , values of a range of organic acids in the three solvent systems are given in Table I. Whatman No. 2 paper (chromatography grade) was used in obtaining these R , values. Some of the lower fatty acids are included in this table. Isocitric acid does not lactonize during chromatography in the acid solvent; it only does so if evaporated to dryness with a strong mineral acid. The R, values of organic acids in a whole range of solvent systems are given by Block et al. (1958).

TABLE I RI VALUESOF ORGANIC ACIDSIN THF~EE SOLVENTSYSTEMS WITH WHAW

Organic acid Monobasic a-Hydroxybutyric &Hydroxybutyric Glyceric Glycolic G1yoxylic Lactic &Hydroxypropionic pyroglutamic QUiXliC

Dibasic Citramalic Glutaric a-Ketoglubric Ketomalonic Mslic Ma1onic Meaotartaric

Rl in solvent (1)

RI in solvent (2)

0.80,0.89 0.73 0.28,0.32 0.51 0.67,0.78 0.73,0.82 0.48 0.18

0.79,0.91 0.75 0.33,0.40 0.55 0.11,0.56 0.69,0.82 0.79,0.87 0.63 0.25

0.59 0.78 0.59 0.35 0.49 0.66 0.19

0.62 0.79 0.61 0.06 0.49 0.60 0.24

0.64

NO. 2 PAPER'

Rl in

RI in solvent

R j in

R , in solvent (3)

Organic acid

(1)

solvent (2)

Methylsuccinic 0.63,0.74 Oxalic 0.56 Succinic Tartaric 0.46 0.51 Tartronic 0.28, tailing Tribaaic 0.57 Citric Isocitric 0.42,0.54 0.51 oxalosuccinic 0.44 Tricarballylic Unsaturated 0.36 Aconitic 0.40 Acrylic Citraconic 0.14,O. 27 streak Fumaric 0.32 Itacon ic Mesaconic 0 31 0.27 Shikimic

0.79 0.47 0.71 0.26 0.39

0.81 streak 0.72 0.26 0.33

0.39 0.20 0.35

0.40 0.40 0.52 0.67

0.41 0.43 0.49 0.68

0.18 0.18 0.31 0.24

0.75 0.86 0.72 0.83 0.77 0.87 0.32

0.75 0.91 0.71 0.82 0.79 0.84 0.39

0.23

(3)

solvent

0.27 0.25

-

0.37 0.41 0.37 0.37 0.37

DETERMINATLON OF ORGANIC ACIDS

357

Spray reagents for paper chromatography of the acids. The following reagents have been found generally useful in the author’s laboratory for the detection of organic acids. The results of the application of these sprays are shown in Table 11. The letters at the head of the column in this table refer to the various spray reagents listed. The paper should be dried before applying the sprays, ( a ) As a general spray for developing all the acids, as acids, the following has proved to be generally satisfactory (Hargreaves, 1951): Dimethyl yellow (25 mg.) and bromophenol blue (75 mg.) are dissolved in 200 ml. of 96%ethanol and adjusted to pH 7.0 by the addition of 0.1 N NaOH (approximately 1.75 ml.). On spraying the paper with this solution the acids appear as a yellow spot on a bluish background. This spray will generally reveal as little as 10-20 pg. of acid. [See column (a1) in Table 11.1 ( b ) With alkaline solvent systems (e.g., system 3 above) spray reagent ( a ) does not always give clearly defined spots and the background color changes rapidly, therefore for these systems the following treatment of the chromatograms is preferable (it is rather expensive because of the xylose required): One gram of xylose is dissolved in 3 ml. of water and to this is added, 1 gm. of aniline and the solution is made up to 100 ml. with methanol. The paper is dipped in this liquid, hung up to drain for 10 to 15 minutes, and then heated to 105°-1100C. for 5 to 10 minutes. Brown spots appear on a pale yellow background. With most acids 10 pg. or more will react (most of the other special reagents mentioned in this section are not so sensitive, 50 to 100 pg. being required for definite results). Rather more acid is required for glutaric and glyceric acids. The paper can be kept as a permanent record. The reagent is stable for one or two months, thereafter it gradually darkens ( Saarnio et al., 1952). When solvent systems containing ammonia have been used, a sensitive method for revealing the “acid” spots is to dip the chromatogram in Nessler’s reagent; the ammonium salts of the acids appear as brown spots on a faint yellow background. The Nessler’s solution should contain only half the usual amount of KOH, and an acid-washed paper (e.g., Whatman No. 54) must be used. After dipping in Nessler’s reagent the paper should be washed immediately in running water. (c) The paper is rapidly dipped in a solution of silver nitrate ( 3 ml. of a 50%aqueous solution) in acetone (200 ml.). The paper dries quickly and is then sprayed with alcoholic NaOH ( 4 ml. saturated aqueous solution in 100 ml. of 95%ethanol) after which it is fixed in 5%sodium thiosulfate and washed and dried ( Trevelyan et al., 1950). ( d ) Two solutions are involved: 0.075%bromocresol green and 0.25%

TABLE I1 S P R A Y TESTS FOR PAPER CHROMATOGRAMS OF ORGANIC ACIDS ~

Test and color reaction- in solvent systems (1) and (2)

(f) Organic acid Monobaaic a-Hydronybutyric PHydroxybutyric G1y ceric

s

(bluish green background)

(c)

(d)*

(el

Pink White Pink Yellow Pink with white purple edge White Pink

Daylight

Blue Grey-green Blue-grey

Yellow-brown

Yellow

Light green

Green, grey edge Duck-egg green Grey-blup Light green Green Grey-green

Pink

-

Glyoxylic

Pink

Black

Yellow

Brown

Lactic 8-Hydroxypropionic Pyroglutamic Quinic

Pink Pink Pink Pink

Black

Pink Pink Pink Pink with purple edge

White white White White

Brown, immediate White White Grey White

Purple

-

white

Grey

Glutaric

Pink

-

white

White

a-Ketoglutaric

Brown

White

Ketomalonic

Black Pink with purple edge Green Black

Pink with purple edge Pink with yellow edge Yellow with purple edge Yellow

Brown

Malic

Pink

-

White

Brown, immediate White

Malonic

Pink

-

White

White

Pink with purple edge Yellow

Second color

Green Green Light green

Glycolic

Dibasic Citramalic

First color

Yellow

-

Ultraviolet fluorescence

White White White

Pink Pink Pink

-

(Y)

Tan

-

-

-

-

-

Grey Yellow

-

Light green -

Green

Green-blue

White

Light green

Green

White

Green

Green-blue

Pink Yellow in center

-

Blue

-

Green-blue

Green

Green-blue

Mesotartaric

Pink

Black

Methylsuccinic

Pink

-

Oxalic

Pink

-

Succinic

Pink

-

Tartaric

Pink

Black

Tartronic

Pink

-

Tribasic Citric Isocitric

' 0

Oxalosuccinic Tricarballylic

Yellow with purple edge Yellow with purple edge Yellow with Black purple edge Yellow with purple edge

Unsaturated Aconitic

Green

-

Acrylic Citraconic Fumaric

Green Green Green

-

Itaconic

Green

-

Mesaconic

Green

-

Shikirnic a

-

-

Pink with purple edge Pink with yellow edge Purple (elongated spot) Pink with yellow edge Pink with purple edge Yellow Yellow-rose, pink edge Pink with purple edge Pink with purple edge Pink with yellow edge Yellow with green edge Green Green Yellow with green edge Yellow with green edge Yellow with green edge Yellow with green edge

White

White

White

White

White

White

White

Blue-green White

-

Green Blue

White

White

Blue, inmediate -

White

White

Pink

White

White

White

White

White

Pink

-

Grey-blue

Brown

White

-

Grey-green

Brown White

White, brown edge White

White, purple edge -

Brown

-

Blue, immediate

Light green

Green Grey-blue Blue

Light green

Pink

-

Green

white

White

-

Green

Brown Brown Brown

Tan White White

Tan White White

-

Green Green

Brown

White

-

Green

Brown

White, pink edge White

White

-

Green

Brown

White

Pink

-

Light green

A dash in the table indicates that no definite color change occu~s. they fade rapidly especially the edging color.

' The colors must be noted immediately after spraying;

Blue-green

-

380

A. C. HULME

+

bromophenol blue in absolute ethanol, and 0.5% KMnO, 1%Na,CO, 10H,O in distilled water. Before spraying, the two solutions are mixed in equal proportions (the mixture is only stable for 5 to 10 minutes). Optimal coloration of acid spots occurs 2 to 5 minutes after spraying the chromatogram (PBskovL and Munk, 1960). ( e ) An aqueous 2%solution of KMnO,. The paper is lightly sprayed and then rapidly washed in running water (preferably hot) until the pink color disappears. The background color of the paper should be a light tan. Spots of acids which have a double bond (e.g., shikimic and fumaric) remain dark brown and hydroxy acids are white against a tan background. ( f ) Ammoniacal silver nitrate-equal volumes of 0.1 M AgNO, and 0.1 N NH,OH mixed immediately before use. The sprayed chromatogram is dried at room temperature away from direct sunlight. The paper should then be examined in daylight and in ultraviolet light (Buch et al., 1952). Best results are obtained if the paper is kept In the dark for several hours before viewing. ( g ) Spray heavily with aqueous 10%potassium ferrocyanide and allow to dry. Then spray the paper with 0.5%ferric ammonium sulfate in 70%ethanol and allow it to dry in air. Mark the spots and note the color (first color in the appropriate column in Table 11). The chromatograms are next treated for 5 to 10 minutes at 100OC. during which time the background color changes from a light blue to a darker blue. The final stage is to spray the chromatogram with aqueous 101% ammonia when the background is bleached and the color of the spots change to the second color given in Table 11. The paper should be re-examined after a day or two since the colors may not develop immediately when acids are present in small amounts (less than about 50 pg.) (Martin, 1955). Oxalic and tartronic acid give a bright blue color after spraying with the ferric ammonium sulfate at levels as low as 10 pg. ( h ) The chromatogram is sprayed with a saturated solution of sodium metaperiodate diluted with 2 volumes of water. After standing 20 minutes it is resprayed with a solution of 50 mg. of sodium nitroprusside and 50 mg. of piperazine in 12 ml. of W ethanol. The paper is then heated for 5 minutes at 100OC. (Cartwright and Roberts, 1955). This test appears to be specific for the alicyclic acids. Quinic acid gives an orange-yellow color, shikimic acid a yellow color which develops without heating, and dihydroshikimic acid gives a greenish-yellow color. Tests with reagents ( c ) , ( g ) ,and ( h ) may be applied to the back of a chromatogram on which the positions of the acids have already been found by spraying with an indicator spray only.

DETERMINATION OF ORGANIC ACIDS

361

(i) A saturated solution of ammonium vanadate (Buch et al., 1955) sprayed onto a chromatogram gives a yellow color with most acids but a brick-red color with tartaric acid; this appears to be specific for this acid. Several other spray reagents are given by Ranson (1955) and by Davies ( 1953). Some of the short-chain fatty acids are included in Table I1 since they will appear, if present in the material under examination, on paper chromatograms of the organic acids, Pyroglutamic acid is included as it may appear with the organic acids as an artifact produced from glutamic acid during certain preparative procedures. D.

QUANTITATIVE

DETERMINATION

The acids are separated by ion exchange chromatography, followed, if gradient elution does not effect complete separation, by silica1 gel

treatment of mixed fractions as described above for qualitative analysis. With ion exchange chromatography the fractions from the Dowex 1 column must be evaporated completely to dryness (to remove the volatile eluting acids); care must be taken not to continue heating long after evaporation is complete since malic acid may be lost if heating is prolonged. The contents of the tubes are then dissolved in a few milliliters of C0,-free water and titrated with 0.005 N baryta as described by Hulme and Wooltorton ( 1958). Accurate determination of acids, even when present in microgram quantities, is possible provided the identity of each acid has been established. Furthermore, provided a standard mixing vessel is used for the eluting acid, the results with gradient elution are extremely reproducible (see Hulme and Wooltorton, 1958). When separation has been effected on silica gel columns, excess solvent is evaporated from the fraction tubes without heat, i.e., using the multiple air blast oven with the heater switched off. The acids are then dissolved in C0,-free water and titrated as before. It is advisable to check the identity of the acids on paper chromatograms after titration. Solvent system ( 1 ) may be used and the barium salts of the acids (formed during the titration) will break down to the free acids during the run with only a slight tendency to streaking on the part of the free acids. The indicator (thymol blue) used in the titration does not interfere with the acids spots on the chromatogram since it moves almost to the solvent front. Examples of the separation of mixtures of acids on ion exchange and silica gel columns are given in the form of flowing chromatograms in

362

A. C. HULME

Figs. 3-6.The percentage recoveries associated with these chromatograms are given in Table 111. Strictly quantitative results are not possible using the above techniques with such semivolatile acids as glyceric, glycolic, lactic, and glyoxylic (this acid may be determined as a keto acid, see p. 370). The specific N

-

Micro equivalents. c

0

FIG. 3. “Flowing” chromatogram of a mixture of acids, obtained by gradient elution from an ion exchange column.

methods for these acids reviewed by Wolf (1955) should prove useful here until the method of gas-liquid chromatography (see Section VII) has been perfected for the organic acids. Phares d al. (1952),using partition chromatography on Celite columns, claim recoveries of 80-9M for some of the acids just discussed, but complete separations were not achieved so that the recovery figures were sometimes “composite” ones. Methods have been described from time to time in which the acids have been determined by separation on paper chromatograms followed

383

DETERMINATION OF ORGANIC ACIDS

by cutting out the acid spots and either weighing them or measuring their area (Lederer and Lederer, 1957). The present author has not, however, found such methods to be very precise; different acids may give different sized spots especially when originally present in a mixture. Finally it should be mentioned that attempts have already been made to separate and determine the whole range of organic acids by means

N

0

P

0,

i

8i-p1 PYWCLUIAUIC. P WIDDOXY BUTYRIC. / HYDROXI rnorionic.

-8 a

/

ClTDAUALlC.

0,

g 8s.

I

I

<

I

U11SO-IADlADIC.

!-I

IRICARBALLVLIC.

MALOMIC.

*

I

of gas-liquid chromatography by converting them first to their methyl esters (see Bayer, 1958). Unfortunately the quantitative conversion of the individual acids of a complex mixture to the methyl esters has not yet been achieved; hydroxy and unsaturated acids provide a problem here. When this problem has been solved in one way or another there is no doubt that gas chromatography will provide the simplest and most rapid method for the separation and quantitative determination of the organic acids.

-

Micro quivrlcntr.

N

82

*

0

1

I

L 0 SUCCIYIC

MCIWYL SUCCIYIC

8

3 -

s

--

ITICONIC

i -nlSlCOYlc

8 FIG. 5. “Flowing” chromatogram of a mixture of acids, obtained by gradient elution from an ion exchange column. 10-

-c

4-

it

ui

-$6-> .-0

t-

$4-

-

2-

-

2

-

-

\ c

40

Frrction Na

FIG. 6. Chromatogram of four acids on buffered silica gel run in methyl isobutyl ketone.

364

365

DI!XXRMINATION OF ORGANIC ACIDS

TABLE I11 PERCENTAGE RECOVERIES OF ORGANIC Acws FROM IONEXCHANGE AND SILICAGEL COLUMNS Per centage recovery

Percentage recovery Organic acid

Ion exchange

Silica gel ~

95

Aconitic 95 a-Hydroxybutyric Severe loss @-Hydroxybutyric Severe loss Citramalic 96 100 Citric 98 Isocitric 93 Fumaric 104 a-Ketoglutaric Severe loss Glyceric Severeloss Glycolic Severeloss 94 Olyoxylic Severeloss Itaconic 103 Malic 100 98

Ion exchange

Organic acid ~~

~~

Silica gel

~~

Malonic 110-115 Ketomalonic 96 100 Mesaconic 8-Hydroxypropionic Severe loss 98 Pyroglutamic 102 Quinic 102 Shikimic 101 Succinic 80 Methylsuccinic 99 Tartaric 98 Mesotartaric 94 Tartronic 06 Tricarballylic

V. Sugar Acids (Group 3) The preparation of extracts for the examination of the sugar acids is exactly the same as that described for acids of groups 1 and 2. Indeed, the same extract may generally be used both for the determination of the organic acids and the sugar acids. For the structure and nomenclature of the sugar acids the reader is referred to textbooks such as that of Pigman and Goepp (1948). Essentially, the sugar acids are the oxidation products of the sugars in which one or both of the terminal aldehyde or alcohol groups are oxidized to carboxyl groups. A difficulty in the determination of these acids is that they tend to form lactones in acid solution. Some sugar acids (e.g., the aldonic acids in which carbon atom 1 is involved in the carboxyl group) form lactones more easily than others. In extreme cases an equilibrium mixture of the acid and its lactone is always present in acid solution. The uronic acids are discussed by Anderson and Sands (1945), the saccharinic acids (the lowest members of which are acetic and lactic acids) by Sowden (1957), and there is a brief description of the paper chromatography of the sugar acids in a general paper by Kowkabary (1954). For a general discussion on the determination of sugar acids the reader is referred to Bell (1955). It is clear from Bell's article and from the

386

A. C. HULME

literature generally that the systematic determination, qualitative and quantitative, of the naturally occurring sugar acids (and, with microorganisms it is probably only a matter of time before all the theoretical sugar acids come under this heading) has not yet been attempted. Once again, then, it will only be possible to examine a few acids and thereby indicate general methods which may be used as a starting point for the solution of a particular problem. A. QUALITATIVE ANALYSIS

Probably the simplest method of detecting the sugar acids is by filter paper chromatography, By an examination of their R , values in several solvents and by group-specific color reactions with spray reagents the common acids may then be identified. The less common acids can often be allocated at least to their type by these methods [see, for example, Whiting and Coggins ( 1960), for 2-ketogluconic acid]. As the first step in the analysis, the filtrate and washings from the Dowex 50 column (p. 349) are passed down a column of Dowex 1 in the acetate form to absorb the sugar acids, along with the organic acids (see p. 349). It is often convenient in practice to examine the acids of groups 1, 2, and 3 together from this point onward. The sugar acids fit into the scheme of gradient elution as described for the organic acids (groups 1 and 2) and Fig. 7 illustrates the elution of members of all these groups from an anion exchange resin in the acetate form (Whiting and Coggins, 1960). In this case Amberlite resin IR-400 (acetate form) was used but the flowing chromatogram for Dowex 1 (acetate form) presents a similar picture. In Fig. 7, acids I, 11, and IV were not identified; acid I11 was identified as 2-methyl-2,3-dihydroxybutyricacid; acid V was identified as 3-methyl-2,3-dihydroxybutyricacid; acid VI was tentatively identified as 2-ketogluconic acid; acid VII as digalacturonic acid; acid VIII as benzoic acid; acid IX as mucic acid (this acid is relatively insoluble and generally crystallizes out in the tubes during collection) ; acid X as trigalacturonic acid. In cases where a number of acids overlap into several fractions, the combined fractions may be further fractionated on columns of silica gel, or by paper chromatography. Examples of the separation of sugar acids on ion-exchange columns are given in Fig, 8. Solvent systems especially suitable for the paper chromatography of the sugar acids are as follows: (1) Butanol-formic acid-water as solvent (1) for the acids of groups 1 and 2. (2) n-Propanol-NH,OH-water as solvent (2) for the acids of groups 1 and 2. (3) Phenol, 300 gm.;water, 100 ml.; formic acid, 4 ml. The R, values of some of the sugar acids in

-

ULU Molic

0.5N

40

/itramalic\

1

I

1

60

80

100

I I

0.75N LON

-

J

/Succinic

20

m

I

I

120 140 Fraction No.

I

2N

I

160

1111

3N4N%N

Acetic acid

1

I

I

I

180 200 220 24C

6N Formic acid

Concentration of eluant

FIG.7. Chromatography of acids in cider on an anion exchange column. (After Whiting and Coggins, 1960.)

FIG.8. Chromatography of sugar “acids” on Dowex 1 (acetate form). Gluconic acid ( A ) : the free acid; ( B ) : an artifact produced by evaporation with formic acid; ( C ) : the lactone. 367

368

A. C. HULME

these solvents are given in Table IV and the color reactions after spraying the chromatograms with various reagents (see below ) are shown in Table V. As already stated, acids which form lactones, e.g., gluconic, gulonic, and saccharic acids will tend to give two spots in acid solvent systems, the lactone traveling faster than the corresponding acid. Spray reagents for detecting sugar acids. Details of the reagents used in the results given in Table V are as follows: Reagents (c)-(g) are those described for acids of groups 1 and 2. Reagent (i) was developed by Abdel-Akher and Smith (1951) for sugar lactones and esters but, TABLE IV

R, VALUESOF SUGARACIDSIN THREESOLVENT SYSTEMS WITH WIUTMANNo.

