Section cutting in Microscopy

SECTION CUTTING I N M I C R O SC O P Y SECTIONCUTTING IN MICROSCOPY H. F. STEEDMAN PL,D. D.partnent aJZoolagy, The Uf...

Author: Hugh Fletcher Steedman

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SECTION CUTTING I N M I C R O SC O P Y

SECTIONCUTTING IN MICROSCOPY

H. F. STEEDMAN PL,D. D.partnent aJZoolagy, The Ufiv+tity,

Glagou

uNrvERsl

OF MA}TITOEA

trsRARY

BL A C K W E L L S C T E N T I F I CP U B L I C A T I O N S OXFORD

'

@ Bla&ue Stidtfc Pttlicatio$ Ltd., 1960 Thit bookb capyight.It ndI not betyodued bI N! b.tu ia vhole or b ?an eiftort pmisior. Applkdtiotruith rcgddto coryigllt thosu beaddresed to th. t ublid*s. Publbhedtin lt$eo$lt it the Udte Statetof Abdka by Chad$ C Thq6, Prblishn, Jor j27 Eqt Laud( Avnt.. S?niefeld,nli@is. Publhhedrinultaqosl! in CaMddby th. Ryqilt Pt6s, Qaeil SneetWen, Tot^to 2.

Dr"ilD

& SO& !D

CONTENTS PW

Chptcr

r Ribboniry in6lbation nedia

rEEOirTrc,t1,coNsrDnalroNs Comtituos ofthe living orgunm

-

3 I]lr&es of rwo md of iniltrated tisus 4 Infltration nedia eftect5

-

J

-

7

6 r8

26 !^Rr 3

W* block naling od block trinning Ribbon arrergenent on paper and oo skl* PaESn wd Este!vd rt6o ?oly6te vd Water solublewdes Wax secriotu:fauLs,reasorsad rene&o during cutting :nd aftet Low vlscosityellulose ritrate lrozei setioru dd gelatia secriob urca-fomnde$de rsin Douhle dbedding Isothemal teclniqus Simuheeo6 fsrioD dd sA inflnariotr vd infilrgtior withou! fsiion L{tra thh *ctiom for the electon nico*ope Seciionadhesioned adhesivd Softding material in the block; hot work; choice of medim; tuchdical devics i vacu@ indlnatior; presute blocking ?J Microtome ed midotome lnivcs 26 Ti@ schotule ud dificlit objccts Dimcdt oljecls closary of tefrs Techaicalbooks Supplids

Chepter 8 ,, e ro ,, rr ,, 12 ,, 13 ,, 14 ,, 15 ,, 16 ,, 17 ,, r8 ,, re ,, :o ,, 2r ,, zz ,, 23 ,, 24 ,, ,, ,,

37

_J6 71 8r 88 96

r53 rJ8 fi6 r69

PREFACE This book i, wnttd for the nn rotohist. Behind its feLs od theorieslie thirty yem of prrtie, of teaching,ud of reseachin niootony. Infordation is provided on hfdtratior nedia, section drting teclDiqud, midoloft lnife shqoiog, lhe production of broko-glss knive, adhoion of seaions,md tmy othd rcquireaenls ofthe microtonist ud histologist.Details ofold ud well-tried tedDiqres ofsection dtting aie givo $ well s ntr ud experinmtal nethods, but the book shodd not betegat&d o a conpodim ofall thc koom infiltration nedia or ofal de ruiatior,! of nethod ued in thc cutting of ti$uc scctios. lr is holed howw6 that it uy be of be b all wlo cunectiotu.vhaleeerd,cir.ge. o, crydioe. To the m who wishe only to ot good seaiom the part ofthc book de"oted to prrctical details, vith is Mbered paagapls, my be of osistmce. Shodd he requne some aout of boic pdnciplesmd oftheorie dr chaptersot theoretical cosidmtioro will add interestto hs work. sone ofthe retho& wlich I have deigncd, od ued for 6ve to ten yds, are prblished hae for the tust time. It is bdievcd that thcy oler up fresh possiliJitie. Though condmcd to a fcw pagcsthcir prodution rcqdrcd noy tiousmrJs of erprimo*, md they eppeu hre ody after comiderableuage. cmerou d@uegmdt dd sup?orlhs bcd givd to me lor nore tho twenty6ve y*s by Profc. M. Yonge, ad it is with luppioes drat hn kiadne$ ir recogniad here. It is also a plc*uc to thurL dr pubhhen for drir helpftl md comtruaivc discu$io$ ofthe book. H. F. STEDMAN Cln'got MdI '96a

