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