Sugar acid Arabonic lactone Galacturonic Gluconic Glucuronic Gulonic-ylaotone Mucic Saccharic

2 PAPER'

R , in solvent

R, in solvent

R , in solvent

(1)

(2)

(3)

0.29 0.06 0.09 0.09

0.42 0.09, 0.26 0.37

0.53

0.10 0.00

0.0s

0.33 0.38 0.22 0.23

0.13

0.26, 0.33'' 0.11 0.44

0.00 0.11

Where two values are given the highest one is that of the lactone.

* Artifact produced on evaporating acid with formic acid.

by a preliminary hanging of the papers in a closed jar containing a dish of ethereal diazomethane, the acids are converted into the methyl esters with which the following treatment gives a blue or mauve color. The dried paper is sprayed with a freshly prepared solution of alkaline hydroxylamine (equal volumes of 1 N methanolic hydroxylamine hydrochloride and 1.1N methanolic potassium hydroxide). After drying again for 10 minutes the paper is lightly sprayed with a 1%solution of hydrochloric acid containing 1-% femc chloride. Spraying without the preliminary treatment with diazomethane will, of course, distinguish between the lactones and the free acids. Reagent (k), described by Gee and McReady ( 1957), is a filtered, saturated solution of basic lead acetate. The paper is rapidly dipped in the solution, blotted to remove excess reagent, and then heated for a few minutes. Gee and McReady also suggest another reagent which gives tan spots on a white background in the presence of 10 Fg. or more of free uronic acid. The paper is dipped in this reagent (2 gm. aniline, 2 gm. trichloroacetic acid hydrate dis-

TABLE V SPRAYTESTSFOR PAPWCHROMATOGFLOES OF SUGAR ACIDS Test and color reaction in solvent (1)

Sugar acid

--

3 Arabonic lactone 0

(4

(4

(4

(el

Gluconic

Pink Black Green White Pink Yellow- Pink White brown Pink Black Yellow White

Glucuronic Gulonic-7-lactone Mucic

Pink Black Pink Black Pink -

Pink Green Pink

Saccharic

Pink Black

Yellow White

Galacturonic

White White White

Daylight

Ultraviolet 5uorescence

Pink Yellow, Yellow, brown edge pink edge Yellow, Yellow pink edge White Yellow

-

pink

White

White

White

Pink

First color

-

Grey-blue -

Grey-blue

Second color Blue-green Green Grey Grey-blue Grey-blue Grey-blue Grey

(2)

(k)

Orange Brick-red

Orange

-

Tan

-

Faint yellow

-

370

A. C. HULME

solved in 100 ml. of ethyl acetate), dried in air for 15 minutes, and then heated for 5 minutes at 95OC.

B.

QUANTITATIVE

DETERMINATION

There does not appear to have been any systematic attempt at a quantitative determination of the individual acids of a mixture of sugar acids. Khym et al. (1957) describe the quantitative separation of glucuronic acid (kept entirely in the form of the free acid by solution in 0.02 M NaOH) and galacturonic acid from one another and from sugars by the use of a column of Dowex 1 (acetate form). It would appear that a quantitative method might be developed by separation of the sugar acids on ion exchange columns followed by paper chromatography (highly hydroxylated compounds such as these acids might be difficult to remove quantitatively from silica gel). The acids and their lactones could then be quantitatively eluted from the paper and determined either by the orcinol method (Brown, 1946) or the anthrone method of Dreywood (1946). The latter method, originally somewhat unreliable, has been perfected for sugars, for example by Yemm and Willis (1954),and could undoubtedly be adapted for sugar acids. The periodate method for the determination of carbohydrates should also be applicable here (Hirst and Jones, 1949; Bradfield and Flood, 1961 ) .

VI. Keto Acids (Group 4) The chief difEculty in the detection and accurate determination of the keto acids in plants and microorganisms lies in the instability of certain members of the group (e.g., acetoacetic and oxalacetic acids) and the rapid turnover of these acids in the tissue. Isherwood and Niavis (1956)have made a careful study of this problem for the a-keto acids. The present author has tried many of the published methods for the determination of the acids (see Wolf, 1955; Ranson, 1955; Smith, 1960) and has found the method described by Isherwood and Niavis (1956)to be the most satisfactory method available. These authors investigated only pyruvic, oxalacetic, and a-ketoglutaric acids. The present author has modified the method somewhat and has used it also for acetoacetic, oxalosuccinic, ketomalonic, acetone-dicarboxylic, and glyoxylic acids. For the three keto acids first mentioned and also oxalosuccinic, glyoxylic, and ketomalonic acids good quantitative results are possible but even the 2,4-dinitrophenylhydrazone of acetoacetic acid is unstable and only approximately quantitative results can be obtained. Free acetone-dicarboxylic acid and its 2,4-dinitrophenylhybzone are extremely unstable

DETERMINATION OF ORGANIC ACIDS

371

in solution and the method can be used only for the detection of this acid (Hulme, 1954b). Because of the great importance of the keto acids in general metabolism and the dsculties encountered in their quantitative determination by the older methods, working details will now be given for the modified method of Isherwood and Niavis, &st in the organisms and then in the media in which they are grown. A. DETERMINATION IN MICROORGANISMS

I . Extraction and Qualitative Examination The organism should be frozen in solid CO, or liquid nitrogen. (For fruit tissue, up to 1 kg. has been treated satisfactorily in this way.) Grind the frozen tissue immediately (if necessary with acid-washed sand, etc.) at low temperature (-loo to -15.C.). If liquid nitrogen is used it will generally be found possible to grind directly in a mortar without the need for an abrasive. All subsequent manipulations must be carried out rapidly. Weigh out an aliquot (if large amounts of tissue are being used) and transfer to 1to 2 volumes of 0.6 M metaphosphoric acid in a Waring blendor. Blend for 1to 2 minutes keeping the temperature as low as possible (about -3OC.) without freezing out the metaphosphoric acid. If an emulsion is formed during blending it may be dispersed by adding a little ether before centrifuging. With bacteria and small fungal hyphae, spores, etc., it may be possible to combine the two stages and grind the material directly in a mortar with the metaphosphoric acid. This should be done in a chilled mortar in a room at 1OC. After grinding and/or blending, still at l0C., filter off any large debris through glass wool and wash the residue with metaphosphoric acid. Centrifuge well at low temperature (OOC.) and, to the supernatant liquid, add 10 to 20 ml. of a 1%solution 2,Cdinitrophenylhydrazine in 5 N H,SO, depending on the amount of keto acid present, Bring to room temperature and let stand for 30 minutes. Extract the hydrazones with four successive portions (0.4 volumes) of ethyl ether and shake the combined ethereal extracts with a slight excess of saturated NaHCOs so that the mixture becomes alkaline (pH 8.4).Separate the aqueous layer and shake the extract with 15 ml. of saturated NaHCO, to remove the last traces of hydrazone. The alkaline extraction removes the keto acids and leaves behind in the ether excess phenylhydrazine and any nonacid keto compounds. (If the aqueous carbonate extract of the 2,4-dinitrophenylhydrazonesis turbid at this stage it should be centrifuged.) The combined aqueous phases are acidified with cold 2 N H,SO, to pH 2.0 and then extracted three times with chloroform-

372

A. C. HULME

ether (85:15, v:v). The combined chloroform-ether extracts are evaporated to dryness in vacuo at room temperature. For qualitative examination, the residue is dissolved in a minimum volume of ethanol, spotted-up on paper chromatograms, and run in the solvent systems as described below, The yellow spots formed by the hydrazones are more clearly visible when viewed under ultraviolet light, and it is an advantage for the qualitative examination of the chromatograms to spray them with 1N NaOH which turns the spots deep brown and shows up clearly spots that would otherwise be very faint, The alkali-treated spots must be viewed immediately after spraying since this color change is transitory.

’

2. Quantitative Determination For quantitative separation and estimation of the keto acids, by the following method, which is a modification of the method of Isherwood and Cruikshank (1954), the residue of the combined chloroform-ether extracts (above) is dissolved in phosphate buffer at pH 7.0. An accurately measured quantity is spotted-up on Whatman No. 3 filter paper which has been buffered in 0.2 M sodium phosphate buffer (pH 6.2). As much as 100 pl. can be used provided the “spot” is kept small by drying in a blast of cold air during application to the paper. Two solvent systems are used, ( a ) the nonaqueous phase of tert-amyl alcohol-ethanolwater (50:10:40, v:v); ( b ) n-butanol-ethanol-0.5 N NH,OH (70: 10:20, v:v). System ( a ) is suitable for glyoxylic, pyruvic, oxalacetic, ketoglutaric, and ketomalonic acids; it requires a run of 40 to 44 hours and the paper should then be dried without heat. The yellow spots of the phenylhydrazones are ringed, preferably in ultraviolet light to show the full area of the spot, and cut out. The hydrazones are dissolved out of the paper in 0.2 M sodium bicarbonate and the light absorption read at the appropriate wavelength (see Table VI). It is an advantage to take readings over a region of 5 to 10 mp on each side of the “maximum” and record the highest readings. The wavelengths given in Table VI were obtained from absorption curves of pure, freshly prepared 2,4-dinitrophenylhydrazones (Clift and Cook, 1923; modified by Hulme, 1954b) dissolved in 0.2 M sodium bicarbonates4 The amount of the keto acid in the extract of the spots from the chromatograms is read off from standard curves constructed from measurements of the absorption of pure samples of the phenylhydrazones. When run on paper chromatograms immediately after preparation, pyruvic acid phenylhydrazone gives only one spot (RI 0.68 in Table VI) but after a short time in solution an ‘The older method of dissolving the hydrazones in 1% sodium carbonate NaOH is inadmissable because, in the course of thereby producing the “&ah shift,” breakdown of the hydrazones is greatly accelerated.

+2 N

373

DETERMINATION OF ORGANIC ACIDS

isomer also appears on the chromatograms. Fortunately, the maximum molecular absorption in bicarbonate is the same for both isomers and in quantitative analysis the two spots are cut out and dissolved together for subsequent examination in the spectrophotometer. The percentage recoveries obtained by running sampks of freshly prepared 2,4-dinitraphenylhydrazoneson chromatograms, dissolving out

a,

TABLE VI WAVEI.ENGT€IS OF MAXIMUMABSORPTION OF 2,4-DINITROPHENYLIfYDRAZONES OF KETO ACIDS IN SOLVENT SYSTEM ( a ) (20OC.)

VALUES

AND

~ _ _ _ _ _ _ _ _ _

~

Acid

Wavelength of maximum absorption (ma)

R, value

1

Acetoacetic Acetone dicarboxylic Glyoxylic a-Ketoglutaric Ketomalonic Oxalacetic Oxalosuccinic Pyruvic Pyruvic isomer

Breakdown to acetone 0.51 0.62 0.29 0.46 0.35 0.68 0.83

375" 375" 365 380 370 380 380 370 370

' Value obtained in solvent system ( b ) , TABLE VII PERCENTAGE RECOVERIES OF 2,4-DINITRoPHENYLHYDRAZONES OF KETOACIDS FROM PAPERCHROMATOGRAMS IN SOLVENT SYSTEM ( a ) Acid Glyoxylic a-Ketoglutaric Ketomalon ic Oxalacetic Oxalosuccinic Pyruvic

Percentage recovery 95 103 99 103 99 99

the spots, and reading in the spectrophotometer, as described, above are shown in Table VII. Isherwood and Niavis (1956) give recoveries of pyruvic, oxalacetic, and ketoglutaric acids added to plant material, which was then subjected to the extraction procedure described above, as 70-m. The 2,4-dinitrophenylhydrazone of acetone-dicarboxylic acid and, to a less extent, acetoacetic acid readily break down during the bicarbonate extractions; they break down even during the run on paper. However, if

374

A. C. HULME

the extraction procedures are carried out rapidly, the hydrazones dissolved in phosphate buffer at pH 7.0 and the chromatograms run in solvent system ( b ) on Whatman paper No. 3MM, preferably at 1 O C . rather than at the usual m0C. (Table VIII), then very approximate quantitative figures may be obtained. The whole range of the phenylhydrazones of the keto acids cannot be run in solvent system ( b ) because they will not all separate in this system. A recent paper by Shimi and El Dein (1960) describes the estimation of keto acids during the mycological production of citric and oxalic acids TABLE VJII Rr

KETO ACIDS Two TEMPERATURES IN Two SOLVENT SYSTEMS

VALUES O F 2,4-DWITROPHENYLHYDRAZONES OF

HUN A T

R, values Solvent ( a ) on paper No. 3

Acid Acetoacetic Acetone dicmboxylic

2OOC.

Complete breakdown to acetone

Solvent ( h ) on paper No. 3 MhI

1°C.

20OC.

1°C.

0.57"

0.54"

0.52

0.25"

0.0'Jb

0.13

Slight breakdown to acetone.

' Very slight breakdown.

' Slight breakdown to acetoacetic acid and acetone.

from cane molasses, but the washing of the precipitated 2,4-dinitrophenylhydrazones with bicarbonate advocated here must lead to serious losses.

B. DETERhlINATIOX IN MEDIA The media should be filtered through muslin on a Buchner funnel, or centrifuged. To the filtrate (or supernatant) metaphosphoric acid is added at the rate of 12 gm./100 ml. of medium. Centrifuge if necessary and add 20 ml. (or more if required) of 2,4-dinitrophenylhydrazinein 5 N H,SO,. Then proceed as under " A above.

VII. Fatty Acids (Group 5 ) A. METHODSOF EXTRACTION FROM ORGANISMS Many methods have been published for the extraction of lipids from various tissues (see, for example Folch et nl., 1951; Blankenhorn and Ahrens, 1955; Insull and Ahrens, 1959). James (1960) gives a summary

DETERMINATION OF ORGANIC ACIDS

375

and an appraisal of the various methods and the various solvent mixtures used for extraction. It is suggested that the following method should be satisfactory for microorganisms though modifications might be necessary in particular circumstances. Such modifications might be made after consulting the papers mentioned above. 1 . Short-Chuin Volatile Fatty Acids! (C,-C,) These acids because of their volatility must be determined on the fresh (i.e., not dried) material. Grinding should be carried out at OOC. The short-chain acids are best handled as the free acids since the esters are more volatile and therefore more easily lost. The free acids separate better by gas-liquid chromatography (GLC ). Grind the fresh material with chloroform or carbon tetrachloride with enough 0.2 N H,SO, to keep just acid (to liberate any acid present as a salt). Extract the ground residue twice more with solvent. Evaporate the combined extracts until dry in a mild blast of nitrogen at room temperature. Dissolve in a minimum of chloroform for introduction to the GLC column. For the subsequent GLC of these short-chain fatty acids see James (1960). Examples of the separation of the short-chain fatty acids and their methyl esters are given in Figs. 12 and 13 (later). In certain cases the short-chain acids may be isolated as their sodium salts. James and Martin (1952) describe a simple method for obtaining, from the salts, the acids free from water (the presence of which interferes with the separation on the GLC column) and then distilling them onto the gas chromatogram at room temperature. It should be noted that fatty acids should never be stored free from solvents except in sealed ampills under nitrogen.

2. Long-Chain Fatty Acids (C,-C,, ) The dried material may be extracted with a variety of solvents (if sodium salts are present, acid may be necessary to liberate the free acids). Various methods are described by James (1960). Here again, probably the best solvents to use for microorganisms would be chloroform or chloroform-methanol ( 2 : 1, v:v), or carbon tetrachloride since this will minimize simultaneous extraction of other organic acids which might interfere with subsequent separations on the gas column ( Bayer, 1958). The solvent is then evaporated under reduced pressure and the clried product saponified by hydrolysis for 3 hours under reflux with a tenfold excess of methanolic KOH. The methanol is removed in vacuo (below ‘These acids may also be determined directly by silica gel chromatography as describcd in Section 111 under organic acids (see Isherwood, 1946; Scarisbrick,

1955).

376

A . C. HULME

50°C.), water added, the liquid extracted with petroleum ether, acidified, and re-extracted with petroleum ether, etc., exactly as described by James (1980, p. 18). When GLC is to be used as the final stage in the estimation of the fatty acids, all solvents used in extraction, etc., should be carefully distilled before use, and particular care should be taken that the petroleum ether used is free of aromatics and olefins.

B. EXTRACTION FROM MEDIA The separation of fatty acids for analysis from media is a difficult problem not only because of the variety of media both liquid and solid used, but because, especially with the lower fatty acids, the partition between media and fat solvent is not sufliciently in favor of the solvent for rapid quantitative extraction. So far as the author is aware this problem has not been investigated systematically and the best that can be done here is to make suggestions of the lines along which it might br solved. Consider first the difficulties involved: (1) The medium in which an organism is grown is generally extremely bulky in comparison with the total amount of the organism. ( 2 ) Because of the complexity of the medium no simple method of precipitation, etc., of unwanted constituents is possible. ( 3 ) A concentration of the media by evaporation of water in vucuo at relatively low temperatures is impossible because: ( a ) During such evaporation the action of enzymes secreted into the medium by the organism will be accelerated and changes in acids due to this cause may be considerable. ( b ) Volatile acids will be lost during the process; this difficulty might well be overcome by making the medium faintly alkaline before evaporation. ( c ) Most media contain sugars, peptones, salts, etc., which yield a sticky mass on evaporation and this mass will still contain considerable quantities of water. “Vac-ice” drying is not satisfactorv because, as concentration proceeds, the mass of solutes present will lend to a thawing out followed by “spitting” of the viscous liquid. Another difficulty, namely interference in the subsequent analysis of solventsoluble compounds such as the sterols, can be overcome by sublimation of the esters of the fatty acids in the final stages of preparation (see latcr, Stoffel et al., 1959). The three following methods take into account some of these difficulties and one or other of them, or combinations of them, should lead t o ;I satisfactory separation of fatty acids from most types of media. (1) The procedure of Folch et al. (1951) which was developed for brain tissue. With media the first step is the liberation of any combined fatty acids by acidifying with H,SO,. The media should then be extracted several times with chloroform-methanol (2:1, v:v). After separation of

DETERMINATION OF ORGANIC ACIDS

377

the lower layer, it should be washed with large volumes of water to remove nonlipid material. This procedure can all be done at OOC. Finally the chloroform extract is evaporated to dryness at low temperature. The residue should be saponified and the fatty acids finally recovered in petroleum ether as described for the fatty acids extracted from organisms. The disadvantage of this method is that most of the volatile shortchain acids will be lost largely because of their solubility in water. (2) The method devised by Blankenhorn and Ahrens (1955) for the extraction of the products of triglyceride digestion in man. This involves extraction, after acidification with HCl to pH 3.5,with a solvent system of equal volumes of water, ethanol, ethyl ether, and heptane followed by further separations in a Craig countercurrent distribution apparatus using several different solvent systems. A novel point in the procedure of Rlankenhorn and Ahrens is the use of anion exchange resin (sodium form) at an intermediate stage in the separations to trap fatty acids in the more aqueous of the two phases while nonacid lipid material is dissolved out into the nonaqueous (solvent) phase. (3) To the medium add two volumes of ethanol (to precipitate enzymes and other compounds of high molecular weight) and filter or centrifuge off the precipitate. Make the filtrate fairly alkaline with NaOH (to convert any free volatile fatty acid to the sodium salt), Evaporate off the ethanol in oacz~ubelow 4OOC. Freeze and then carefully “vac-ice” dry the alcohol-free liquid as far as possible. Acidify to pH 2 with HC1 to liberate the free acids and extract with chloroform-methanol (2:1, v:v) or carbon tetrachloride. Evaporate the solvent extract to dryness in oacuu at low temperature, or by means of a mild blast of nitrogen. Saponify, etc., as described in Section VII, A, 2. Finally it should be noted that the above methods are not completely satisfactory if volatile fatty acids are present, and the lower members of this group when present in sufficiently large amounts are best determined by steam distillation after acidification. A suitable method is given bv Scarisbrick ( 1955). AND QUANTITATIVE DETERMINATION C. QUALITATIVE

1 . General Classic methods of determination of the volatile fatty acids (saturated and unsaturated) up to capric (C,,), together with special methods for individual acids, are described in detail by Scarisbrick (1955). The reader is referred to this comprehensive treatise. Spectroscopic methods are dealt with, for example, by Hilditch et al. (1951) and by Singh et al. ( 1956).