PART r: HISTORY OF SECTION CUTTING MEDIA

RIBBONING

INFILTRATION

M3DIA

It would appeu lron the raord: that tie fint coc oftrue sectid.lnins o@rled in rr4: wha Stilliog lcft a pie ofspiDal cord oo the wiodow-sili ofhis laboatory ovehight. It wa froz€n to a solid l1o.k in the noning, dd from it he otelatively tnin sdiotu d! slics {o( mjcoscopic ue. So starteda proces which la led ro rie detailedteclaiqe of todal The *ly attemlts to hold biologiel m*erial wliie it w* sliced with a sharp lnife lroduad a wiety of encaing nefto&, conp.rable widr the pushing of a nulIe into a piee ofchy. Sucha he&od giv$ e*ena1 support odn ad ir ws cleady recogn;ud nidwey tlrongh the nineteenthcentury thar inildarion neriodj by which the tises were supponedint maly s vefl d er:temlly were neesary if relialle tissuescctiorswre to be ob.ained. This udestarding Icd to the production ofa numbcr ofdiFcrot inf,ltration hases eachwith i* om specialadvotags od disadvutages,nd ii ; converidt ar ilis point to divide thee mediainto two nd! tyles: ribboDiis medi. dd non-nbbodig nedia, :ad to *mine their historical developndt sep$rely. The t rh 'ribboning: lrre btu the adlsion to cachother of succesiveslics or strtiobr s @t on a midotode so that a lobg ch.in or ribbon ofsecrios is prodrced. A materid lut lorwud for this pupse wd soa! (FleMing, 1873; Kadyj, r87e). Tirsueswere t.ko into a wter od alcohol solution of so.p, ed atui sone evrpootiotr bad t Id p1a6 thc soapbec@e hud enoughto cut into thiD sectios, the mterial be;ngconpletely inf,lcated. Beautiful setios could be obtaincdin riis Ijj]mer but iheir ..lh6ioD to gls Didoscope slids vs sopoor that the nethod ws soonabddorEd. Its llace wd takd ly dr pa.afin R* proes, ud this ha remined the nost videly ued cve! sin@. It rd jnrodtued by Klcbs (r86e) sd w4 alsolued by Giobrccht io Septmber r88r, by BfficHi dd Bloc\me in Decdbd r88r, dd by c.Dle i! 1882.Eaci aplared to have vorked indclad@dy. In liis nelnod inflrariou of lhe ri$us i! ody pusible aftd they hevebeal comllcrely dehydFred.rhe gnded alcoholswhich de ued, the su solvdt tuongh vhich thc tiss@shlst p*s bdore the nolta wu ea pemete tho, od the noltm vd itsclfhavc adr ! 1owvscosity dbt inllrrario! n .chievedvery quicuy. In ddition tie cuttirg ofsecrio$ in puaffr wx ls relatively dy, asis the nanipul.rion oflhe ribboir or se.tios .ftcrwrds. Parafin wx h$ tvo mjor wealnBses,howevei The 6rsr of th6c is its rathu high mclling point J,-.6o' C, od rhe rcood is its conglete inconpatabitry wirh