378

A. C . HULhlE

Undoubtedly, the best method now available for the systematic qualitative and quantitative determination of the whole range of fatty acids is that involving gas-liquid chromatography. This new analytical tool was first developed by James and Martin (1952) and a description of the principles of the method and of the apparatus required, especially in relation to fatty acid analysis, has been given by Jamcs (1960) and a l s o by Farquhar et ul. (1959). As with all chromatograms, one phase of iin immiscible pair is held stationary on a column and the second phase is moved continuously through it. Any mixture applied to the top of the column and washed through by the moving phase will separate into individual components provided those components possess sufficiently different partition coefficients in the two phases used. The essential units in a gas chromatography apparatus are a column of suitable material on which the stationary phase is deposited, a suitably regulated gas stream to “flow” the acids under investigation along the column, and a sensitive. detector by means of which the acids leaving the column may be picked up. The compounds (in this case acids) being “flowed” along the column must of course be volatile at the temperature at which the column is held. For fatty acids and their esters (prepared from the acids-see below) many workers use columns of Celite (diatomaceous earth) impregnated with greases or polyesters, argon as the flowing gas, and an ionization detector (using strontium80 as the ionizing agent) to measure the concentration of ionized vapor (of the acid or ester) in each zone leaving the columns. Usually also in the latest form of apparatus the inform at’ion supplied by the detector is amplified and fed into a chart (potentiometric) recorder. The recorder chart appears at the end of a run with peaks representing individual components of the fatty acid (or ester) mixture, the area beneath the peaks representing the amount of each constituent present (see Fig. 9 ) . This record chart is indeed quite comparable with the “flowing chromatogram” obtained “manually” from silica gel or ion exchange columns (see p. 364). At Ditton Laboratory, Dr. D. F. Meigh, who has developed the gusliquid chromatography apparatus shown in Fig. 10 for the estimation of volatile substances of all kinds from fruits and for the determination of fatty acids in the peel of apples, etc., uses Celite as the support for the stationary phase, mixtures of hydrogen and nitrogen as the flowing gas, and a very sensitive flame ionization detector (Meigh, 1960). A modified form of this detector suitable for the GLC of fatty acids is shown in Fig. 11. The order in which fatty acids will leave the column is, with a given stationary phase, no different whether argon or hydrogennitrogen mixtures are used. The disadvantage of the flame ionizer over other detectors of comparable sensitivity ( e.g., the argon ioniziition

DETERMINATION OF ORGANIC ACIDS

379

monitor) is that the acids under investigation are burned and cannot subsequently be recovered. It is not necessary to describe in detail the construction and operation of a gas-liquid chromatography ( GLC ) analyser; detailed working instructions are given in the papers already referred to (James, 1960; Farquhar et al., 1959; Meigh, 1960). The preparation of material in a suitable form for placing on the column (usually from some form of micropipette) has already been described.

FIG. 9. Copy of recorder chart obtained during the separation of saturated and unsaturated fatty acids on a Celite column impregnated with silicone “elastomer E301” run at 200°C. Peaks 1-8: saturated acids Ca,, C1?,C,,, C,“, C,,, CIS,Clu, Cz0; peak 9: 10-undecanoic acid; peak 10: oleic acid; peak 11: linoleic acid; peak 12: 12hydroxystearic acid. (Supplied by D. F. Meigh.)

James and Martin (1952) have described a recording burette for the determination of the acids leaving the column (and being a titration method, it can only be used with free acids). This saves the cost of a recorder but the auxiliary apparatus is fairly complicated and the results have to be integrated (see Fig. 12) to give the final clear pattern of separated acid peaks which is achieved directly with the detector and recorder arrangement mentioned above. A brief consideration will now be given to suitable stationary phase materials for the separation of short-chain free acids and short- and long-

380

A. C. HULME

FIG. 10. Photograph of gas chromatography apparatus showing the column enciisccl in its heater with the flame ionizer immediately above (center of picture) and the recorded and automatic bwitcli gcar ( t o left of picture). Dcsigned by D. F. Meigh.

DETERMINATION OF ORGANIC ACIDS

381

chain fatty acid esters and to the preparation of these esters preparatory to placing them on the column. As already mentioned, Celite (No. 545, Johns Manville Company, Ltd.) is now almost exclusively used for supporting the stationary phase in GLC. Brick dust has been used but at the /AIR

OUTLET. G L A S S WOOL

.IS0

M E S H GAUZE. ROTATING FLAME LIGHTER, 3 MM. LOOP NICHROME WIRE.

Nr. I2 HYPOOERMIC *NEEDLE.

TPUT TO AMPLIFIER.

W

G fj

L

A

S

S

TUBE FROM COLUMN.

l i VOLTS. VOLTS.

,XIAL SOCKET.

FIG. 11. Flame ionization detector designed by D. F. Meigh. The eluting gas, which dso providcs the flame, is ii misturc of hydrogen and nitrogen ( 5 0 / 5 0 ) .

high temperature required (200OC.) for adequate separation of the higher fatty acids, mineral matter present in the dust may catalyze undesirable changes in the compound under examinGtion. The Celite must be conditioned by treatment with acid and alkali before use to remove traces of materials which again may produce unwanted catalytic changes. Working details of the preparation of the Celite and of the preparation

Naof

36

3

2

4

4

5

5

5

-

3c0

m to

u

Eaperimentol curve

0

I0

20

30

40

50 Tim

in

60

70

80

90

I00

rninufes

FIG. 12. Separation of the short-chain free fatty acids by GLC. Temperature 137°C.; column length, 11 ft.; nitrogen pressure, 76 cm. Hg above atmospheric; stationary phase, DC 550/10!&stearic acid; flow rate. 18.2. ml. per minute; column efficiencv, 2000 plates. Detection by antomatic recording burette. (From James, 1960. )

DETERMINATION OF ORGANIC ACIDS

383

and conditioning of the columns are given, for example, by Farquhar f?t al. (1959). The employment of both nonpolar (silicone and Apiezon greases) and polar (organic acid polyesters of diethylene glycol) gives a valuable “check on the identity of the acids (or their esters) under examination since unsaturated acids leave the column in a different order from their corresponding saturated acid depending on whether a nonpolar or a polar stationary phase is used; the unsaturated acids are retained to a greater extent by a polar stationary phase. Some workers prefer a relatively coarse Celite, 60-80 mesh, while others use a fine particle size, 100-140 mesh or even 140-170 mesh. At present there appears to be no advantage in any particular limit of particle size. James (1960) states that it is not convenient to attempt complete resolution of mixtures containing both short- and long-chain fatty acids on one column. As already remarked the short-chain acids are best separated as the free acids, since their low volatility prevents loss during handling. The free long-chain acids do not separate well and should, therefore, be esterified before placing on the column. The esters of the complete range of acids can be separated on one column if arrangements are made to raise continuously the temperature of the column during a run. This is difficult to do in a reproducible manner without expensive, completely automatic procedures. Other factors involved in such a procedure are mentioned in Section VII, C, 3, c. Generally it appears better to treat the long- and the short-chain acids separately. In special circumstances it may be useful to make a preliminary separation of complex lipid mixtures into groups by silicic acid chromatography before resorting to GLC. The use of silicic acid chromatography for this purpose is described by Hirsch and Ahrens ( 1958). It must not be overlooked that the analysis by GLC of microorganisms or the media in which they are grown is a difficult problem. Unlike the material used in studying body fats, or plant and animal tissues particularly concerned with the elaboration of fats, the total amount of fatty acids and their compounds present in microorganisms and their media may be a very small fraction of the total material. It may not be possible to separate completely the fatty acid compounds from the rest of the material present (see methods of extraction, Section VII, A and B ) . Indeed it is likely that at least some organic acids other than fatty acids may be present in the final extract prepared for fatty acid analysis. If sufficiently volatile under the conditions used in the GLC separations, these acids may appear on the GLC chromatograms; this is especially likely if the fatty acid sample has been subject to methylation.

384

A. C. HULME

So far as the author is aware, this problem has never been investigated, so no precise guidance can be given here. Microbiologists who decide to use GLC to investigate the fatty acid metabolism of their organisms will have to pioneer the field! Before proceeding to a discussion of the methylation of acids and the separation of the esters by GLC, a selection of some stationary phase TABLE IX COMPOSITION OF SOMESTATIONARY PHASESUSEDIN THE SEPAHATION OF FATTYACIDSAND THEIR ESTERSBY GLC

Stationary phase

Type

Temprrature range ("C.1

10% Steario acid in DC-550 silicone

Polar

10@140

15% Sebacic acid in dioctyl sebacate Dioctyl phthalate

Polar

Up to 150

Low melting paraffin wax Apiezon grease L Silicone grease E 301 Polyethylene glycol adipate Diethylene glycol succinate polyester

Mainly polar Nonpolar

60-150

Nonpolar

Up to 300

Nonpolar

u p to 200

Polar

u p to 200

Very polar

u p to 200

80-150

Remarks Suitable for free acids (C1-Ce). If formic acid present, add 10% orthophosphoric acid Suitable for free acids (C,-Cd Suitable for methyl esters of acids C1-Cs Suitable for methyl esters of acids CI-Cs Suitable for methyl esters of acids C6-CIu Suitable for methyl esters of acids Ca-CZn Suitable for methyl esters of acids up to Can Suitable for methyl esters of acids up to Cia. Speed and resolution excellent

Refcrcncc See James (1960) See Raupp

(1959) Sce James (1960) See James (1960) See James (1960) See Meiglr (1960) See James (1960)

Lipsky and Landown (' (1 959)

compounds, their characteristics and uses will now be presented (Table IX) . 2. Preprution of Methyl Esters for Separation by GLC After the acids have been extracted from the tissue, and, if necessary, saponified and separated into groups (see methods of extraction) they may be converted to their methyl esters, before being presented to the GLC columns, by the following two standard methods. It may be advisable to free the esters, when prepared, from contaminants by sublimation (Stoffel et al., 1959) before presentation to the column.

DETERMINATION OF ORGANIC ACIDS

385

a. Interestcrification with Methanolic HC1. This is described in detail by James (1960). Stoffel et al. (1959) describe an excellent micromethod for the preparation of methyl esters by this process. For this method of esterification the starting material must be quite free from water. It has the advantage that a preliminary saponification is not necessary. b. Esterificution with Diazomethane. Working details of this method are given by James (1960) and more generally by Vogel (1956). For this method the sample need not be absolutely dry. Diazomethane is, however, very toxic and also tends to explode so that special precautions must be taken in its use. The disadvantage of method (u) is that if glycerides are present in the fatty acid mixture, the h a 1 product contains not only the methyl esters but also nonsaponifiable substances from the original lipid. However, if Stoffel and associates’ (1959) sublimation of the esters is carried out this objection does not apply. Method ( b ) has the disadvantage that the sample must first be saponified with alkali (see, e.g., James, 1960, p. 12) which may lead to isomerization. Diazomethane may lead to pyrazoline formation and a loss of polyunsaturated acids. The choice must be made to suit the particular circumstances, but, in the present context, the method of Stoffel et al. would probably be the first choice.

3. Results Obtained by the Use of GLC a. Qualitatice Amlysis. Retention volumes and retention times ( these are equivalent constants) of compounds on a column go a long way toward establishing the identity of the compound, In this respect they are similar to the position of organic acids, etc., when leaving ion exchange columns in gradient elution and to R, values in paper chromatography. Just as marker spots of known compounds can be run alongside “unknown” compounds on paper chromatograms, so known acids and esters can be run with “unknown” acids and esters on gas chromatograms as an aid to the establishment of the identity of the “unknown” acids and esters. Where the acid(s) are unknown or unavailable, runs on gas chromatograms are so reproducible under any given conditions that identification may be achieved by comparison with published chromatogram record charts, a whole range of which are now available for fatty acids (James, 1960). One of the great advantages of GLC is the rapidity with which the solutes pass through the column and are recorded as peaks on the recorder chart. Retention times for the various acids on various stationary phases are given, for example, by Farquhar et al. (1959), Lipsky and Landowne (1959), and James (1960). On silicone or Apiezon greases, acids up to C,, run through at elevated temperatures

386

A . C. I-IULME

(see Table IX) in about 1 hour, For these stationary phases and for some polyester-impregnated columns the time to run through is approximately doubled for each C, increase in chain length of fatty acid. Speed is increased with increase in the temperature of the column but this has disadvantages (to be discussed later). The ideal stationary phase is one which gives good separation with minimum retention. Lipsky and Landowne (1959) have found a succinate polyester of diethylene glycol to be excellent in this respect. James (1960, p. 25) gives a useful table showing the effect of molecular structure of the fatty acids on retention volumes. An aid to the identification of unsaturated acids is to make two runs, one on a nonpolar (grease) and one on a polar (polyester)-impregnated column. This will be clearly seen in the figures which follow. James (1960, p. 45) gives useful information on the identification of fatty acids by GLC. Here it should be mentioned that the amounts of starting material required for GLC are very small-usually less than 1 mg. for the whole sample. As little as 0.2 pl, of mixed esters can be used where mixtures of pure compounds are concerned (Farquhar et al., 1959). Column efficiency depends on sample size only when the latter exceeds the critical level at which the distribution coe5cient becomes dependent on the weight of the solute, i.e., when the solvent (stationary phase) is changed by the presence of the solute (sample), overloading with the latter may cause band skewing. Typical separations (flowing chromatograms) of fatty acids and their esters are shown in Figs. 12-17. b. Quantitative Determination. Before accurate quantitative results can be obtained, it is necessary to know whether the ionization current recorded on the moving chart is directly proportional to the amount of fatty acid methyl ester applied to and eluted from the column. This “linearity of response” must be assured over a fairly wide range of peak heights of individual components of a “calibration mixture” of acid esters (10-100% of full-scale deflection) when the column is not overloaded, With sensitive detectors where the maximum load is necessarily low, it is essential in calibration to use samples of known concentration in a solvent such as chlorobenzene. Usually it is a fairly simple matter to adjust conditions so that a linear response is obtained. Details of IIOW to make the necessary corrections, and suggestions of suitable ester mixtures for calibrating instruments, are given both by James (1960) and Farquhar et d. (1959). To obtain the amount of each acid represented by a peak, the peak area is measured. This may be done in several ways (James, 1960). The

387

DETERMINATION OF ORGANIC ACIDS

host accurate is by cutting out and weighing the peak areas after determining the relationship between area and weight of paper, or by means of a planimeter. Details are given by James on how to deal with overlapping peaks. When it is desired to know the absolute amount of acids in a mixture, the load placed on the column must be known accurately, so that the

T i m in minuter

I

7 I2

10

9

I 20

30

40

x)

60

m

80

Tnm in minutes

FIG. 13. Upper: Separation of methyl esters of the short-chain ( C r G ) acids. Temperature, 78.6"C.; pressure, 14 cm. Hg above atmospheric; stationary phase, dioctyl phthalate; flow rate, 50 ml. per minute. Peak identification: (1) air, ( 2 ) formic, ( 3 ) acetic, ( 4 ) propionic, ( 5 ) isobutyric, ( 6 ) n-butyric, (7) a-methylbutyric and isovaleric, ( 8 ) n-valeric, ( 9 ) 3-methylbutyric, ( 10) isocaproic, ( 11 ) n-caproic. Lower: Separation of methyl esters of the short-chain ( C A , )acids. Temperature, 100°C.; pressure, 14.5 cm. Hg above atmospheric; stationary phase, dioctyl phthalate; flow rate, 10.1 ml. per minute. Peak identification: ( 1 ) air, ( 2 ) formic, ( 3 ) methanol, ( 4 ) acetic, ( 5 ) propionic, ( 6 ) isobutyric, (7) trimethylacetic, ( 8 ) n-hutyric, ( 9 ) isovnleric, ( 10) n-valeric, (11) isocaproic, ( 12) n-caproic. (From James, 1960.)

total peak area obtained can be related to the total load. For many purposes it is sufficient to know the total peak area for a given range of acids (e.g., Clo-C22) and to calculate the percentage of any component. In order to measure minor constituents accurately it may be necessary to carry out analyses at more than one level of loading. c. The Accuracy and Reproducibility of Qua.ntitatiue Determinations

388

A . C . HULMk:

by GLC. It is somewhat difficult to obtain figures horn the literature of the over-all accuracy of recovery and determination for a complex mixture of fatty acids. Lipsky and Landowne (1959) give results for a standard mixture of eight methyl esters of fatty acids which suggest that the over-all accuracy when known mixtures of pure compounds are concerned

.tl 4

6

22

u 20

Time

in

mnules

FIG, 14. Separation of 80 pg. of fatty acids from humnn fecal lipids. Temperature, 197°C.; argon pressure, 76 cm. Hg above atmospheric at inlet, ntmospheric at out1c.t; column length, 4 ft. straight tube, stationary phase, Apiezcin L viicuum stopcock grease. Detection by argon ionization monitor; commercial version ( Pye Scientific Instruments). Peak identification: ( 1) solvent (light petroleum), ( 2 ) n-decanoic, ( 3 ) n-dodecanoic, ( 4 ) branched triclccanoic acids, ( 5 ) 11-tridecanoic, ( 6 ) branched tetradecanoic, ( 7 ) monounsatia;itecl tetradcciinoic, ( 8 ) ti-tetr:idecanoic, ( 9, 10) branched pentaclecanoic acids, ( 11 ) n-pentadecanoic, ( 12 ) monounsaturated hexadecanoic, ( 13) branched hexadecanoic, ( 14) n-hexadecanoic, ( 15) highly branched heptadecanoic, ( 16, 17) branched heptadecanoic acids, ( 18) n-heptadecanoic, ( 19 ) linoleic, ( 2 0 ) olcic, ( 2 1 ) positional and configurational isomrrs of oleic, ( 2 2 ) stciiric.. (From James, 1960.)

is very high though there may be 1-2% errors (positive or negative) for some of the individual components, clue, no doubt, to “carry-overs” from one band to another. An aid to the identity and quantitative determination of the unsaturated fatty acids is to compare chromatograms run after bromination or hydrogenation of the methyl esters. Details of the procedure involved are described by Farquhar et al. (1959). James (1960) says that such comparisons as have been made between analysis by GLC and other methods, such as the alkali isomeriz;ition

389

DETERMINATION O F ORGANIC ACJDS

technique, show excellent agreement, It is of interest that such methods as alkali isomerization determine acid types but not specific acids whereas the gas chromatogram determines the amounts of individual acids provided they can be resolved from all other components. Separation of com-

Time in minutes

FIG. 15. Separation of 80 pg. of mixture similar to that shown in Fig. 14. Temperature, 180°C.; argon pressure, 57 cm. Hg above atmospheric at inlet, atmospheric at outlet; column length, 4 ft., straight tube; stationary phase, polyethylene glycol adipate. Detection by argon ionization monitor, laboratory model. Peak identification as in Fig. 14. (From James, 1960.)

A , ,A,

-

.-5 :>O 0.2 .tlJl

,

0.I

,

~

0

0

10

20

30

40

50 60 70 80 90 Time in minutes

100 110

120

FIG. 16. Separation of long-chain fatty acid esters. Temperature, 270°C.; pressure, 43 cm. Hg above atmospheric; gas flow, 29 ml. of N2 per minute; filling 4 gm. Celite 545/Apiezon L, 9 : l ratio; column, 115 x 0.4 cm., amount introduced, approximately 2 mg. Detection by Co? after combustion. The lower components, which eluted before Czrmethyl ester, originate from the impure triacontanoic acid. (From James, 1960.)

ponents and spectrophotometric analysis of the constituents, such as in the method of Hilditch et al. (1951), are more laborious and time consuming than GLC when once the chromatographic apparatus has been set up.

390

A. C . HULhlE

Finally, certain factors affecting the use of the gas chromatographic method for fatty acids should be mentioned. (1) The accurate delivery of liquids into a gas stream is not easy. This becomes an important factor with highly sensitive detectors. The iise of micropipettes has been mentioned. Another more accurate method is that described by Scott (1958) in which a specially designed micropipette is applied to the top of the column by means of a wire. This gives an accuracy of delivery of f1-2% using a 25-pl. pipette. Concentrated and pure samples of esters must be diluted with a suitable solvent I

-

2

- 0.5.-9

3

c

f

4

6

7

12

0

30

10

40

Time in minutes

FIG. 17. Sepurntion of a standard mixture of long-chain fatty acid estcrs. 'Tomperature, 200°C.; column length, 6 ft.; 60-80 mesh; helium; stationary phnsc, SIICcinate polyester of diethylene glycol (LAC-4-R777); How rate, 186 ml. pcr minute. Peak identification: ( 1) air, ( 2 ) methyl laurate, ( 3 ) methyl myristate, ( 4 ) methyl palmitate, ( 5 ) methyl palmitoleate, ( 6 ) methyl stearate, ( 7 ) methyl oleate, ( 8 ) methyl linoleatc, ( 9 ) methyl linolenate, ( 10) methyl arachidnte, ( 1 1 ) methyl behenate, ( 12) mcthyl hcxacosanoate. ( Aftcr Lipsky and Landownc, 1959. )

( e.g., chloroform or chlorobenzene ) before being placed on the columns in order to give a sufficiently large volume for accurate pipetting. ( 2 ) Some breakdown of the stationary phase is inevitable with the high temperature necessary for the flowthrough of the higher fatty acids. The temperatures and stationary phases given above minimize this, With high temperatures also it is more difficult to obtain reproducible results. ( 3 ) Unsaturated and higher saturated acids are liable to break down at high temperatures. This is minimized by decreasing the time necessary for acids (esters) to pass through the column by the use of stationary phases ( such as the succinate polyester of diethylene glycol ) which have a low affinity for the fatty acids (esters) under examination.

DETERMINATION OF ORGANIC ACIDS

391

( 4 ) There may be some interaction between solvent and solute during passage down the column. ( 5 ) It is obviously important to use especially pure organic solvents during the preliminary extraction of the fatty acids from the material under study because of the micro scale of the work. Small amounts of contaminants, if present in the extraction solvents, could appear as interfering peaks in the final chromatograms.

VIII. Conclusion It will be obvious to the reader that, in spite of the general title of this chapter, there are some notable gaps in the list of acids dealt with and which are elaborated by microorganisms (see, e.g., Martin, 1960), particularly in relation to the aromatic, hydroaromatic, and heterocyclic acids (Crombie, 1960). No apology is made for this because the number and range of naturally occurring acids identified will undoubtedly in. crease as the techniques described here achieve general use. No methods are yet available for the systematic examination of the aromatic, hydroaromatic, and heterocyclic acids although the electrophoretic methods being developed for aromatic compounds in general by Pridham (1959) and by Halmekoski ( 1959) may prove to be useful in this field. During the planning of the chapter it was envisaged that the last section would consist of a model quantitative analysis of all the acids in a microbiological “brei.” As writing proceeded the author became more and more appalled at his temerity in suggesting such a possibility. It became clear that the working out of such a scheme might take six months or more and it was finally abandoned. Microbiologists have a great deal of pioneer work to do before such a comprehensive analysis becomes the routine matter which it undoubtedly can become. If the foregoing account of the new techniques now available or under development induces them to grasp the nettle with confidence that the pain will be short lived and the benefits immense, then the object of the author in writing it will have been achieved. REFERENCES Abdel-Akher, M., and Smith, F. ( 1951). J. Am. Chem. SOC. 73, 5859. Anderson, E.,and Sands, L. (1945). Advances in Carbohydrate Chem. 1,329. Bayer, E. ( 1958). I n “Gas Chromatography 1958” ( D . H. Desty, ed.), p. 341. Buttenvorths, London. Bell, D. J. ( 1955). I n “Modem Methods of Plant Analysis” (K. Paech and M. V. Tracey, eds. ), Vol. 11, p. 1. Springer-Verlag, Berlin, Blankenhorn, D. H., and Ahrens, E. H. (1955). J. B i d . Chsm. 212,69. Block, R. J,, D u r n , E. L., and Zweig, G. (1958). “A Manual of Paper Chromatography and Paper Electrophoresis,” 2nd ed., p. 169. Academic Press, New York.

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A. C. HULME

Bradfield, A. E., and Flood, A. E. ( 1961). In press. Bradfield, A. E., and Penney, M. (1948). J. Chem. SOC. p. 2249. Bradfield, A. E., Penney, M., and Wright, W. B. (1947). J. Chem. Soc. p. 32. Brimley, R. C., and Barrett, F. C. (1954). “Practical Chromatography.” Chapman and Hall, London. Brown, A. H. (1946). Arch. Biochem. 11, 269. Buch, M. L., Montgomery, R., and Porter, W. L. (1952). Anal. Chem. 24, 489. Buch, M. L., Dryden, E. C., and Hills, C. H. (1955). J. Agr. Food Chem. 3,960. Bulen, W. A., Varner, J. E., and Burrell, R. C. (1952). Anal. Chem. 24, 187. Cartwright, R. A., and Roberts, E. A. H. (1955). Chem. &. I n d . (London) p. 230. Clift, F. P., and Cook, R. P. (1932). Biochem. J. 26, 1800. Cochrane, V. W. ( 1958). “The Physiology of the Fungi.” Wiley, New York. Crombie, W. M. ( 1960 ). “Encyclopedia of Plant Physiology” ( W. Ruhland, ed. ), p. 889. Springer-Verlag, Berlin. Davies, D. D. (1953). J. Exptl. Botany 4, 174. Desty, D. H., ed. ( 1958). “Gas Chromatography 1958.” Buttenvorths, London. Dreywood, R. (1946). Ind. Eng. Chem. Anal. Ed. 18,499. Farquhar, J. W., Insull, W., Rosen, A., Stoffel, W., and Ahrens, E. H. (1959). Nutrition Reos. ( S u p p l . ) 17 ( 8 ) , Pt. 11, 1. Folch, J., Ascoli, I., Lees, M., Meath, J. A., and Le Baron, F. M. (1951). J. Biol. Chem. 191, 833. Foster, J. W. (1957). Texas Repts. Biol. and Med. 16, 79. Gee, M., and McReady, R. M. (1957). Anal. Chem. 29,257. Halmekoski, J. ( 1959). Suomen Kemistilehti B3%,170. Hanes, C. S., and Isherwood, F. A. (1949). Nature 164, 1107. Hargreaves, C. A. ( 1951). Quoted as personal communication in “Techniques of Radiobiochemistry” (1956), by S. Aronoff, p. 119. Iowa State College Press, Ames, Iowa. Hilditch, T. P., Patel, C. B., and Riley, J. P. (1951). Analyst 76, 81. Hirsch, J., and Ahrens, E. H. (1958). I. Biol. Chem. 233, 311. Hirst, E. L., and Jones, J. K. N. (1949). J. Chem. Soc. p. 1659. Howe, J. R. (1960). J. Chromatog. 3, 389. Hulme, A. C. ( 1951). J. Exptl. Botany 2, 298. Hulme, A. C. (1954a). Biochim. et Bbphys. Acta 14, 36. Hulme, A. C. (1954b). Biochim. et Biophys. Acta 14, 44. Hulme, A. C., and Wooltorton, L. S. C. (1957). J. Sci. Food Agr. 8, 117. Hulme, A. C., and Wooltorton, L. S. C. (1958). J. Sci. Food Agr. 9, 150. Insull, W., and Ahrens, E. H. (1959). Biochem. J. 72, 27. Ishenvood, F. A. (1946). Biochem. J. 40, 688. Ishenvood, F. A. ( 1954). Brit. Med. Bull. 10, 202. Ishenvood, F. A,, and Crnikshank, D. H. (1954). Nature 173, 121. Ishenvood, F. A., and Niavis, C. A. (1956). Biochem. J. 64, 549. James, A, T. (1960). Methods of Biocheni. Anal. 8, 1. James, A. T., and Martin, A. J. P. (1952). Biochem. J. 50, 679. Khym, J. X., Zill, L. P., and Cohn, W. E. (1957). In “Ion Exchanges in Organic and Biochemistry” (C. Calmon and T. R. E. Kressman, eds.), p. 392. Interscience, New York. Kowkabary, C. N. ( 1954). Aduances in Carbohydrate Chem. 9,304. Lederer, E., and Lederer, M. ( 1957). “Chromatography,” 2nd ed., Elsevier, Amsterdam, Holland.

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Lipsky, S. R., and Landowne, R. A. (1959).Ann. N.Y. Acad. Sci. 72,559. Lugg, J. W. H., and Overell, B. T. (1947).Australiun J. Sci. Research Ser. A 1, 98. Magasanik, B. (1957).Ann. Rev. Microbiol. 11, 221. Martin, S. M. (1955).Chem. 6 Ind. (London) p. 427. Martin, S. M. (1960).In “Encyclopedia of Plant Physiology” (W. Ruhland, ed.), p. 605. Springer-Verlag, Berlin. Meigh, D. F. (1955).In “Modern Methods of Plant Analysis” (K. Paech and M. V. Tracey, eds. ), Vol. 11, p. 403.Springer-Verlag, Berlin. Meigh, D. F. (1960).J . Sci. Food Agr. 11, 381. Palmer, J. K. ( 1955).Conn. Agr. Expt. Sta. Bull. 589. Partridge, S. M. (1949).Biochem. J . 44,521. Phkov6, J., and Munk, V. (1960).J. Chromatog. 4,241. Phares, E. F., Mosbach, E. H., Denison, E. W., and Carson, S. F. (1952).Anal. Chem. 24, 660. Pigman, W. W., and Goepp, R. M. (1948). “Chemistry of the Carbohydrates.” Academic Press, New York. Pridham, J. B. ( 1959).J. Chromatog. 2, 605. Pucher, G. W., Vickery, H. B., and Leavenworth, C. S. (1934).I d . Eng. Chem. Anal. Ed. 6, 190. Ranson, S. L. (1955).In “Modern Methods of Plant Analysis” ( K . Paech and M. V. Tracey, eds. ), Vol. 11, p. 539.Springer-Verlag, Berlin. Raupp, G.P. (1959).Angew. Chem. 71,284. Saarnio, J., Niskasaari, E., and Gustafsson, C. ( 1952 ). Suomen Kemistilehti B25, 25. Salton, M. R. J. (1953).Biochim. et Biophys. Acta 10, 512. Scarisbrick, R. ( 1955). In “Modern Methods of Plant Analysis” (K. Paech and M. V. Tracey, eds.), Vol. 11, p. 444. Springer-Verlag, Berlin. Scott, R. P. W. (1958). In “Gas Chromatography 1958” (D. H. Desty, ed.), p. 263. Buttenvorths, London. Shimi, J. R., and El Dein, S. N. (1960).J . Sci. Food Agr. 11, 592. Singh, J., Shah, S . , and Walker, T. K. (1956).Bwchem. J . 62,222. Smith, I. ( 1960). “Chromatographic and Electrophoretic Techniques,” Vol. I, Chaps. 14 and 15.Heineman, London. Sowden, J. C. (1957).Advances in Carbohydrate Chem. 12, 36. Stark, J. B., Goodban, A. E., and Owens, H.S. (1951).Anal. Chem. 23,413. Stoffel, W., Chu, F., and Ahrens, E. H.(1959).Anal. Chem. 31, 307. Synge, R. L. M. (1946).Analyst 71, 256. Trevelyan, W. E.,Proctor, D. P., and Harrison, J. S . ( 1950).Nature 166,444. Vogel, A. I. (1956). “A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis,” 3rd ed., p. 971. Longmans Green, London. Whiting, G. C., and Coggins, R. A. (1960).J. Sci. Food Agr. 11, 337. Wolf, J. (1955). In “Modem Methods of Plant Analysis” (K. Paech and M. V. Tracey, eds. ), Vol. 11, p. 478.Springer-Verlag, Berlin. Yemm, E. W.; and Willis, J. (1954).Biochem. J. 57, 508.

This Page Intentionally Left Blank

AUTHOR INDEX Numbers in italics show the page on which the complete reference is listed.

A

Arai, H., 282, 284, 291 Ardern, E., 194, 220 Arigoni, D., 297, 340 Arima, K., 19Q,209, 221 Amstein, H. R. V., 302, 310, 317, 339 Aronow, L., 251,256 Ascoli, I., 374, 376, 392 Askonas, B., A., 117,125 Atherden, L. M., 118, 125 Atkin, L., 69, 72 Audus, L., 85,105 Azuma, Y.,60,73

Aartiu, D. B., 297,341 Abdel-Akher, M., 368, ,391 Abdel-Tawab, G. A., 121, 125 Abderhalden, E., 193, 219 Abe, M., 282,284,291 Abramson, C., 251, 255 Abramson, S., 170, 187 Ackermann, W. W., 119,125 Adnir, M. E., 4, 73 Adams, R., 145, 169, 187 Ahrens, E. H., 374, 376, 377, 378, 379, 383, 384, 385, 386, 388, 391, 392, B 393 Alexander, L., 80, 103, 105 Backus, E. J., 233, 254 Alexander, 11. A., 32, 72 Baddiley, J., 313, 339 Alg, R. L., 61, 63, 64, 75, 140, 142, 143, Bader, J. P., 119, 125 144, 157, 158, 166, 172, 189, 190, Baggett, B., 118, 125 191 Bahr, H., 79,87,105,106 Allen, H. F., 175,188 Baker, L. E., 114, 120,125 Allen, M. B., 208, 220 Baldacci, E., 258, 263, 266, 269, 270, Allport, D. C., 321, 322, 338, 339 271, 272, 273, 274, 275, 277 Amberg, H., 85, 95, 96, 97, 105 Balderas, L., $18, 125 Amici, A., 274, 277 Balis, M. E., 251, 256 Anchel, M., 294, 334, 339,342 Ban, Y., 314, 340 Andersen, A. A., 142, 188 Barban, S., 121, 125, 126 Anderson, E., 115, 125, 365, 391 Barbesgard, P., 324, 341 Anderson, E. H., 235,253 Baron, S., 120, 128 Anderson, E. P., 249, 253 Barratt, R. W., 42, 72 Anderson, H. V., 285, 291 Barrett, A. S. D., 5, 72 Anderson, K., 145, 188 Barrett, F. C., 355, 392 Anderson, R. E., 139, 142, 188 Barrett, J. P., Jr., 171, 188 Anderson, W. W., 6, 15, 26, 29, 30, 31, Bartlett, M. C., 124, 126 33, 39, 40, 44, 48, 49, 55, 57, 58, Barton, D. H. R., 315,339 67, 73 Bassett, E. W., 305, 312, 318, 339 Angier, 11. B., 229, 255 Batchelor, H. W., 142, 188 Anglemier, A. F., 68, 72 Baudet, J., 207, 220 Annear, D. I., 25, 27, 33, 34, 35, 36, 38, Bauer, J. H., 166, 167, 189 Bayer, E., 363, 375, 391 41, 42, 63, 72 Appleman, M. D., 54, 38, 70, 72 Beattie, J., 114, 125 395

396

AUTHOR INDEX

Becher, A., 80, 107 Beckett, L. G., 5, 15, 17, 72 Beger, H., 91, 105 Behrman, E. J., 216,219 Bekker, Z. E., 328,339 Bell, D. J., 365, 391 Benedict, R. G., 26, 27, 28, 30, 35, 72, 273, 274, 278 Bennett, L. L., Jr., 242, 248, 250, 254 Bentley, R., 297, 302, 305, 320, 331, 339, 340 Bentzen, O., 145, 188 Bergmann, D. E., 31, 72 Berliner, D. L., 117, 118, 125, 129 Bemasconi, R., 281, 290 Bernheim, F., 194, 219 Bemlohr, R. W., 338, 340 Bettendorf, G., 319, 342 Beutelspacher, H., 274, 277 Biaggi, C., 200, 219 Binns, V. M., 229, 230, 252, 254 Birch, A. J., 297, 305, 306, 307, 315, 319, 320, 324, 329, 340, 342 Birkhaug, K., 36, 59, 72 Birkinshaw, J. H., 295, 324, 340 Bishop, C. B., 121, 128 Bishop, M. W. H., 39, 74 Bjorklund, B., 119, 125 Black, J,, 182, 188 Blance, G. E., 319, 340 Blankenhorn, D. €I., 374, 377, 391 Block, R. J., 355, 391 Bloom, F., 117, 128 Bloom, H. H., 146,188 Bloom, W.,117, 125 Blumenthal, H., 7, 73 Blumson, N. L., 313, 339 B@e,J., 36, 59, 72 Boerkc, E. E., 149, 189 Bogart, W. M., 4, 35, 45, 46, 68, 74 Bogen E., Y43, 188 Bohlen, N. G., 148, 189 Bohonos, N., 230, 233, 254, 255 Bolande, R. P., 119, 125 Bollet, A. J., 279, 290 Bollinelli, R., 140, 191 Bond, V. P., 122,126 Bonting, S. L., 121, 127 Booth, J. H., 229, 255

Borman, A,, 279, 291 Bourdillon, R. B., 153, 176, 188 Boryczka, A., 234, 254 Boyd, T. C., 233,254 Boyland, E., 200, 219 Brack, A,, 288, 289, 292, 294, 319, 334, 340 Brackman, P. S., 145, 190 Bradfield, A. E., 352, 370, 392 Bradley, C. F., 116, 128 Bradner, W. T., 231, 254 Brady, B. L., 25, 72 Brandly, C. A., 145, 191 Brant, H. G., 144,190 Brbchot, P., 38, 73 Breed, R. S., 24, 72, 92, 105, 258, 277 Brenner, M. P., 285, 291 Bretz, H. W., 52, 72 Brian, P. W., 337, 340 Brimley, R. C., 355,392 Bringmann, G., 92, 105 Britt, J. J,, 297, 340 Broadwater, G. C., 144, 190 Brockman, R. W., 245, 248, 249, 250, 254 Brockmann, H., 301, 340 Broda, E., 121, 125 Brooks, S. C., 234, 255 Brown, A. H., 4, 9, 49, 73, 370, 392 Brown, B. T., 282, 290 Brown K., 80, 104,105 Brown, M. E., 338,341 Bryant, J. C., 119, 120, 122, 126 Bryson, V., 85, 105 Buch, M. L., 360, 361, 392 Buchanan, J. M., 248,255 Buchanan, L. M., 142, 170, 189, 192 Buchanan, R. E., 258, 277 Buckley, S. M., 229, 232, 233, 245, 255, 256 Bulen, W. A., 352, 392 Bu’Lock, J. D., 297, 302, 305, 312, 314, 319, 321, 322, 330, 334, 338, 339 Bunim, J. J., 279, 290 Burnet, F. M., 119, 125 Burrell, R. C., 352, 392 Burrows, M. T., 110, 113, 117, 125 Burt, A. M., 112, 123, 125 Burton, R. B., 280, 290

397

AUTHOR INDEX

Busby, D., 61, 72 Bush, I. E., 280, 290 Butcher, R., 82, 105 Butel, A., 140, 191 Butte, J. C., 297, 340 Buttolph, L. J., 177, 178, 188

C Callison, E. G., Jr., 153, 191 Calman, R. M., 174,190 Campbell, D. H., 19, 61, 63, 72 Canonica, L., 203, 204, 219 Carey, G., 87, 98, 106 Carpenter, E., 114, 125 Carr, E. A,, Jr., 143,188 Carrel. A.. 110. 113. 117. 120, 125 Carson, S..F., 349, 362, 393 Cartwright, R. A., 360, 392 Castor, C. W., 117, 125 Cataldi, M., 78, 86, 87, 90, 91, 98, 99, 105 Cavalieri, L. F., 229, 256 Cawley, W., &O, 95, 91,105 Chambers, K., 305, 340 Chambers, R. D., 114,125 Champy, C., 116,125 Charipper, H. A., 114, 127 Chatigny, M. A., 149, 150, 159, 164, 168, 170, 178, 188, 189, 190, 192 Cheney, L. C., 233, 255 Cherry, W. R., 112, 122, 125 Chevrefils, W., 143, 188 Chiga, M., 7, 73 Cho, C., 36, 37, 72, 75 Christian, T., 26, 45, 72 Chu, F., 376, 384, 385, 393 Cieciura, S. J., 129, 128 Citek, F. J., 153, 188 Clarke, D. A., 231, 232, 233, 245, 247, 254, 255, 256 Clark, M. E., 146, 189 Clift, F. P., 372, 392 Cochrane, V. W., 343, 392 Codner, R. C., 305, 306, 341 Coffey, G . L., 233,255 Coggins, R. A., 351, 366, 367, 393 Cohen, E. P., 112, 123, 125 Cohen, S. S., 244, 254