2

SectionCutti g in Midaeopl

watel Boih thesetend io mate tisues h*d od distoned. A diid lics iE its strotg crystallineproperti*, wlnch at tins tend to prodlce bloc*s of low honogmeity This leds to ?oor scctioing ud ribboning. The nethod also.e-ov* fas fron the Attenpts havebmn nade very stedily sjnceiL' inception to q. dee faul* One olthe ftst oodifietioro to bc nade ws tiat of Schulgin(r88t who reconnended thc useol erein u a paraFn w* rdditive to redrce exessiae.rysrallinity ln r 88J craf Speeadvisedt,heue ofheat treatnet for the smc Purposedd elsoto inlrove itr ribboning propefties. Para6h wx is heated &tiIit tlms broM La the cold conditioo thn heatedlu shom nuked los of dFt line con&tion, dd h6 a The yeas imdiately followirg the tm of tire twotieth cotury were narked lry nodinetiom of dr laom nethods r.tLer tlun by uy sdling isovatiod. Itr rlor Johuton advocateda nirture of putrrn w:a *phalt dd pao rubber. This reducedthe crlstallinity ofthe parafrn wc ud alsoinpro'ed its .dhesionrct only to iselfbut also to shiny strfads suchd imcct cuticle. The ncx thirty yeus uw modifcatiod ofrdafn Rd by vdiou.dditives such as beewx, rubbe.. J.po v,1 baybeny w*, lard, etc., viih the object aLeady indicatedabove,but ia 1936a nixture wo put forwd by Lehowich to redue rhe hy&ophobic propeni:s ofpaEffln ud. It cosistcd ofPamffa wu 8o, stearicacid zq dietiyloe glyol r'5, ed@oladiqe r'5- The so.!-vx mixture had the advmtage th.t it rvs morc wate! tolerut tlh pfte praffn wo<, but thi Gatuc alonetls not goved suficiot to oake it a popula nethod or onc which is widely usd. ID r93e watelrlm ued par.6jr steaic *id" spermeti, od baybery war aud h r94r chy suggsted a niItue of pua6n oar rubbcr, be6Rd, sPem.@ti ud 'Nevillite' ioproved de conistecy of Nflillite' 5. The ituorpo€tion of the resin The givetr fomulae, dd very ruy otle*, my ail be coddded nodifations ofthc !@ffi! \w teclnique, but drc nost inportot bralawy frcn this matcrial canc ia r935 wha Cuder ed glycol stearate:loe This wulikc mterial is soluble in 9J% rlcohol, dd is nore tolerant of wter ftd is pxa6r wx, od aaditional ua sLcnts suchx xylm md be*"l md cllorofom were no lolse. reqfted. The yer r9p saw vh$ apleed to be a very inportut new naterial avanabb 'C{bo\,tex' and to the hicrotoEisr It ws . mtd+oluble wd-liLe material c.[ed its usefor sectioncuttiis vd put fomdd by Richddr, Andcrsonud Haoe. It ; a po$hedylene glyml onplcte\ soluble in water in all ?loportios. Its useoqnt tbat graded alcohol solutiou od cl@ing agdts such s x-yloe wq€ no loryer Equftd, dd that 6&d d$6 could go straightiDto it froo water, or fton nixttm ofthe ws with water. At tust sight ihis seemedto the poffn-weary microtonisr a theoretiel dren fulilied, but it soonbecaft dear tlat the w* *s not oniy solublein water it w.l sotublein wey othe c1s of fluid" including alcohols,eters, ethm, ketora, and hydlo@bons. Merclry vA the only [email protected] ploperty ofextcnsiye solubility led to gleat dificulti6 io the nattdng ud adhesiorof sectiom,ud fnally to tLc ear\ abudomot of thn biltntion mdfun for goeral histologicalpuposes.

x.bl)onig Wltatiai MuM

3

Othcr workcs *perinotcd with thoe water solublepoJyethyloe glycols but no nodification re lrodu@d rhich led ro ceitain edheior otsections,and rsultj were .o vrirble od dL.lpoirrirs Lbr wJler-.ol-bk !ol)clhyttue gt.coL baer,1trIy ,e1ed ro adertuJtcrhe g"De-l sup$ioriD of parlrTr w* oc is nixturo. h n e}?"crd tbarrlu \irrriob wrll be n"irtaired :nol , uter ,ol..bh $,x L prod(ed $hici L roluol"b $, reroDtv. Ftrther worL on glycol sremrer wu dore by Steedmu (r9a7) in which the tomule put foruard vs bded upon dicthyloe glycol distearateod monostqate, to vhich ws added ethl ftllulose ro tougho the nas, dd otor oi1 atrd sredic eid. Thc mkture sas very ouch larder dE p&6n vq, wu more v,ier roleladt ud h"J r lovo r"ldng poir!. lr did lor requne\rlqe a a clcains,g'br. buL .olvcnb:u,hs eJooal.ohor.or etblrebegl)col moDoe$ylelhe' s ererccom"bded. It v6 not d esy to use as parafia dd secriotrnartening occasionallygave sone Polyetblenc glycols de obtainablein d exilive resq and when estaficd with frny.cid, tlry tom - ri6 of wu', whil,hoq, vdorr d.Srcs of wkr solubliLy or vater tolerdce. The disteuate of 4oo polluhy)oe glycol ro suggcted * m intitr.tion nediun (Stddnan rq:z). It shos coniderable wter tolcrucc, od yet is insolublein watea dd iir iow mefting point of37 40" C produce ls heathardoing ud het atifaca dr:n uy oths wax available.It is solublein 9J% alcohol,and wili cut at a mom tehlEatw of r7-2o" C.