Cohen, T., 315, 339 Cohn, W. E., 344, 370,392 Cole, Q. P., 229, 255 Coleman, M. B., 146, 189 Colla, C., 200, 205, 219 Colledge, E. H., 159, 190 Collins, F., 87, 105 Comnschi, G. F., 266, 269, 271, 277 Cook, B., 119,126 Cook, R. P., 372,392 Cooke, G. M., 143, 191 Cooper, E. J., 67, 74 Cooper, P. D., 112, 123, 125 Copeland, V., 248,250,254 Corbaz, R., 268, 277 Corcoran, J. W., 297, 340 Corey, E. J., 290,291 Cormack, J., 85, 95, 96, 105 Corman, J., 26, 27, 2&,30, 35, 72 Costa, F., 234, 254 Couling, C. W., 148, 188 Courtade, R., 57, 69, 76 Cowan, S. T., 61, 62, 63, 64, 65, 72 Crawford D. L., 68, 72 Crombie, W. M., 391, 392 Cron, M. J., 233, 255 Cruikshank, D. H., 372, 392 Cullen, W. P., 234, 255 Cummings, M. M., 58, 73 Cutting, W. C., 115, I25

D Dagley, S., 206, 211, 219, 319, 340 Dahlgren, C. M., 142, 192 Dale, L. O., 34, 57, 58, 75 Daniels, J. B., 120, 125 Darlow, B. A., 162, 171, 188 Dauben, W. G., 314, 340 Davidson, 3. N., 121, 125 Davidson, W. L., 133, 188 Davies, C. N., 142,, 188 Davies, D. D., 361, 392 Davies, J. P., 114, 128 Davis, E. V., 112, 120, 122, 127, 129 Davis, J. M., 249, 254 Dawson, F. W., 175,188 Decker, H. M., 142, 153, 155, 156, 166, 177,188,189, 192 DeHaan, P., 85, 105

AUTHOR INDEX

Deinema, M. H., 330,340 Dekking, F., 141,188 Demain, A. L.,316,340 De Martini, F., 82,104: 105 Dementjeva, G.,87,98,106 Demoll, R.,79, 105 De Moss, R., 302, 340 Demuth, F., 113,125 Denison, E. W., 349,3@2y393 Dern, R. J., 159,188 Desty, D. H.,3.15, 392 DeVoe, S. E., 233,254 Dewey, V. C.,229, 230,2'52, 254, 255 Diamond, L. K.,229, 254 Dickie, H.A.,134,188 Di Marco, A.,329,340 Dimmick, R. L.,018,72 DiPaolo, J. A.,231, 254 Dobberstein, H.,40, 41, 42, 611, 75 Doerschuk, A. P., 332, 341 Dolan, M. M., 138,188 Dondero, N.,88,98,105 Donovan, F. W., 305,340 Dorfman, R. I., 118, 125, 290, 291 Doskocil, J., 329, 341 Doskocilova, D., 329, 341 Dougherty, T. F.,117, 118, 125, 129 Drescher, R., 82, 105 Dreywood, R., 370, 392 Droblyanets, E.,81, 107 Dryden, E. C.,381, 392 Dube, E. W.,145, 189,187 Dube, F. J. C., 145, 169,187 Dubos, R. J., 55, 72 Dunklin, E. W.,174, 176,188, 190 Dunstan, G.,104, 106 Durrum, E. L., 355,391 Dushmnn, S., 11, 12,13,72 Dybkaer, R., 159, 189

Edwards, S. W., 319, 341 Ehrlich, J., 232,233,234,254,255 Eilers, N.J., 234,256 Elbein, A. D., 302, 341 Elberg, S. S., 32, 41, 43, 45,48, 48, 73 El Dein, S. N., 374,393 Elder, R., 97,105 Eldridge, E., 94, 105 Eliasek, J., 194, 220 Elion, G. B., 230, 245, 254, 255, 256 Elser, W.J,, 9, 17,33,72 Elvidge, J. A., 194,190,219 Emmerling, O.,193, 219 Emmons, C.W., lf3-4, 191 Engel, L. L., 118, 125 Engel, P., 115, 126 Engley, F. B., 61, 72 English, J. P., 229,255 English, R. J., 297,306,340 Enright, J. J., 38, 41, 42, 45, 50, 52, 56, 88, 75 Eppstein, S. H., 279, 284, 285, 291 Erichsen, S., 118,129 Ettlinger, L.,268, 277 Evans, N. R., 302, 340 Evans, R. A,, 198, 200, 208, 219, 220 Evans, V. J., 111, 112, 115, llS, 120,

122,126,128,129

Evans, W. C., 1D4, 195, 198, 197, 198,

199, 200, 201, ao8, 208, ao9, 210, 211, 213, 214, 218, 219, 220, 221, 319, 340 Evjen, A,, 36, 59, 72

F

Fadeeva, N. P., 328,339 Fagraeus, A,, 117, 126 Fahlman, B., 145, 169,187 Falco, E. A,, 200, 255 Farber, S., 229, 254 E Fardig, 0.B.,233,255 Eagle, H.,112, 113, 119, 120, 121, 123, Fanner, P., 37, 47, 76 125,126,928,254 Farmer, V. C.,lQ4, 220 Earle, W. R., 111, 112, $15, 119, 124 Farquhar, J. W.,378, 379, 3&3, 385, 122,126,128,120 388, 388, 392 Eaton, M.D., 120,125 Farrer, S. M., 145,190 Ebeling, A. H., 110, 113, 117, 128 Farris, E.J., lm,189 Ebner, K. E., 118,126 Faunce, K., Jr., 32, 41, 43, 45, 46, 48, Eddie, B., 1133, 190 73

399

AUTHOR INDEX

Fawcett, D. W., 124, 126 Federoff, S., 119, 126 Feindendegen, L. E., 122,126 Fekety, F. R., 145,190 Feldmann, S., 40, 41, 452, 61, 75 Fell, H. B., 109, 129 Feretti, L. D., 320,341 Femley, H. N., 194, 200, 201, 200, 214, 220 Ferris, D. H., 1 3 , 1 8 9 Fetzer, W. R., 14, 72 Fewster, M. E., 211, 219 Fiecchi, A., 203, 204, 205, 208, 219, 221 Field, J. B., 2 3 , 254 Fiese, M. J., 134, 189 Fieser, L. F., 280, 291 Fieser, M., 28'0, 291 Finlay, A. C., 269, 277 Finn, R., 85,105 Fiotamonti, M. C., 119, 126 Fischer, A., 117, 191, 124, 126 Fischer, G. A., 249, 255 Fish, C. H., 182, 189 Fisher, H. W., 120, 126 Fisher, M. W., 233, M,254, 255 Fisher, P. J., 24, 25, 26, 51, 61, 63, 75 Fishman, M., 117, 126 Fitton, P., 324, 340 Fitzpatrick, J. P., 148, 189 Flaig, W., 268, 27'4, 277 Fleischman, R., 121, 126 Fletcher, L, G., 120, 122,127 Flickinger, M. H., 24, 215, 29, 38, 76 Fling, M., 333, 341 Flood, A. E., 370, 392 Flosdorf, E. W., 1, 2, 4, 3, 8, 9, 10, 11, 12, 14, 18, 19, 20, 40, 5+7, 64, 72, 73 Flynn, L. M., 235, 254 Fodor, P. J., 317, 341 Folch, J., 374,376, 392 Foley, G. E., 113, 125, 228, 229, 230, 252, 254 Ford, J. H., 234, 254 Foster, E. M., 30,75 Foster, J. W., 240, 247, 254, 255, 330, 337,341,344,392 Foster, R., 78, 80, 84, 93,106 Fowler, G. J., 194,220 Francis, M. D., 121,126

Francis, T., Jr., Ell, 126 Frank, M. A., Sr., 157, 158, 191 Fried, J., 118,128,279,291 Frisby, B. R., 162, 189 Frobisher, M., Jr., 28, 54, 58, 73 Fry, R. M., 2, 13, 24, 25, 27, 28, 29, 32, 34, 35, 36, 38, 42, 43, 44, 48, 64, 60, 67, 69, 73 Fujimoto, Y., 233, 256 Fulton, W. C., 121, 127

G Gafford, R. D., 194, 197,220 Gaillard, P. J., 116, 126 Gaines, M. L., 117, 129 Gaines, M. L., Jr., 117, 126 Gardner, G. D., Jr., 167, lm, 190 Garibaldi, J., 100, 105 Gamer, H. R., 302, 341 Gatenbeck, S., 305, 381, 323, 324, 341 Gates, D. W., 34,57, 58, 76 G a u h , A,, 80, 105 Gausch, J. P., 170, 189 Gause, G. F., 231, 254 Gautheret, R. J., 109, 126 Gee, M., 368, 392 Geile, F. A., 153, 155, 156, 166, 188 Ceiling, E. M. K., 110, 115, 127 Gellhorn, A., 228, 230, 253, 254 Gellman, I., 80, 106 Gerarde, H. W., 122, 126 Gerhardt, P., 124, 126 Geser, A,, 60, 73 Gey, G. O., 111, 1112, t14, 115, 122, 126, 127, 128 Gey, M. K.,114, 115, 122, 126, 127, 128 Giardinello, F. E., 120, 122, 127 Gibson, J., 157, 189 Gilardi, E., 274, 277 Gilman, A., 225, 254 Gilman, E. N., 52, 76 Ginsberg, H. S., 119, 120, 126 Giolitti, G., 273, 274, 277 Giufse, N., 118, 128 Glendenning, D. M., 178, 191 Glick, C.A., 153,188 Goepp, R. M., 365, 393 Gogolak, F. M., 164,189 Gold, E., 120, 126

400

AUTHOR INDEX

Gold, S. S., 148, 189 Goldberg, H. S., 262, 277 Goldberg, L. J., 149, 189 Goldin, A,, 245, 254 Gonzales, F., 114, 126 Goodban, A. E., 355, 393 Goodner, K., 28, 29, 30, 37, 59, 74 Goodwin, T. W., 330, 341 Gordon, A. S., 114, 127 Cordon, J. J., 305, 306, 341 Gortner, W. A., 56, 76 Gots, J. S., 251, 255 Could, B. S., 309, 341 Graff, S., 112, 123, 126 Graham, R. P., 4, 9, 42, 73, 74 Gray, P. H. H., 194, 220 Gray, P. P., 69, 72 Greaves, R. I. N., 4, 8, 10, 11, 12, 13, 17, 24, 25, 27, 28, 29, 32, 34, 35, 36, 38, 42, 43, 44, 48, 63, 66, 67, 69, 73 Greenburg, J,, 247, 255 Gregoriou, G. A., 290, 291 Gregory, H., 297, 302, 312, 330, 334, 340 Greiff, D., 7, 40, 73 Grein, A., 263, 272, 274, 277, 278 Gremillion, G. G., 148, 149, 164, 167,189 Griffin, A,, 101, 1 a5 Gross, N. H., 61, fX3, 64, 75, 139, 140, 142, 143, 153, 155, 156, 166, 188, 191 Gross, S. R., 194, 197, 220 Grosser, B. I., 117, 129 Grossfeld, H., 117, 126 Grossman, M., 146, 189 Gruber, G. B., 132, 189 Gubler, A., 281, 282, 286, 287, 288, 291, 292 Guibert, L., 38, 73 Guirard, B. M., 229, 230, 252, 254 Gundersen, K., 216, 220 Gunsalus, C. F., 211, 220 Gunsalus, I. C., 211, 220 Gunter, S. E., 211, 220 Gustafsson, B. E., 167, 189 Gustafsson, C., 357, 393 Gut, M., 290, 291 Guyton, H. G., 170, 189

H Haag, F., 78, 90, 105 Habel, K., 120, 125 Haber, L., 159, 189 Habermann, R. T., 34, 57, 58, 75 Hagan, W. A., 145, 189 Hageman, E., 116, 126 Haines, R. B., 67, 73 Hajdu, S., 280, 284, 288, 291 Hakim, S., 28, 54, 73 Hall, H. H., 26, 27, 28, 30, 35, 72 Hall, H. M., 9, 22, 76 Hall, L. B., 142, 192 Halleck, F. E., 31, 72 Halmekoski, J., 391, 392 Halvorson, H., 99, 100, 1&, 107 Hambleton, A., 141, 189 Hammer, B. W., 2, 6, 17, 73 Handschumacher, R. E., 249, 254, 255 Hanel, E., Jr., 140, 159, 163, 170, 174, 175, 177, 178, 180, 189, 192 Hanes, C. S., 355, 392 Hanson, F. R., 285, 291 Hanson, R. P., 145, 191 Happold, F. C., 194, 195, lW, 211, 212, 219, 220 Harary, I., 217, 220 Hargreaves, C. A., 357, 392 Harris, A., 297, 340 Harris, M. M., 142, 192 Harris, R. J. C., 2, 5, 73 Harrison, J. S., 357, 393 Harrison, M., 78, 83, M, 105 Harrison, R. G., 109, 127 Harstad, J. B., 61, 63, 64,75, 142, 143, 153, 155, 156, 166, 177, 188, 189, 191 Hart, D., 177, 178, 189 Hartman, F. W., 175, 189, 190 Hartsell, S. E., 52, 72 Hashimoto, Z., 195, 220 Hata, T., 233, 255, 256 Haws, E. J,, 324,341 Hayaishi, O., 195, 196, 197, 212, 219, 220, 221 Hayano, M., 290,291 Haymaker, W., 110, 115, 12.5, 127 Haynes, H., 177, 178,188

401

AUTHOR INDEX

Haynes, W. C., 24, 215, 38, 73 Hays, E. E., 19, 73 Hearn, H. T., 175, 188 Heaton, M. W., 117, 129 Heckly, R. J., 6, 15, 16, 17, 18, 26, 29, 30, 31, 32, 33, 39, 40, 41, 43, 44: 45, 46, 48, 49, 53, 55, 57, 58, 59, 67, 73 Hedberg, H., 117, 127 HedBn, C. G . , 157, 170, 189 Heger, E. N., 4, 35, 45, 48, 68, 74 Heinmetz, F., 55, 73 Heller, G., 30, 32, 35, 44, 49, 74 Heliman, L. M., 114, 126 Hemmons, L. M., 24, 25, 26, 31, 46, 51, 54, 61, 64, 75 Henderson, D. W., 144, 164, 170, 189 Henderson, M. E. K., 194, 220 Hennum, L. A., 29, 33, 42, 4.6, 76 Herbst, W. P., 225, 255 Herreid, E., 19, 76 Herrera, T., 67, 74 Hertz, R., 115, 129 Herz, J. E., 279, 291 Hesse, R. H., 118, 128 Hesseltine, C. W., 24, 25, 38, 73, 230, 255 Hesseltine, G. W., 273, 274, 278 Heukelekian, H., 78, 81, 83, &8, 94, 98,

105 Hewitt, R. I., 230, 255 Hey, M., 312, 330,340 Higgins, G. M., 115, 127 Hilditch, T. P., 377, 389, 392 Hill, J. M., 9, 74 Hillegas, A. B., 233, 254, 255 Hilleman, M. R., 119, 127, 157, 189 Hills, C. H., 361, 392 Hilmoe, R. J., 13, 14, 32, 41, 74 Hirsch, H. M., 338, 341 Hirsch, J., 383, 392 Hirsch, U.,332, 341 Hirschberg, E., 228, 230, 253, 254 Hirst, E. L., 370, 392 Hiscox, E. R., 49, 51, 74 Hitchings, G. H., 229, 230, 245, 254, 255, 256 Hobby, G . L., 269,277 Hockenhull, D. J. D., 194, 211, 220, 328,333, 334,341

Hodges, C . V., 225, 255 Hodgman, C. D., B,74 Hohnl, G., 78, 85, 87, 987, 98, 102, 103, 104, 105, 106

Hohnl, W., 94, 101, 105 Hoffman, R. K., 174, 175, 176, 188, 189, 191, 192

Hofmann, K., 318,341 Hogan, A. G., 235, 254 Holbrook, A. A., 175, 189 Holden, H. F., 9, 74 Holker, J. S. E., 324, 341 Holzman, G., 18, 74 Hooper, I. R., 233,255 Hoover, C. R., 116, 126 Hopkins, W. J., 50, 51, 55, 56, 68, 76 Hornibrook, J. W., 18, 25, 29, 32, 33, 35, 38, 74

Homing, M. G., 297, 341 Horowitz, N. H., 333, 341 Horsfall, F. L., 166, 16r7, 189 Horsfall, F. L., Jr., 119, 126 Horton, R., 133, 190 Hoshi, T., 233, 255 Howe, J. R., 3515,392 Huebner, R. I., 133, 189 Huggins, C., 225, 255 Hughes, D. E., 216,220 Hull, L. W., 2, 4, 10, 49, 73 Hull, R. N., 112, 122,125 Hulme, A. C., 349, 350, 351, 352, 354, 355, 361, 371, 392

Hultquist, M. E., 256, 256 Hummeler, K., 133, 188 Humphrey, J. H., 117, 125 Hundemann, A. S., 175, 189 Hursky, V. S., 113, 128 Hurst, N., 146, 189 Hutchings, B. L., 229, 255 Hutchison, D. J,, 235, 251, 255, 256 Hutton, R. S., 13, 14, 20, 23, 31, 32, 41, 74, 75

Hyatt, E. C., 152, 191

I Ingebrigsten, R., 117, 125 Ingols, R., 81, 105 Ingram, D. C., 116,127 Ingram, F. R., 146, 189

402

AUTHOR INDEX

Insull, W., 374, 378, N 9 , 383, 385, 386, 388, 392 Irwin, J. O., 161, 190 Isaacs, A., 119, 128 Isherwood, F. A., 344, 352, 355, 370, 372, 373, 375,392 Ishidate, M., 283, 287, 288, 291 Ishii, H., 283, 284, 287,291 Isono, M., 211, 220 Issaiev, B. M., 274, 278 Iwasaki, T., 38, 72 J Jackson, E. B., 25, 34,74 Jackson, R. W., 28, 27, 28, 30, 35, 72 Jackson, T. W., 118, 128 Jacobs, H. R., 19, 74 Jacobs, S., 95, 106 James, A. T., 345, N4,3715, 376, 378, 379, 382, 383, 384, 385, 38@, 387, 3.88, 389, 392 Janes, C., 20, 76 Janssen, R. J., 175, 188 Januszewicz, I., 56, 69, 74 Jemski, J. V., 144, 190 Jensen, F. C., 116,129 Jensen, H. L., 216, 220 Jensen, K. A., 147, 189 Johansson, K. R., 139, 187 Johnson, A. W., 303,341 Johnson, D. A., 233, 255 Johnson, D. L., 233, 255 Johnson, M., 85, 106 Jones, E. R. H., 297, 314, 3115, 334, 340, 341 Jones, G. E. S., 115, 127 Jones, J. D., 211, 220 Jones, J. K. N., 370, 392 Jones, K. L., 264, 277 Jones, M., 121, 122, 126, 127 Jordan, H. C., 121,127 Jordan, H. S., 152, 191 Jordan, R., 95, 106 Jordan, W. S., Jr., 120, 126

K Kachmar, J., 82, 106

Kadull, P. J., 133, 192

Kahn, R. H., 117, 127 Kaiser, F., 280, 291 Kalle, G. P., 251, 255 Kamada, H., 233,256 Kamiya, T., 2&2,284, 291 Kanamori, K., 233, 255 Kane, J. H., 209,277 Kaneda, T., 297, 340 Kantorowicz, O., 147, 189 Karmen, A., 29'7, 341 Kamemaat, J. N., 285, 291 Kasparova, J., 329, 341 Katagiri, M., 197, 219, 220 Kato, E., 233, 256 Katz, A., 281, 291 Katz, E., 302, 329, 333, 341 Kawasaki, J., 36, 37, 72, 75 Kaye, S., 174, 189 Keane, J. F., Jr., 39, 74 Keeney, E. L., 148, 189 Keil, J., 305, 340 Keller-Schierlein, W., 268, 277 Kelley, G. A., 145, 191 Kelley, G. G., 248, 2,50, 254 Kelly, A., 324, 341 Kelly, A. R., 175, 189 Kelly, S. M., 9, 74, 146, 189 Kemp, C. E., 26, 27, 28, 30, 35, 72 Kenner, G. W., 305,340 Keuning, F.J., 117, 127 Keutmann, E. H., 280, 290 Khym, J. X., 344, 370,392 Kidder, G. W., 229, 230, 252, 254, 255 Kilby, B. A., 197, 220 Kimball, A. C., 57,64,73 King, J. R., 53, 74 Kirsop, B., 24, 25, 38, 56, 74 Klarmann, E. G., 174, 190 Klein, G., 240, 255 Klein, G. C., 58,73 Kleps, R. N., 132, 190 Kligman, A. M., 138, 188 Kling, D. H., 117, 127 Kluyver, A. J,, 194, 211, 220, 311, 341 Kniazuk, M., 143, 162, 166, 178, 191 Knox, W. E., 319, 341 Kobylinski, P. G., 138, 188 Koch, F. C., 19, 73 Kochetkova, G. V., 231,254 Kocor, M., 320,324,340

403

AUTHOR INDEX

Koffler, H., 302, 341 Kohberger, D. L., 233, 254, 255 Kohler, H., 118, 128 Koklov, A. C., 282, 278 Koller, L. R., 1'77, 190 Kom, E. D., 248,255 Komberg, A., 248,255 Korzybski, T., 262, 278 Koscheleva, N. A., 328, 339 Kowkabary, G. N., 363,392 Krainsky, A., 269, 278 Krassilnikov, N. A., 260, 268, 278, 309, 341 Kriss, A. E., 274, 278 Krueger, A. P., 1&, 147, 164, 166, 190 Krueger, B. J., 146, 153, 188 Krupa, G., 230, 255 Krzeminski, L. F., 323, 341 Kucera, C. J., 120, 122, 127 Kuchayeva, A. G., 268,278 Kuchler, R. J., 112, 120, 121, 122, 123, 124, 127 Kudo, S., 233, 256 Kiister, E., 258, 272, 273, 274, 277, 278 Kugelman, M., 234, 255 Kugler, H., 9, 22, 76 Kuritzkes, A., 285, 291 Kurylowicz, W., 36, 59, 74, 262, 278 Kutzner, H. J., 264, 268, 277, 278 Kuwabara, H., 117, 127 Kuzniecow, A., 36, 59, 74

1 Lackey, J., 78, 80, 81, 8T, 94, 98, 1013, 104, 106, 107 Ladimer, I., 182, 188 Lagsdin, J. B., 19, 22, 76 Lamanna, C., 158, 159, 17Q, 190 Lampen, J. O., 229,255 Lamy, F., 120, 127 Landa, S., 194, 220 Landheer, C. A., 330,340 Landowne, R. A., 384, 385, 386; m, 390, 393 Lanman, G., 118,125 Lannon, T. J., 199,209,220 Larson, B. L., 116, 126 Law, L. W., 245, 249, 253, 254, 255

Leach, B. E., 234,254 Leach, R. H., 13, 50, 51, 52, 53, 69, 74 Leavenworth, C. S., 344,393 Le Baron, F. M., 374, 378, 392 Lechevalier, H. A., 268, 278, 309, 342 Lederer, E., 345, 363, 392 Lederer, M., 3415, 383, 392 Lees, M., 374, 376, 392 Legallis, F. Y.,120, 128 Lehman, J. J., 55, 73 Leif, W. R., 144, 184, 166, 190 Leifson, E., 17, 74 Leigh, H. M., 284, 291 Leigh Osborn, H. M., 285,291 Leighton, J., 122, 127 Lemcke, R. M., 26,27, 74 Lense, F. T., 170, 189 Lentz, C.P., 3314, 341 LePage, G. A., 245,256 Leschinskaya, E. N., 37, 60, 74 Leslie, I., 121, 125, 127, 128 Lessel, E., Jr., 270, 278 Lester, W., Jr., 174, 190 Levenstein, I., 114, 127 Levine, H. B., 134, 190 Levine, M. G., 117, 127 Levy, A. A., 200,219 Levy, M., 121, 126 Lewis, M. R., 110, 115, 117, 120, 125, 127 Lewis, W. H., 120,127 Lidwell, 0. M., 147, 153, 176, 188, 192 Lieberman, I., 120, 121, 127, 248, 251, 255 Liebman* 79, w, Io6 Lincoln, J., 78, 80, 84,93, 106 Lincoln, N. S., 133,190 Lind, A., 157, 190 Lind, P. E., 119, 125 Lindbergh, C. A., 112, 123, 127 Linstead, R. P., 194, 196, 219 Lipsky, S. R., 384, 385, 386, 3P3&, 390, 393 Littman, M., 81, 82, 88,106 Liu, T. Y., 316,341 Lloyd, R. S., 174, 190 Lockett, W. T., 194,220 Lockwood, L. B., 330, 331, 342 Loewus, F. A., 9, 74 LoGrippo, G. A., 175,189,190

w>

404

AUTHOR INDEX

Long, E. R., 13,8,190 Long, J. H., 116,127 Lovelock, J. E., 39, 74, 176, 188 Lowe, A, E., 146, 189 Lowe, G., 297, 341 Lowery, C. E., 2417, 255 Lowery, J. A., 230, 255 Lu, K. H., 122, 127 Luckiesh, M., 177, 190 Ludwig, E. H., 120, 129 Lueck, B., 95, 106 Lugg, J. W. H., 355,393 Lurie, M. B., 144, 190 Lux, L., 115, 127 Luyet, B. J., 31, 39, 74 Lybing, S., 325, 341 Lynch, J. M., 182,188 Lyttle, D. A,, 284, 291

M McAlpine, R. J., 234, 255 McCalpin, H. A., 112, 123, 128 McCarthy, R. E., 229, 230, 252, 254 McCarty, K. S., 112, 123, 126 McClean, I. W., Jr., 112, 123, 128 McCormick, J. R. D., 332, 341 McCrone, M. A., 121, 127 McCulloch, E. C., 173, 190 MacDonald, J. C., 301,341 McFall, E., 122, 127 McGraw, J. J., 2, 19, 76 Machamer, H. E., 233,254 Mack, W. N., 148,188 MacKay, I., 176, 790 McKenna, J. M., 117,128 McKeon, J. E., 332, 333, 342 Macleod, H. L., 333, 341 McLimans, W. F., 112, 120, 122, 127, 129 Macpherson, I. A., 114, 129 McPherson, W., 80, 106 McQuilkin, W. T., 119, 126 McReady, R. M., 368, 392 Magasanik, B., 122, 127, 347, 393 Mahlandt, B. G., 39, 67, 74 Maister, H. G., 4, 20, 23, 35, 45, 46, 68, 74, 75 Mallmann, W. L., 146, 188 Mann, F. C., 115, 127

Mann, K. M., 285,291 Mann, R. L., 302, 341 Manson, L. A., 121, 127 Mantel, N., 245, 254 Marcus, P. I., 122, 128 Margreiter, H., 302, 339 Marlowe, M. L., 120, 121, 127 Marshall, B., 338, 341 Martin, A. J. P., 375, 378, 379, 392 Martin, R., 82, 106 Martin, J. H., 233, 254 Martin, S. M., 360, 391, 393 Martinovitch, P. N., 116, 127 Marumo, H., 233, 256 Massy-Westropp, R. A., 297, 306, 340 Masuo, E., 283, 284, 287, 291 Matelsky, I., 177, 188 Matsumae, A., 233, 255 Matt, C., 57, 76 Maximov, A. A,, 117, 127 Maxwell, R. E., 242,255 May, C. W., 147, 148, 191 Mayama, M., 274, 278 Mayyasi, S. A., 120, 127 Mazur, P., 39, 67, 74 Meath, J. A., 374, 376, 392 Mechalas, B. J., 31, 72 Meeks, R. C., 285, 291 Meigh, D. F., 347, 378, 379, 384, 393 Meister, P. D., 279, 284, 285 291 Melnick, J. L., 120, 127 Mendelsohn, W., 116, 127 Mercer, R. D., 229, 254 Merchant, D. J., 112, 117, 120, 121, 122, 123, 124, 127 Merger, C., 133, 162, 190 Meryman, H. T., 2, 5, 9, 39, 40, 74 Meyer, K. F., 133, 190 Meyer, R. K., 116, 128 Meyers, C. E., 143, 191 Meyers, R. P., 50, 75 Michel, R., 114, 128 Middlebrook, G., 55, 72, 164, 190 Mikol, E. X., 133, 190 Miles, A. A., 161, 190 Miller, A., 317, 341 Miller, C. P., 48, 74 Miller, G. A., 305, 306, 341 Miller, L. L., 121, 127

405

AUTHOR INDEX

Miller, 0. T., 159, 163, 170, 174, 175, 178, 180, 192 Miller, R., Jr., 28, 29, 30, 37, 59, 74 Miller, R. P., 34, 74 Miller, W. S., 140, 142, 157, 158, 166, 191 Mills, C. C., 143, 191 Misra, S. S., 161, 190 Mitchell, I., 14, 75 Mitchell, J. H., Jr., 38, 41, 42, 45, 80, 52, 56, 68, 75 Mitchell, R. N., 152, 191 Miyakawa, M., 167, 190 Miyao, K., 338, 342 Montgomery, R., 380, 392 Montgomery, T. L., 115, 129 Moore, A. E., 248, 255 Moorman, H. E., 153,188 Morch, E., 145, 188 Morgan, H. R., 119, 125 Morgan, J. F., 119, 121, 127, 128 Moritz, U., 117, 127 Morris, D., 316, 317, 339 Morris, E. J., 139, 176, 181, 190 Morris, S. I., 132, 190 Morton, H. J., 121, 128 Mosbach, E. H., 349, 362,393 Mosbach, K., 305, 306, 320, 321, 341 Moser, H., 85, 106 Moses, W., 69, 72 Moss, M. L., 139, 142, 143, 188, 191 Moss, P., 214, 219 Motsavage, M. A., 138, 188 Mott, L. O., 34, 57, 58, 76 Mouromseff, G., 177, 190 Mowat, J. H., 229, 233, 254, 255 Mudd, S., 1, 2, 4, 8, 9, 10, 11, 18, 19, 20, 49, 73 Muggleton, P. W., 26, 37, 40, 41, 43, 47, 75, 76 Mullican, C. L., 167, 177, 190 Munk, V., 360, 393 Munyon, W. H., 112, 121, 122, 123, 124, 127 Murphy, J. E., 282, 291 Murphy, J. F., 206, 220 Murphy, J. T., 175, 188 Murphy, M. E., 134, 188 Murray, A. W., 280, 285, 287, 292

Murray, E. G. D., 24, 72, 92, 105 Murray, H. C., 279, 284, 285, 291 Murray, J., 174, 190 Murray, M. R., 116, 128 Musgrave, 0. C., 297, 340

N Nabors, C. J., Jr., 117, 129 Nadel, E. M., 247, 255 Nagy, R., 177, 190 Nakamura, M., 104, 106 Nawa, H., 282, 284, 291 Naylor, H. B., 14, 24, 26, 27, 29, 30, 35, 45, 75 Nei, T., 39, 56, 60, 75 Neidle, A,, 317, 341 Neilands, J., 100, 105, 106 Newman, J. P., 38, 54, 57, 76 Newman, W. C., 37, 75 Niavis, C. A., 370, 373, 392 Nickel, V. S., 242, 255 Nickell, L. G., 112, 123, 128 Nielson, E. D., 285, 291 Niskasaari, E., 357, 393 Nitta, K., 233, 256 Nomi, R., 274, 278 North, E. A., 37, 75 Novak, F. E., 172, 190 Novelli, G. D., 338, 340 Novick, A,, 85, 106, 123, 128 Nozaki, Y., 283, 284, 287, 291 Nyka, W., 143, 191

0 Obayashi, Y., 36, 37, 72, 75 O’Connell, P. W., 285, 291 O’Dell, B. L., 235, 254 Okada, M., 283,287, 286, 291 Okami, Y., 231, 233, 255, 256 Okita, G. T., 282, 290 Okuda, T., 233,256 Okumura, T., 283, 284, 287, 291 Oleson, J. J,, 229, 255 Oliver, A. J., 157, 192 Ollis, W. D., 305, 3Og, 341 Olsen, E., 81, 106 Oppenheimer, J, H., 114,128

406

AUTHOR INDEX

Peterson, J. E., 152, 190 Peterson, W. H., 50, 52, 67, 74, 75 Pfeifer, V. F., 35, 45, 46, @8, 74 Pfeiffer, D. C., 9, 74 Phares, E. F., 349, 362, 393 Philips, F. S., 225,2447, 254 Phillips, C. R., 174, 189, 191 Phillips, G. B., 140, 144, 159, 153, 167, 170, 172, 174, 175, 17'7, 178, 180, 190, 191, 192 Phillips, R., 88, 98, 98, 105, 106 Pidcoe, V., 143, 190 Pienta, P., 329, 341 Piez, K. A,, 121, 126 P Piggott, W. R., lBa, 191 Pigman, W. W., 368, 393 Paff, G. H., 117, 128 Pigoury, L., 57, 69, 76 Pagano, J. S., 145, 190 Pike, R. M., 133, 134, 135, 137, 138, Pai, S. E., 28, 54, 73 148, 192 Painter, R. B., 122, 126 Pinkerton, H., 7, 73 Palermiti, F. M., 233, 255 Piper, F. J., 153, 188 Palmer, J. K., 349, 393 Pittillo, R. F., 234, 240, 254, 255 P'an, S. Y., 289, 277 Plotkin, S. A,, 145, 190 Pappis, G. D., 117, 128 Poole, J. P., 128 Parke, D. V., 201,207,220 Popova, L., 329, 341 Parker, R. C., 124, 126 Popp, L., 79, 106 Parker, R. F., 117, 129 Porter, J. N., 230, 255 Parkes, A. S., 2, 75 Porter, K. R., 117, 128 Parks, R. E., Jr., 230, 255 Porter, W. L., 360, 392 Parr, W. H., 198, 200, 20s; 219, 220 Potter, E. F., 15, 75 Parsons, E. I., 28, 54, 73 Powell, A. D. G., 324,341 Partridge, S. M., 349, 393 Pratt, H. K., 18, 75 Pasieka, A. E., 121, 128 Pressman, D., 19, 61, 63, 72 PgSkovfi, J., 360, 392 Price, R. T., 116,129 Pasquier, J. F., 36, 59, 74 Pride, E., 324, 340 Pastemak, C. A,, 249, 255 Pridham, J. B., 391,393 Patel, C. B., 377, 389,392 Pridham, T. G., 273, 274, 278 Patel, M. D., 208, 219 Pringsheim, E., 78, 87, 90, 91, 92, 98, Paul, J., 119, 121, 122, 128 Paulin, N., 271, 273, 278 99, 101, 102, 103, 106 Proctor, D. P., 357, 393 Pavlovic, H., 114, 128 Proctor, M. H., 213, 220 Payne, M. A., 110,128 Prokofieva-Belgorskaya, A., 329, 341 Pechet, M. M., 118, 128, 279, 290 Proom, H., 13, 24, 25, 26, 31, 46, 51, Pennella, P., 329, 340 54, 61, 84,75 Penney, M., 352, 392 Pucher, G. W., 344,393 Peppler, H. J., 67, 74 Puck, T. T., 120, 122, 126, 128, 176, Perkins, J. J., 163, 173, 190 Perlman, D., 118, 128, 279, 291 188 Perry, M. E., 120, 125 Pullman, A., 207,220 Peters, P. A,, 121, 127 Pullman, B., 207, 220 Peterson, D. H., 279, 284, 285: 290, 291 Pullman, T. N., 15'9,188

Orkin, B. A., 196, 219 Osato, T., 233, 256 Osbom, H. M., 285,291 Ostroikhov, A. A., 32&, 339 Otten, L., 28, 31, 75 Ove, P., 120, 121, 127, 251, 255 Overell, B. T., 395, 393 Overhulse, P. R., 175, 190 Owen, S., 85, 106 Owens, 0. von H., 122,128 Owens, H. S., 355, 393 Oyama, V. I., 121,126

AUTHOR IN'DEX

Pulvertaft, R. J, V., 114, 128 Pumper, R. W., 117, 128

Q Quackenbush, F. W., 323,341 Quiney, A., 102, 192

R Rabson, A. S., 120,128 Rahn, O., 68,75 Raistrick, H., 295, 309, 340, 341 Rake, G. W., 120, 122,127 Ramage, W. D., 4,9,42,73 Ramakrishnan, C. V., 331,341 Ranson, S. L., 345, 349, 355, 361, 370, 393 Rao, K. V., 234, 255 Rappaport, C., 121, 128 Rappaport, H. P., 128 Raupp, G. P., 384,593 Rawson, R. W., 114,128 Ray, R. L., 172, 191 Raymond, W. F., 153,188 Read, S., 1415, 169, 187 Record, B. R., 31, 75 Reddish, G. F., 17'3, 191 Rees, R. J. W., 147, 148, 188, 189 Regna, P. P., 269, 277 Reichard, P., 249, 255 Reichel, J., 1, 2, 19, 40,75, 76 Reichstein, T., 280, 281, 285, 290, 291 Reid, D. D., 133, 191 Reid, J. J., 214, 220 Reilly, H. C., 232, 233, 2&, 255, 256 Reinecke, L. M., 284,285,291 Reio L., 325, 341 Reitman, M., 81, 63, 64, 75, 140, 141, 142, 143, 144, 155, 156, 157, 158, 166, 167, 177,190,191 Remy, C. N., 248,281,255 Renis, H. E., 302, 341 Renz, J., 288, 289, 291, 292 Repke, K., 282, 291 Rew, R. R., 1 4 , 188 Reyniers, J. A., 164, 167, 191 Reynolds, L. I., 172,192 Rhian, M. A., 20, 23, 39, 67, 74, 75

407

Rhoades, H. E., 24, 29, 32, 33, 34, 54, 61, 75 Rhoads, C. P., 233, 245, 256 Rhodes, M., 24, 25, 26, 51, 61, (33, 75 Ribbons, D. W., 197, 210, 220, 319, 340 Richards, J. H., 314, 320, 340, 341 Rickards, R. W., 297, 340 Rieder, S. V., 302, 341 Rightsel, W. A., 112, 123, 128, 233, 254 Rigler, N. E., 233, 254 Riley, J. P., 377, 389,592 Riley, M. C., 143,191 Riley, R. L., 14'3, 191 Rixton, F. H., 177,190 Roach, K., 13, 75 Roberts, E. A. H., 360,392 Roberts, R. L., 13, 14, 32, 41, 74 Robertson, A., 324, 341 Robinson, H. J., 143, 162, 166, 178, 191 Roblin, R. O., Jr., 229, 255 Robson, E. M., 15, 17, 75 Roche, J., 128, 128 Rockenmacher, M., 6, 15, 26, 29, 30, 31, 33, 39, 40, 44, 48, 49, 55, 57, 58, 67, 73 Roe, E. T., 330, 331, 342 Roe, F. J. C., 176,191 Roegner, F. R., 233,254 Rogers, D. E., 143, 191 Rogers, H., 81, 76 Rogers, L. A., 12, 17, 29, 45, 46, 56, 75 Rogoff, M. H., 199, 203, 204, 205, 208, 207, 210, 214, 216, 220 Romano, A. A., 234,255 Roof, B. S., 199, 209, 220 Rose, G. G., 119, 128 Rosebury, T., 164, 166, 191 Rosen, A,, 378, 379, 383, 385, 386, 388, 392 Rosenfield, R., 231, 254 Rosenthal, S. R., 37, 55, 59, 75 Rothberg, S., 219, 220 Routien, J. B., 269, 277 Rowe, T. W. G., 5, 7, 10, 75 Ruchhoft, C., 81, 82, 87, 103, 106 Rukina, E. A., 274, 278 Russell, P. B., 230, 255 Russell, D. W., 338, 341 Ryan, A. J., 319, 324,340 Ryan, K. J., 118, 128