NoN-mloNc

INrILri,{rloN MDD

The developnmt of tJree oerhods $arted with fteeziis tisuel, asalrady bdicated on p. r. Thi5 tool llacc in 1842,but b' 1878Hmillon had rcconmendcda ieezing mcrhodin rvhich the tisue v* 6rst iniltrated with a srgr ad gu soturion.Thn m!1ed good sectioc to be obtainedvidl a freeiig hicrotome, but peuetrationof the tissre3with the sws-guh sohtior took . iong tiDe. It cd judy !e called d aqreors infltration nediun ud wirh ir secrionsstil containiig firty nate.ial were coily obt,ined. Frorn its inception the nctiod hd alwlys bea.rin uc eirher fol histologicalnaterl:l containiogfars or lor tisue such* liganentln nuh@ which becobe too iard to cut fho put tlrough hot vd !t!16. A gelatinimpregmtio! proces w"s put fol9ud for rhe sane pupor s rhe gmsuge nethod (Soll2s,i88a), dd owirg to ttre sonewhat lower vi3cosiryof. solulion "fgel"tia the p-cs took lex rine tfio the sugr-gm. Ar suuhit lad irs adherdts md ir stil in lse today, tholgh nodi6ed. For rhe dcnodtiarion offaB ir lud rhe disadvstegethai a.enain morut ofheat w* required,od this pernitted the escalc ofsone ofthe fatty nat*ial. Pcrlups the Eos! populd non-ribboning nelhod olal vd thc @loidin proces (Duvd, 1879).Celoidin $A tle proprictary nme ofa type ofce]lulosenitate. This substuce wc dnsolvedin equal par* of absolutealcohol od erler, ud specimds .oe del-ldtaredrd brougLtslo ,l,obol<,lFr. )o.jo bdore bffg pl,c;d iD lhc ccloidin solurioi. Two or threesoluriod wse !sed; rtu!, nediu ud rhick. WheD

t

Se.nd Cadingifl Mi6o$au

6ra[y brcught ifto the tlLk solution thc spcimen ws allowed to stay sone day, wecls, u *o notts, depdding @ its siz, ud ir ws 6olly hadoed in clJorofom, or ia clio.ofom vapoui. Ator ihjs teatnet thc block wo placedb 8o/6 zlcohol for stonge.The block could !e ot in this condition fron the :1ohol solution, or it could bc tako through c orofom into edamood oi1 od cut in the oily condition. Soth nethods had then adhddb. Both netho& produced ercellot stciom, though thicker tLo thoe ot b p*6n wu, od alsowith no traca offat left within the ti.sss. Afta sctioning the elluiosc aitnte wc rot redoved from ttu tissucs,asis !ua6r wax, ud a a eult the hdividorl detailsofa celloidin secim look s6@dts, fios, nore clorly knit togetha thal thoseofa !!ra6n ird setion. This ncthod, with littlc mjo! diferercs, sti[ rehains ve./ trfii, panicdarly with hard material suc! s rth'opodr, woody botrticd speihe$, erc. It ows its continued udolnes and c\dn to ii5 uique prolerty of foming a v*y 6m gel, cvo who the weiel* to volwe !,tio olnitrocelulose to solvent is * low r zo to 80. Thc vqknqs oftlE rctLod lio in the tine ta.ko for bf,lnation: thc dissolution of 6ts; and io the umgmmt od conttol of'ectiorx The mterirls aud methodsoutlioed abovewse zli in ne by reoo, ed conri Ed for nmy yem without majc altentioo u imprcvenos. Agd Ns ?ut fom{d d o infltration me&u (Chatton, re23) aDd it hs the advmtagethat it form a very 6m gel which lmds ieelfto the ldehation of ot}er liquids, such* nolta pmdn w*, without cessive shrinlage. Agd tuy aho be 6ed s d hf,ltratior mediun for rhe cutting ofsoil scctions where its solubility in wat* md touglas after seningshow to advnuge Grudol', N. ud weissFogh, re5z)produccd elearon niaoscope led to rdewed ilterest The fn! olmetcialy bfltratioo oedia. This approa.h wx fored ;n dd intdive voik on dorthou or upon electron niclocope .)tologists laaue practice showd that gmdt drrt.lliDe nateridi su.l a paafin w wae quite muitable for very drln seaiom such* roo-:ooA. The old *terials w* aeftlly invatigated ud foud muiahle, nnlond.e ro the ed it vas thd tbat nodeo plstics 6nt beme of rliDly The poryoetlayJates, a grcup of cl* gl*lile ro;ro, beetu dsely iovstisated in reae (NeEj]d e, al) dd section in thee materialshrve beenprodued o tlin $ 7oA {sFstled, re53). Materi.ls suh * nethyl poliaetiacrylate od rbutyl polynethatrylate are ued mday eithu siogly or ir onbinatioa dd by appropri.te wiationl @y give sectio6 from 5oA upwrds. As wnL tle light Eidoscope the preoe ofthe iniltr:tiod tu&un in thc tirsud aad surrooding the *ctio le.dr to diGcultis in obs@tion od resolotion who ultrathin *ctiom ae be;ngcxmircd with the eleto nicroscope,dd sone worker prefer ro roovc dre oedim after the sectioc tave tea cut. T!$ led to the Prodoctior of a subJ.imting nedim rophu-uphthalene by Claudc ud !ull@ (I9a6) which had the advartageof@ttely leaviig the se.tid when thc latt* wc subjected to vaanm. Tli; ldtly Ele of neth&rylates, bur i' ge@l the conplete rcnoval ofa suppo*iry nedtun le& to a collapc off,ae strutures, &o!gh n my give thc rs!. bdedt ofdininished b.cksro@d dd ttcrdolc ofiadeard.o