408

AUTHOR INDEX

S

Scott, R. P. W., 390, 393 Scott, W. J., 13, 30, 34, 36, 44, 45, 50, 51, 52 53, 63, 66, 67, 69, 74, 75 Saamio, J., 357, 393 Scotti, T., 266, 269, 271, 273, 274, 277 Sabachewsky, L., 248,255 Seamen, A. R., 114, 128 Sack, J., 7'8, 90, 106 Saito, A., 290, 291 Sears, 0. H., 54,58, 70, 72 Sebek, 0. K., 290, 291 Salser, J. A., 251, 256 Seeger, D. R., 229, 256 Salton, M. R. J., 338, 341, 348, 393 Salvador, R., 140, 191 Seeger, G. E., 114, 126 Salzman, N. P., 121, 122, 128 Seeley, D. B., 269, 277 Samant, H. S., 324, 340 Segeler, J. C., 164, 192 Segler-Holzweissig, G., 274, 277 Sampaio, A. A. deC., 119, 128 Seifert, H. E., 153, 191 Sands, L., 365, 391 Sanford,.K. K., 111, 112, 119, 126, 128 Semb, J,, 229, 255 Shackell, L. F., 2, 17, 75 Sano, M . E., 115,129 Shaffer, H. C., 145,191 Sano, R., 233, 255 Shah, S., 377, 393 Sant, R. K., 50, 52, 75 Shannon, J. E., Jr., 111, 112, 122, 126, Santer, U. V., 332, 333, 342 128 Sartorelli, A. C., 245, 256 Sharp, J. G., 68, 75 Sasakawa, Y.,280, 291 Shave, E. S., 26, 27, 28, 30, 35, 72 Sato, G., 120, 126 Shay, A. J., 233, 254 Satoh, D., 283, 284, 287, 291 Shechmeister, I. L., 177, 178, 191 Savage, G . M . , 234, 256 Shepard, C. C., 147, 148,191 Sward, K., 118, 125 Shima, T., 233, 255 Scarisbrick, R., 375, 377, 393 Schabel, F. M . , Jr., 235, 242, 245, 254, Shimao, T., 37, 75 Shimi, J. R., 374, 393 2S6 Shimizu, G., 233, 256 Schaberg, A., 124, 128 Shirey, W. N., 31, 74 Schnd, D., 48, 74 Shreeve, W. W., 122, 126 Schapiro, E., 116, 129 Shtemov, V. A,, 174, 190 Scheidy, S. F., 26, 32, 76 Shull, G. M., 269, 277 Scher, S., 208, 220 Scherer, W. F., 40, 75, 111, 112, 128 Shulman, L., 117, 129 Sigg, H. P., 281, 290 Scheuring, L., 78, 85, 102, 106 Sikyta, B., 329, 341 Schikora, F., 78, 82, 106 Silverman, M., 302, 334, 341 Schilling, E. L., 1210, 122, 126 Silverman, W. B., 342 Schindler, O., 280, 291 Siminoff, P., 113, 128 Schley, D. G., 174, 191 Simms, E. S., 248, 255 Schmidt, B., 4'0, 41, 42, 61, 75 Simpson, J. R., 218, 219 Schmidt-Kastner, G., 333, 341 Simpson, M. S., 248, 250, 254 Schmitz, H., 233, 255 Sims, P., 196, 219 Schoening, C. N., 34, 57, 58, 75 Sinclair, R., 121, 127, 128 Schofield, P., 297, 306, 315, 324, 340 Schroeder, W. H., 68, 75 Singer, S., 245, 254 Schulte, H. F., 132, 191 Singh, J., 377, 393 Sinha, S. K., 1435, 191 Schultz, H. W . , 6.8,72 Schulze, H . O., 121, 125, 126 Sisler, F. D., 204, 220 Sistrom, W. R., 197, 221 Schuurmans, D. M., 120, 127 Sitek, K., 36, 59, 74 Schwartes, G . , 288, 292

AUTHOR INDEX

Sivak, A,, 329, 341 Sjolander, N. O., 332, 341 Skadhauge, K., 145, 188 Skaliy, P., 142, 192 Skerman, V., 87, 98,106 Skipper, H. E., 242, 245, 248, 250, 254, 256 Skold, O., 249, 255 Skrinde, R., 82, 106 Skuja, H., 78, 106 Skyring, G., 98, 106 Slaytor, M., 306, 340 Sleeper, B. P., 198, 208, 221 Sloan, R. A., 141, 191 Smadel, J. E., 25, 34, 74, 157, 183, 191 Smalley, H. M., 305, 340 Smirnova, A. D., 328,339 Smit, J., 81, 106 Smith, A. U., 2, 75 Smith, B. S. W., 144, 196, 211, 214, 219, 220, 221 Smith, F., 368, 391 Smith, G. W., 15, 76 Smith, G. S., 138, 191 Smith, H., 297, 306, 315, 319, 324, 340 Smith, H. G., 338, 340 Smith, I., 345, 370, 393 Smith, J. N., 200, 221 Smith, J. M., Jr., 229, 256 Smith, M. S., 251, 255 Smith, N. R., 24, 72, 92, 105 Smith, P. A., 14, 24, 26, 27, 29, 30, 35, 45, 7 5 Sneeden, R. P. A., 281, 291 Snell, E. E., 229, 230, 252, 254 Snell, J, F., 307, 329, 342 Sobin, B. A., 269, 277 Sokolski, W. T., 234, 256 Solberg, A. N., 145, 191 Solomons, I. A,, 269, 277 Solotorovsky, M., 143, 162, I€@,17&,191 Somers, A. R., 183, 184: 192 Somers, H. M., 183, 184,192 Sonkin, L. S., 142, 192 Sowden, J. C., 365, 393 Spalla, C., 263, 266, 269, 271, 272, 274, 277 Sparks, M. C., 248, 250, 254 Spaulding, E., 85, 107

409

Speck, M. L., 50, 75 Speck, R. S., 164, 168, 170, 178, 192 Spendlove, G. A., 182, 189 Spiegel, H. E., 280, 285, 287, 292 Spiner, D. R., 175, 178, 192 Splittstoesser, D. F., 30, 75 Stahl, S., 114, 128 Stamp, T. C., 24, 25, 28, 35, 42, 43 51, 57, 58, 61, 76 Stanier, R. Y., 196, 197, 198, 200, 206, 208, 211, 212, 216, 219, 220, 221, 244, 256 Stark, J. B., 355, 393 Stark, W. M., 302, 341 Starkey, R., 80, 99, 105, 106, 107 Steel, R., 331, 341 Steenson, T. I., 214, 215, 221 Steffen, G. I., 9, 17, 33, 7 2 Stein, C. D., 34, 54,57, 58, 61, 76 Stein, L., 139, 142, 188 Stenderuk, A., 177, 192 Stephenson, J. L., 5, 15, 39, 76 Sternberg, S. S., 247, 254 Sterzl, J., 117, 128 Stevens, K. M., 117, 128 Stevens, R. E., 225, 255 Stewart, H. L., Jr., 115, 129 Steyn, H. S., 32, 76 Stickings, C. E., 325, 342 Stillman, E. G., 57, 76 Stillman, J. W., 14, 76 St. John-Brooks, R., 238, 277 Stock, C. C., 227, 229, 232, 233, 245, 247, 254, 255, 256 Stockton, J. J., 26, 38, 45, 54, 57, 72, 76 Stoffel, W., 378, 378, 379, 383, 384, 385, 386, 388, 392, 393 Stokstad, E. L. R., 229, 255 Stormer, K., 194, 221 Stoker, M. G., 146, 192 Stokes, A. M., 133, 190 Stokes, F. J., 11, 73 Stokes, J. L., 54, 55, 58, 67, 76, 83, 86, 87, 89, 94, 97, 98, 102, 103, 107 Stoll, A., 288, 289, 291, 292 Stone, D., 290, 291 Stone, R. W., 199, 201, 206, 220, 221 Storey, P. B., 143, 191 Straka, R. P., 54, 55, 58, 67, 76

410

AUTHOR INDEX

Tchan, Y. T., 208, 221 Temple Robinson, M. J., 305, 340 Tenney, R. I., 31, 72 Tenny, M., 80, 107 Thayer, P. S., 229, 230, 252, 254 Thiessen, C. P., 297, 331, 340 Thom, C., 281, 278 Thoma, R. W., 279,291 Thomas, R. A., 9, 17, 33, 72 Thomas, W. J,, 112, 121, 122, 127, 129 Thompson, E. L., 174, 190 Thompson, K. W., 116,129 Thompson, P. E., 234,255 Thomson, J. R., 245, 248, 250, 254, 256 Thomson, P. L., 307, 329, 342 Thornton, H. G., 27, 76, 194, 220 Thow, D. C. W., 55, 76 Tihnuss, D. H. J., 114,129 Titus, E., 280, 285, 287, 292 Tkachenko, N., 81,107 Todd, E. W., 119,125 Tomioka, K., 233, 256 Tomizawa, S., 251, 256 Tomlinson, A. J. H., 140, 141, 192 Toolan, H. W., 248,255,256 Toombs, G., 80, 107 Topping, N. H., 25, 31, 34, 76, 147, 148, 191 Townsend, R. J., 325, 342 Trantham, H. V., 15, 76 T Treccani, V., 200, 203, 204, 205, 206, Tabenkin, B., 330, 331, 342 219, 221 Takeda, Y., 211, 221 Trevelyan, W. E., 357, 393 Takeuchi, T., 233, 256 Trexler, P. C., 167, 172, 192 Talalay, P., 279, 290, 292 Tulecke, W., 112, 123, 128 Tamm, C., 281, 288, 288, 287, 288, 291, Tullner, W. W., 115,129 292 Turner, J. C., 199, 209,220 Tanenbaum, S. W., 305, 312, 318, 339 Turner, R. B., 281, 291 Tanguay, A. E., 76 Turner, W. B., 314, 340 Tarnowski, G. S.,245,256 Tyndall, R. L., 120, 129 Tarzwell, C., 80, 105 Tate, J. R., 114, 128 U Tatum, E. L., 42, 72, 194, 187, 220 Uchibayashi, M., 282,284, 291 Tausson, W. C., 194, 105, 204, 221 Umbreit, W. W., 213, 221 Taylor, D. A. H., 280,290 Umezawa, H., 231, 233, 255, 256 Taylor, P. J., 157, 192 Umezawa, S., 233, 256 Taylor, R., 31, 75 Ungar, J., 37, 47, 59, 76 Taylor, R. O., 157,189 Utahara, R., 233, 256 Taylor, W. W., 55, 73

Strangeways, T. S. P., 109, 129 Strawinski, R. J., 199, 201, 203, 221 Strumia, M. M., 2, 19, 76 Stubbs, J. J., 330, 331, 342 Stull, J. W., 19, 76 Stutts, P., 248, 249, 250, 254 Subbarow, Y., 229, 255 Suda, M., 211, 221 Sueoka, N., 333, 341 Sugawara, R., 233,255,256 Suhadolnik, R. J., 301, 342 Sulkin, S. E., 133, 134, 135, 137, 138, 148, 192 Sultan, L. U., 143, 191 Sumiki, U., 338, 342 Sutherland, I. D., 305, 306, 341 Sutton, L. S., 166, 191 Suzuki, M., 231, 255 Sweat, M. L., 117, 129 Swift, H. F., 6, 17, 37, 58, 61, 64, 76 Swim, H. E., 117, 118, 125, 129 Sylvester, R. F., Jr., 229, 254 Synge, R. L. M., 352, 393 Syverton, J. T., 111, 112, 128 Szemiakin, M. M., 262, 278 Szilagyi, D. E., 175, 190 Szilard, L., 85, 106, 123, 128 Szybalski, W., 81, 106

AUTHOR INDEX

v

411

Warshowsky, B., 175, 189 Wasilejko, H. C., 248, 255 Vagelos, P. R., 297, 341 Wasserman, A. E., 50, 51, 55, 56, 68, Van Bruggen, J. T., 166, 192 76 van Deinse, F., 36, 59, 60, 76 Wasserman, H. H., 332, 333, 342 Van den Ende, M., 147, 153, 192 Watkins, H. M. S., 149, 189 Van Der Slikke, L. B., 117, 127 Watkins, J., 81, $7, 1@3,106 Vandenverff, H., 230, 254,255 Wattie, E., 81, 87, 941, 98, 103, 106 van Drimmelen, G. C., 26, 29, 32, 72, 76 Waymouth, C., 119, 120, 129 Van Metre, T. E., Jr., 133, 192 Webb, S. J., 30, 76 Van Niel, C . B., 311, 3 4 1 Webster, B. R., 305, 340 van Rooyen, C. E., 20, 76 Webster, G. W., 14, 419, 73 van Zijp, J. C. M., 194, 211, 220 Wedum, A. G., 135, 140, 141, 142, 143, Vamer, J. E., 352,392 147, 148, 153, 155, 156, 157, 158, Vaughan, J. R., 229,255 159, 163, 167, 170, 174, 175, 177, Velle, W., 118, 129 178, 180, 183,189, 191,192 Velu, H., 37,69, 76 Weinstock, N., 143, 191 Venvey, W. F., 26, 29, 32, 4f7,57, 67, Weintraub, A., 284, 285, 291 76 Weiser, R. S . , 29, 33, 42, 46, 76 Vickery, H. B., 344, 393 Weiss, E., 164, 192 Vincent, M. M., 116, 129 Weiss, F.A,, 51, 52, 65, 76 Vinson, J, W., 269, 277 Weiss, L., 114, 120, 128, 129 Vischer, E., 279, 286, 290, 292 Weiss, S., 117, 127 Vladimirova, G. V., 231, 254 Welch, A. D., 249, 256 Voets, J. P., 208, 221 Wells, W. F., 143, 164, 191, 192 Vogel, A. I., 385, 393 Wender, I., 199, 203, 204, 205, 206, 216, Volz, F. E., 56, 76 220 Von Euw, J., 285,291 Werber, E., 115, 126 Werner, J. A. 119, 127 Westwood, J. C . N., 114, 129 W Wettstein, A., 279, 286, 290, 292 Wachter, R. F., 52, 76 Whalley, W. B., 297, 324, 340 Waelsch, H., 317, 341 Wheatley, W. B., 233, 255 Wagner, R., 80, 107, 194, 221 Wheeler, G. P., 245, 256 Waitz, S., 98, 103, 104: 107 White, P. R., 109, 129 Wakaki, S . , 233, 256 Whitewell, F., 157, 192 Waksman, S. A., 230, 256, 298, 278, Whiting, G. C., 351,366, 367,393 Wickerham, L. J., 24, 25, 29, 38, 73, 76 309, 338, 342 Wilkin, G . D., 194, 211, 220 Walker, A. D., 194, 211, 220 Walker, N., 199, 200, 201, 204, 209, Wilkinson, J. H., 114, 128 Williams, J. H., 230, 255 214, 215, 221 Williams, R. E. O., 147, 153, 192 Walker, T. K., 377, 393 Williams, R. T., 201, 207, 220 Wallace, W. S . , 230, 255 Williams, V. B., 235, 254 Waller, C. W., 2219, 255 Waltz, H. K., 111, 112, 115, 122, 126, Willis, J., 370, 393 Willits, C. O., 14, 76 128, 129 Wilson, A. R., 248, 250, 254 Wang, T. L., 208, 221 Wilson, C., 102, 107 Warburg, O., 121, 129, 231, 256 Wilson, J., 80, 107 Ward, G. E., 330, 331, 342 Wilson, J. N., 112, 123, 125 Warren, J., 9, 22, 76

412

AUTHOR I N D E X

Wilson, R., 85, 105 Wiltshire, G . H., 199, 200, 201, 204, 221 Windaus, A., 2881, 292 Winder, F. G., 194, 220 Winkelstein, W., Jr., 148, 189 Winkler, K., 85, 105 Winner, H. I., 162, 192 Winnick, R. E., 121, 129 Winnick, T., 121, 122, 126, 127, 129 Winogradsky, S., 86, 98, 101, 107 Winsscr, J., 146, 189 Winstead, J. A., 301, 342 Wiss, O., 319, 342 Wolf, H. W., 14'2, 192 Wolf, J., 345, 362, 370, 393 Wolfe, R., 78, 93, 102, 107 Wolfeboro, N. H., 145, 169,187 Wolff, J. A., 229, 254 Wolochow, H., 164, 168, 170, 178,192 Woodbine, M., 291, 342 Woodside, G. L., 230,255 Wooltorton, L. S . C., 349, 350, 351, 354, 361, 392 Worden, A. N., 166, 192 Wright, E. S., 174,190 Wright, S. E., 282,290 Wright, W. B., 352, 392 Wuhrmann, K., 95, 103, 107

Wurtz, A., 78, 107 Wyckoff, W. G., 19, 21, 76

Y Yagishita, K., 233, 256 Yamada, A,, 283, 287, 288,291 Yamaguchi, M., 18, 75 Yamamoto, T., 233, 256 Yamano, T., 282, 284,291 Yamaoka, S., 233, 256 Yano, K., 199,209, 221 Yardley, J. H., 117, 129 Yemm, E. W., 370, 393 Yoshioka, T., 37, 75 Young, L., 200, 221

Z Zahner, H., 268, 277 Zaffaroni, A,, 280, 290 Zejicek, J., 329, 341 Ziegler, D. W., 112, 122, 129 Zill, L. P., 344, 370, 392 ZoBell, C.-E., 204, 220 Zubrzycki, L., 85, 107 Zweig, G., 355, 391

SUBJECT INDEX a-Aminoadipate, 317 ( a-Aminoadipyl) cysteinylvaline, 316 Acetic, 387 6-Aminopenicillanic acid, 316 Acetone-dicarboxylic, 370 Anaerobic microorganisms, 231 Acetylenic acids, 314 Analysis of acids, 343 Acid hydrazides, 245 Anhydroperiplogenin, 284 Acids, 343 Anhydroperiplogenone, 387 Activated sludge, 81 Animal bites, 138 Active dry yeasts, 25 Animal cage housings, 166 Accident rate, 135, 137 Animal caused laboratory infections, 144 Acetic, 387 Animal-holding devices, 166 Acetoacetic, 370 Animal inoculations, 161 2-Acetylresorcinol, 321 Animal respiratory exposure equipment, ACTH, 124 163 Actidione, 232 Animal to man transmission, 145 Actinobolin, 230, 234, 233, 237, 238, 242 Anthraquinones, 321 Actinomycin, 230, 232, 234, 237, 275, Antibiotics, 111, 293 329 synthesis, 293 Actinomycin C,,301 Anticancer agents, 112, 229 Actinomycin D, 237 Anticancer drugs, 226 Actinomycins, 333 action mechanism, 249 Additives, 40 Antigenicity of lyophilized preparations, Adrenal explants, 115 57 Adrenocortical hormones, 279 Antitumor agents, 223 Adtevac process, 9 characterization of, 234 Aeration and lyophilization, 26 Antitumor antibiotics, 232, 233 Aerobic corrosion of iron pipes, 81 Antitumor screening, 236 Aerosol, 145 Antivirus compounds, 112 Aerosol hazards, 139 Appearance of colonies of Sphaerotilus, Aerosols, 140, 141, 143, 144 88 Agaricic acid, 297, 298 Applications of cell and organ culture, “Aglycone”, 297 123 Air-home infection, 139 Aromatic compounds, 193,201 Air-borne contaminants, 176 Aromatic degradation, 206 Air filters, 153 Aromatic herbicides, 213 Alazopeptin, 233 Aromatic rings, 195 Alpha-particle ionization, 13 Aromatic nitro compounds, 216 Alternate pathways, 206 Aromatic secondary metabolites, 305 Amberlire resin IR-400, 366 Aspergillic acid, 300 Amethopterin, 235, 238, 245 “Attachment factor”, 120 Amicetin, 305, 307 Amino acid derivative, 300 Auroglaucin, 297, 299, 306, 309, 314, Amino acid metabolism, tissue culture, 324, 325 121 g-Azaguanine, 229, 245 413 A

414

SUBJEKT INDEX

Azaserine, 233, 235, 237, 238, 240, 245 L-Azaserine, 300

Carolic acid, 326 Carolinic acid, 326 /+Carotene, 296, 299 Catechol, 195 B oxidation, 319 Bacillus, 90 Categories of taxonomic groups, 266 Bacitracin, 338 Cause of death, 224 Bacteria, extraction, 348 Cause of death by lyophilization, 66 Bacteria used in studying anticancer Celite, 378 agents, 236 Cell concentration, 28 Bacteriological nomenclature code, 266 Centrifugal-freeze-drying, 4 Batch enrichment methods, 86 equipment, 23 Bacteriophages, 253 Centrifugal vacuum freezing, 42 BCG, 59 Centrifugation of infectious agents, 157 Benzene derivatives, 194 Cephalosporin N, 317 Benzoic acids, 208 Characteristics for species, 274 Biochemical activities of animal cells Characterization of antitumor agents, 234 grown in oitro, 113 Chartreusin, 302 Biochemical activity, 55 Chemical injury, 39 Biological slime, 79 Chemical slime, 79 Blendor, 157 Chemostats, 85 Blooms of Sphaerotilus, 93 Chemotherapeutic agents, 223 Broken glass, 138 Chemotherapy screening, 112 Brucellosis, 133 Chlamydobacteriaceae, 91 Buffers, 354 Chlamydobacteriales, 90 Bulked sludge, 84 6-Chloropurine, 245 Bulking of activated sludge, 81 Chlorotetracycline, 332 n-Butyric, 387 Chloramphenicol, 304 Chlorine, 103 Chromatography of acids, 345 C Cinnamic acid derivatives, 304 Cabinets, 150 cis-Aconitate, 297 Cancer, 223 cis,cis-Muconic acid, 195 Cancer chemotherapy, 225 Citramalic, 352 Cancer screening, 227 Citric, 296 n-Caproic, 387 Citric acid cycle, 349 Calcium carbide, 9 Citrinin, 310 Calcium sulfate, 8 Cladothrix, 78, 79, 90, 91, 92 Carbamates, 242 Clonothrix, 79 Carbamyl serine, 300 Classification of actinomycetes, 257, 309 Carbohydrate metabolism, tissue culture, Closed-front hoods, 152 121 Co-distillation, 14 Carboxylic acid derivatives, 296 Collagen, 117 Carcinostatic agents, 227 Colloid mills, 159 Cardenolides, 280 Columbia River, 98 Cardiac lactones, 279 Combinations of drugs, 246 Cardiac steroids, 280 Condensers, 9, 10 Cardiotonic activity, 280 Contaminants, 143 Carlic acid, 326 Contaminated waste, 145 Carlosic acid, 326 Continuous cell culture, 123

415

SUBJECT INDEX

Continuous culture apparatus, 112 Continuous vacuum-drying systems, 23 Cordycepin, 302, 305, 307 Cordycepose, 302, 303 Correlation between inhibitory activity of anticancer drugs, 244 Correlation between microbial and mammalian system, 243, 249 Corrosion, 80 Corticosterone fibroblasts, 118 Corticotropin, 116 Cortisol, 117 Cortisone, 279 Corynomycolic acid, 298 Crenothrix, 79, 93 Cryochem, 20 Cryochem apparatus, 8 Cycloheximide, 232, 234 Cyclopaldic acid, 310, 320 D-Cycloserine, 300 Cysteine, 317 L-Cysteine, 316 Cytotoxic cell culture, 228 Cytotoxicity for mammalian cells, 228 Cultural characteristics of Sphaerotilus, 89 Culture media for Sphaerotilus, 87 Culture-shaking machine, 155 Curvularin, 297, 298 Cuts, 138

D Daraprim, 246 Death rate in lyophilization, 65 Decanoic, 388 Decontamination, 153, 173 Degradation of aromatic compounds, 193 of nicotinic acid, 216 Deoxypyridodne, 245 Deposition of iron, 100 Descending solvent method, 355 Desiccants, 8 Desiccators, 17 Design, 178 Desivac system, 11 Desosamine, 302, 303 * Detecting sugar acids, 366 Determination of keto acids, 371 ’

Determination of organic acids, 343 2,4-Diamino-5- ( 3',4'-dichlorophenyl ) -6ethyl pyrimidine, 230 2,4-Diamino-5( 3',4'-dichlorophenyl ) -6methyl pyrimidine, 245 S,B-Diaminopurine, 230 Dianthraquinones, 321 Diatomaceous earth, 378 Diatretyne I, 294 Diatretyne 11, 294 Diatretynes, 334 6-Diazo-5-oxo-~-norleucine, 233 Diazoketone, 238 Diazomycins, 234 Difficulties in classifying antibiotics, 262 Digitalose, 302 Digitoxigenin, 280, 281, 282, 286 Dihydroarenediol intermediates, 200 Dihydroxybenzene, 195 Dihydroxybenzoic acid, 195 1,8-Dihydroxynaphthalene, 321 Dihydroxyperylenequinone, 32 1 Dipyrrylaldehyde, 332 Direct pumping, 10 “Dispersing agents”, 121 DON, 233, 238, 240, 245 Dowex 1, 349 Dowex 50, 349 Drug resistance, 246 Drug resistant bacterial strains, 236 Dry ice, 9 Drying of bacteria, 2 Drying of unfrozen preparations, 42 Drying temperature, 41 Drying to a constant weight, 14 Dual pumping system, 7 Duct size, 8

E Eburicoic acid, 299 Echimilin, 304, 305, 314 Electric air heater, 153 Electrostatic precipitators, 153 Emodin, 321, 323 Endocrine function in vitro, 116 Enrichment cultures, 84 Enzyme inactivation as the cause of death, 68

416

SUBJECT INDEX

Enzymic induction, 332, 3-Epidigitoxigenin, 287 Epoxy resin paints, 179 Ergot alkaloids, 301 Erythromycin, 297, 298 Estrogens, 118 Ethionine, 238, 240 Ethylene oxide, 174 Evaporative freezing, 4 Extent of drying, 43 Extraction of acids, 347 of keto acids, 371 of lipids, 374

F Fabricated lyophilizers, 17 Factors affecting stream infestations with Splinerotikis, 94 Fast freezing, 40 Fat synthesis, 297 Fatty acid derivatives, 297 Fatty acids, 374 Fetuin, 120 Filter design, 154 Filter paper chromatography, 354 Flame ionization detector, 381 Flask-shaking machines, 144 Flavoglaucin, 309 2-Fluoroadenosine, 235 p-Fluorophenylalanine, 240 Folic acid antagonists, 238 Follicular hormone, 116 Formaldehyde, 174 “Formation of antibiotics” as a taxonomic characteristic, 258 Formic, 387 “Freeze-drying”, 2 Freeze-drying, 1 Frequentin, 309, 324 Fucose, 302 Fulvic acid, 310 Fungal tetronic acids, 297 Fumagillin, 297, 298 Fumaric, 296 Fumarylalanine, 300 Functions of secondary metabolism, 335 Fungi, extraction, 348 Future of lyophilization, 71

G Gallic acid, 304, 305 Gas chromatography, 345, 363, 378, 375, 379, 389 Gas-fired air incinerators, 153 Gas masks, 170 Gaseous disinfectants, 174 Gentisic acid, 294 Gentisyl alcohol, 334 Geomycin IV, 307 Germfree animals, 167 Germicides, 173 Gentisic acid, 318 Gibboellic acid, 297, 299 Gill nets, 80 Gitoxigenin, 282 Gliotoxin, 300, 304, 310 Gloves, 169 Glutamic acid-y-hydrazide, 240 Glyoxylic acids, 370 Gonadotropin, 116 Gonadotropic hormone, 114 Gradient elution, 349, 361 Griseofulvin, 310 Growth of animal cells in fermentors, 112

H Hazard, 138 Hazards of lyophilization, 61 Hazards of opening lyophilized ampules, 63 Heat input, 11 Hemicellulose, 101 Heptadecanoic acids, 388 Hexadecanoic, 388 High-frequency spark coils, 12 High-speed blendor, 157 Hispidin, 304 Homogenization, 348 Homogentisic acid, 319 Homologs of salicylic acid, 199 Holdfasts, 91 Hood, 149, 152 “Hot Lab” techniques, 182 3-Hydroxyantliranilate, 319 Hydroxylation of aromatic rings, 313 of cardiac aglycones, 281

417

SUBJECT INDEX

3-Hydroxyphthalic acid, 318 Hygromycin, 303 Hygromycin A, 302

I PI, 114 Ice, 10 Ice crystal( s ) , 30, 31, 67 Ideal screening system, 229 Ionization gages, 13 Impaved oxidative metabolism, 231 Incubator, 162 Induced neoplasm, 227 Infectious disease laboratory, 178 Infectivity of lyophilized cultures, 57 Infrasubspecific taxa, 267 Ingestion hazard, 138 Inhibitors in natural supplements, 119 Inhibition of Sphaerotilus, 103 Inoculating loop, 162 Inoculation through the skin, 138 “In-package’’ desiccant, 41 Intermediary metabolism, 293 Intracage cross infections, 144 Ion exchange chromatography, 350, 361 Iridoskyrin, 323 Iron, in metabolism of Sphaerotilus, 98 Iron bacteria, 78, 80 Irradiation, 48 Isalandicin, 323 Isobutyric, 387 Isocaproic, 387 Isolation methods for Sphoerotih, 87 Isopentenylation, 314 Isoprenoid substances, 299 Isoprenoids, 296, 297, 314 Isovaleric, 387 Itaconic, 296 K

Karl Fisher method, 14 alpha-Keto acids, 370 5-Keto-6-deoxy-~-arabohexose, 302 3-Keto-6-hydroxyhexanoic,326 3-Keto-6-hydroxyoctanoic, 326 3-Ketoadipic, 326 alpha-Ketoglutaric acids, 370 3-Ketohexanoic, 326

Ketomalonic, 370 3-Ketooctanoic, 326 Kojic acid, 303

1 Laboratory, 171 Laboratory-acquired infections, 134 Laboratory design, 178 Laboratory safety devices, 186 Laceration, 138 Lactobacillic, 316 Lagosin, 297, 298 Large-scale animal cell production, 122 Large scale use of animal cell cultures, 109 Large scale use of tissue cultures, 109, 110 Laundry disposal, 145 Leading causes of death, 224 Leavening ability of dried yeast, 56 Leptothrix, 78, 79, 90, 92 Leucothrix, 90 Linoleic, 388 Liquid decontaminants, 173 Liquid nitrogen, 9, 10 Liquid wastes, 180 Live vaccines, 59 Long chain acids, 383 Long-chain fatty acids, 375 Luteic acids, 302 Luteoskyrin, 323 Lycomarasmin, 300 Lymphoid tissue, 225 Lyophilization, 1, 2, 5, 25, 143 equipment, 20

M Macrolides, 297, 302 Maintenance of cultures, 27 Malic acids, 352 Malonate, 312 Malonyl-CoA, 312 Mandelic acid, 211 Manganese, 101 Mannisido streptomycin, 388 Manometric method, 17 . Manometric methods for estimating moisture, 15

418

SUBJECT INDEX

Massive cultures of animal cells, 122 McLeod gage, 11, 12 Mechanical samplers, 142' Mechanisms of action of anticancer drugs, 249 Mechanisms of resistance to anticancer drugs, 249 Media for growing lyophilized cells, 55 Medicine dropper pipette, 160 Melanophore-expanding factor, 115 6-Mercaptopurine, 230, 239, 245 Mercury manometer, 11 Metabolic pathway, 195 Metabolism of bacteria, 343 of catechol, 195 of protocatechuric Acid, 195 of the indole nucleus, 212 Metabolite reversal experiments, 240 Metal lyophilizers, 19 Methanol, 337 Method of drying, 40 of freezing, 31 of reconstitution, 49 Methylation, 313 N-Methylfomamide, 242 6-Methylmercaptopurine, 245 6-Methyloctanoic acid, 2198 D-&Methyloctanoic acid, 297 Methyl acachidate, 390 Methyl behenate, 390 %Methylbutyric, 387 alpha-methylbutyric, 38'7 Methyl esters, 3tM Methyl hexacosanoate, 390 N-Methyl-L-glucosamine, 302 Methyl laurate, 390 Methyl linoleate, 390 Methyl linolenate, 390 Methyl myristate, 390 Methyl oleate, 390 Z-Methyl-3-pentyl-pyrrole, 332 0 Methyl salicylate, 3121,317 6-Methyl salicylic acid, 309, 318 Methyl stearate, 390 alpha-Methyl tetronic acid, 296, 326 Mevalonate, 296, 313 Mevalonic acid, 297 Microbial assay of antitumor agents, 232 Microbial screening systems, 229 Microbiological transformation, 281

Mitomycin, 233 Mixed cell cultures, 124 Mode of oxidation, 201 Modular hoods, 152 Modular-type, 150 Moisture, 13 Moisture testers, 14 Molecular vacuum gage, 13 Monitoring, 143 Monkey B vinis, 133 Monolayer cultures of animal cells, 110 Morphological changes in lyophilization, 80 Morphological research, 272 Morphology of Sphaerotilus, 90 Motion pictures, 186 Mouth pipetting, 1% Mutant strains, 231 Mutations, 331 Mycelianamide, 297, 299, 304 Mycophenolic acid, 306

N Naphthalene( s ) , 201, 321 Nemotin group, 330 Nemotinic acid, 298 Neomycin( s ) , 302, 309 Neoplasms, 225 Netropsin, 237, 238 New classes of anticancer agents, 235 Nicotinamide, 319 Nicotinic acid, 216 Nitrogen mustard, 225, 235 Novobiocin, 304, 305 Nucleoside phosphorylase, W9 Nutrition, 26 and age, 26 of Sphaerotilus, 97 Nutritional needs for virus synthesis, 119 Nutritional requirements of animal cells, 119

0 Oleandomycin, 302 Oleandrose, 302 L-Oleandrose, 303 Oleic, 316, 388 Open hoods, 150, 152

419

SUBJEm INDEX

Optimal rate of freezing, 39 Organic acids, 34G, 355 Orsellinic acid, 320 Oxalacetic acid, 3‘70 oxalic, 296 Oxalosuccinic, 370 Oxidation of aromatic compounds, 193 Oxidative metabolism of polynuclear aromatic compounds, 201 beta-Oxoadipic acid, 1:97 Oxytetracycline, 232 P

Palitantin, 309, 324, 325 Palmitate, 390 Palmitoleate, 3190 Paper chromatograms, 351 Paper chromatography, 3@6 Paper machines, 82 Parallel mechanisms, 313 Parathyroid gland, 116 Partition chromatography, 353 Partition coefficient, 353 Patulin, 294, 312, 315, 317, 318, 319,

334 Pellets, 9 Pelletizing, 4 Penicillic acid, 306, 310, 315, 319 Penicillin, 293, 317, 328 Penicillins, 300,316,333 n-Pentadecanoic, 388 Peptide derivatives, 300 Periplogenin, 280, 284 pH, 31 Phalloidin, 300 Phenol breakdown, 197 Phenol oxidases, 315 Phenoxyacetic acid herbicides, 214 Phenylalanine, 210 Phenylhydrazine, 371 Phenylpropane, 303 Phenylquinoline, 310

SPhosphoribosyl-1-pyrophosphate,248 Phosphoric acids, 344 Phthalaldehyde, 310, 320 Physiology of Sphaerotilus, 97 “Pig”, 19 Pigment, 349 Pipe clogging, 80

Pipetting, 138, 159 Pirani gage, 12 Plastic films, lT2 Plastics, 1711 Plucker tube, 12 Plug freezing, 3 Poliomyelitis vaccine, 111 Pollen, 53 Polyacetylenes, 294, 297, 334 Polycyclic aromatic compounds, 207 Polyene antibiotics, 297 Polynuclear aromatic hydrocarbons, 204 Polypeptide antibiotics, 338 Polymalonyl-8-glucose, 302 Polymixin antibiotics, 297 Potentiation of inhibitory activity, 245 Preparation for analysis of acids, 346 of methyl esters, 384 Preservation of Sphaerotilus, 88 of tissue cultures, 40 Pressure gages, 11 “Primary sheath”, 91 Prime precursors, 312 Prodigiosin, 333 Production of epinephrine, 115 Progesterone, 281 Prognosis of cancer, 223 Propionic, 387 Protection against infection, 131 Protective clothing, 168 Protective substances, 3 Protocatechuic acid, 195, 304, 319 PRPP, 248 Pseudo sheaths, 90 Puberulonic acid, 320 Puncture wounds, 138 Purine derivatives, 305 Puromycin, 230 Pyrimidine derivatives, 305 Pyrogallol, 304 e-Pyrromycinone, 306 Pyruvic acid, 370

Q Q fever infections, 133 Qualitative analysis, 366 Quantitative detomination, NO Quinones, 321

420

SUBJECT INDEX

R Rapid cloning techniques, 122 Rate of freezing, 31 of reconstitution, 52 Rational chemotherapy, 226 “Red water”, 80 “Replacement cultures”, 327 Residual moisture content, 43 Resorcinols, 321 Respirators, 170 Respiratory tract exposure, 139 Retention times, 385 Reversal of inhibition, 240 R, values, 3’55 R, values of organic acids, 356 Rhamnosidase, 289 Ribonucleotides, 248 D-Ribose, 302 Room finishers, 179 Rosenono lactone, 297, 299 Rotary shaker, 122 Rotiorin, 324, 325 Rubber gloves, 171 Rubropunctatin, 324, 325 Rubroskyrin, 323 Rules of nomenclature, 257, 266

5 Sacharinic acids, 365 Safety cabinet, 149 Safety devices, 146 Safety hood, 146 Safety manual, 182 Safety programs, 182 Salicylic acid, 198 Sarkomycin, 233 Sarmentogenin, 285 Scillaren A, 289 Sclerotiorin, 309, 324, 325 “Screening”, 223, 293 Screening for anticancer agents, 229 Secondary biosynthesis, 334 Secondary metabolites, 293, 294, 295, 311 Separation of fatty acids, 376 Services, 180 “Sewage fungus”, 83 Sheathed bacteria, 77

Sheath( s ) , 83, 90, 101 Shell freezing, 4 “Shelling machines”, 4 Shikimic acid, 305 Shoes, 169 Short chain volatile fatty acids, 375 Shunt metabolite, 331 “Shunt” products, 330 Silica gel, 352, 381 as dessicant, 9 Silica gel chromatography, 352 Silicic acid chromatography, 383 Simple lyophilizer, 20 Skin contamination, 138 Skyrin, 323 “Slime blossoms”, 79 Slow freezing, 40 Snap-freezing, 4, 39, 41 Solvent systems for paper chromatography, 355 Somatotropin, 116 Sources of infection, 137 Species, 257 Spectrum of antibiotic activity, 284 Spectrum of tumors, 228 Sphaerotilus, 77, 78, W, 91, 92, 102 Spin freezing, 4 Spinulosin, 310 Spontaneous neoplasm, 227 Spray reagents, 357,368 Spray tests for paper chromatogranis of organic acids, 358 Spray tests for paper chromatography, 369 Spread plate procedure, 240 Steam pessure sterilizer, 103 Stearic, 388 Stipitatonic acid, 380 Stream slimes, 82 Streptonigrin, 234 Streptomycetaceae, 270 Streptomyces, 271, 274 Streptomycin, 246, 280, 302, 303, 309, 328 Streptovitacin, 234 Structural types, 295 Subgeneric taxa, 268 Substituted salicylic acids, 199 Sugar acids, 365, 369

421

SUBJECT INDEX

Sugar derivatives, 302 Suggested procedure, 70 Sulfite waste liquor solids, 95 Sulfanilamide, 24‘6 Sulfonamides, 225 Surgical gown, 169 Survival of organisms on lyophilization, 23 Suspending fluids, 31, 32 Suspending medium, 29 Suspending menstrua, 3 “Swamping”, 353 Syringe and needle, 161 Systematic separation of acids, 344

T Terrestric acid, 326 Tespenoids, 313 Tetracyclines, 307, 309, 329, 332 Tetradecanoic, 388 Tetrahydroxydinaphthyl, 321 Tetronic acid, 324, 326 Tetronic acids, 297 Theory of mechanical injury, 39 Thermal decontamination, 173 Thermocouple gage, 12 Thioguanine, 245, 246 6-Thioguanine, 230 Thymidine diphosphomannose, 313 Thyroid function in vitro, 114 Thyroxine, 114 Tissue culture, 112 Tissue culture supplements, 120 Traffic pattern, 179 Transplantable neoplasms, 227 Trash incinerator, 146 Trichothecin, 297, 299 Tridecanoic, 388 Trimethylacetic, 387 Triterpenoids, 297 Triterpine, 314 Tropolone, 32’0 Tropolone derivatives, 310 Tuberculosis, 133 Tumor, 223 Tumor screen, 231 Two dimensional chromatography, 355

Type of organism, 24 Tyrosine, 319

U Ultrasonic disintegrators, 159 Ultraviolet irradiation, 176 Unit processes, 313 Uronic acids, 365 Use of filters in lyophilization, 63 Uzarigenin, 287 V

Vacuum requirements, 5 n-Valeric, 387 “Valid” species, 275 Valine, 317 L-Valine, 316 Valinomycin, 301 Vapor phase decontaminants, 174, 176 Vapor rehumidification, 52 Variability of antibiotic formation, 259 Ventilated suit, 170 Ventilation systems, 145, 181 Viability, 54 Violacein, 301, 302 Viral systems in screening, 2% Viridicatic acid, 326 Viridicatin, 310 Virulence of lyophilized cultures, 57 Virus isolation, 110 Virus vaccine production, 111 Viruses, 25, 40 Volatile acids, 347 Volatile fatty acids, 377 Volume effect on reconstitution, 52

W Warburg theory, 231 Weight loss, 14

X D-Xylusylnemotinic acid, 302

Z Zeokarb, 215, 349

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