Ribkhins wlttdtiat Metia

5

No iniltration medie i, coopletely f.ce 0on vcalner*s, ud good though the ue they re still tu lrom behg rhe completei&al vhich the usu of netlar/ate the electron nidoscope mtrst contiououslyhavc io nind. With nerlao/ic tsim the ruin dificrkie3 lie in hinute swdings or explosiotsororhg dtrring pobneriatioD, d s displa.ing sohe ofthe 6ner struaues ofthc spEimens,,r seedlater, dd in a tendencyto producebubbls. A fither stepms nade when cpoxy reiu were introduced * hf,lnatioo media for ultothin seaiom, by Malrle ad &ch-Andenon (Ipr6) and Glauertdd Glauet G9i6). These resis have the advutage ovq netfadtlates in showhg smaller slrblage on polynerizing, nd also show much bena adhdor They re usedas adhesiv* cometcialln ud this latter f.ct my vel prove of someimPolt r@ in the seaiodng of dificdt ofjcct sucho l"ir, conot 6lrs, etc since r88o thcrcfore mlch prog!6s l$ b€d hade, but within the cobirg ten d twenty yeus it is lossible that more wil1le made tho in thc Previols eighry. This is beausethe prodrcts avatuble6on the oergy ofth plotics chenist hay provc to be nore suitabletlun q'tling y6t seen. RTFER,ENCES Burscrf,! O., d Br-oct]'l,jN. F-,1asr, Bial.cdh4lbldtt. deb;alogie, d6 kdfet a nosons deld Soriete Cs^noN, t9zX,Con?k erdu h&doflodane 88,r99. Cdm!, A., and !@tN, lllll.F., 1946,J. E?t. Med., a3, 499. eu't@, O.1., lqs, Ath- Pdth.,m, 4454. Dw e, 1819, Ja"n. Anat.Phtt., rS, r I s. FJ::rlJ\1!jWG, 1823, Ath nikr. Anat., Bal,9. G^@,1a82,Ahii4fl h'oftrytMnt.Jounot,11. GNBm, r88r, Zooi.&zdg.i 4,Jarrg. Gu@r, A. M. dd GrA@r,k H. 19s8,J- Biorhtlk. aid Biorheht.Cyto|.,4. No. 2. GMy, P., 194r,U.S,Pat@t2,267,rJr. H ^ " " 6 , N . , r d w E s - I o ( ,, 1 . . t 9 5 2 . O i \ o s , 4 , 4 4 .

Jo,l\so\,1., reo3,J. Apll. nid., 6, 2662. Krrv, raj9. Zoal,AE,, 2, 4n. I
PART 2: THEOR-ETICAI CONSIDERATTONS Cr lrR 2 CONSTITT]TNTS OF TI]E LMNG

ORGANISM

rle fou o-jo .on\lirumK o $e liviig sgdln !e Nds. !rde;n. rrq ebd cdbohrdrue. IheJn of rbeni.oroni.t. hi.rJogis.dd qlo,ogd b to ept,e lhr rsrer by wd or .sia 50 fi:t qerioru my be oL o" rhe reruirug .rrbroce. Additimrl fb\u6.i.L a homtr.,. ftLlnd, Duter prcdu.c. c[Lm, dd $

WrrDx ln thc hajority otliving orgmisns, orgm, tnsue od cells,vater is the lrimiral consritlent, and jn the nost widely ued netho& of*crion ftfting ii n renoved froo f,xed material by usirg a selicsof gr.&d alcohob,by wLich t i5 fimly comolerejy ' epbed. T\s .-b.u' Irtior is accomoo"d rs r rulc by "hrul Bedd ditrorrioD ofd;ue relatiotuHls ed cell .ontenrs.T; rhe hicrotomisr the co-plete rcnoval of wais frcm d$u$ i5 gtutraly ro|lowrdby hddeains,!h6 c\.e.si\; h,rdeniis. sd tD y by dificult cutting whd the materialis iofttrated with w* or roin. wld the inliltration mdia, such s pdttu w* or methaa,late rcsin, arc irolubb nr vatq, ed .rc buledly hydrophobic ev.ry eForr b rude to bring material into rho so gr.duafly r,\ar rhe lo$ of vat6 prodees lirde distortion of tisues, btt no natter how oreftly rhjs is donesomeshrinkage clmping, grmularion dd hrdening ofti$us and ells ta.kesplace. The use of qater-solulle resi.r ud wdes elihimr$ sone of th6e udsnabb fc*ues, od in gmeel ifthe highestdegre oftisue fom ir esdrtul belte. rsults will be obtaircd *nrg biltntion ruteriats such as gelarin or ue-fornatd&yde r6iD dE by birg h/drorhobic'ms lcb 6 !!-Eia A did-point betwetr ttrc completelyhy&ophobic r6in5 o! wdes dd rhe coollet ly hy&or ic ws-like rut*ials sudr o r,5m polyernyboe glycoi i found io dr polyethylse giycol sterrateswhere a tolcuce ol v.tef is premr vfthout conplete solubiJiry. This tolcrece emb16 rut*ial tc be eko into molten wx froia a solution oferhyl alcohol conrainingj,ro% warer, od thc prere ofsuch a large momt of mto enalles ti$rs to be intlrrared wirholt Drcducins excssive hrrdorg. The extent to wlictr water-ituolublc, water-to1erut, md wter-soluble nedia * s! fd ;]i!Edon dcrods or iiejob ;n hdd, 3nd . knowledgeofrhe lossitililiej dd lioir of rhe nedia tlr3melves.The employnot of wato solubleh;dia which uy !e gelcd dd d&red imoluble * is thc coe with urea-folnuldehvdei6id. hs a s eJrdealro recotueod n.

Conttittents oJ tlE L ting Otgffibnl

When the renaidry comtitms of dre oimal body are .oBidered - Protei!, f.t rd carbohydmte- ir rould be d accertiblesuggestionliat o biltratior mediud should be useddifioot lion thrt of thc *s!e to be sttdied. Thu iffat were to be observedin the tuishcd rction it would bc usuitable to in61$atedre tissueswitb o $ter su.h a dter wax bmw it would disolve the f,t out of the tissues. !i pro*ise the idllaatior of ore clcs ol naterial suchx protein wit! a difddi naterirl *ch asa wu is connon md seuible, but good resulc my be obtaiaedly i'iltraring \aith a lrotein sucha gclatin. Th; is not dot with vcry gret {iequocy becatservxcs co ir Lctter sectioncntting proFrties fiu do prctei*, od o the D4oity of scdos for nicrcropic sttdy cooist rrirciraly of protein, wd or resir nedia are ued nore tho uy other. Duriry the prc@ses of dehydration"od ln6ltntion widr oottcn wx. somehldenilg of the protein my t*e Place.Ttis lurdening is gocnlly due to shrinkageoftises, which is xccohPdied in most sses !y demsed poresity or tnsues.Tlrc mtdiai to le infiltrated hasto be kept longer in nolie sd th.n is denabb beeut tle tissuesrrc more do:e md conptt thm is tie c* in 1jfc,ud due6re it tlt eslonger lor 6uidr ro perrneatetheo. This again leadsto greater}ldoi4. k Dight lc thooght tlut rcagars wliich *ou1d kccp proteits soft would !e nost desiebl;, lur in prac*e dns is not ahv:ys so. An ddrle ofa fuid which w tcep pmtels qdite soft right iDto ws is orthochlorphenol,but Ji{ soakingti$us in this Buid ad tho bringing them i:rto nolten polyesterwu, tLe vs taks much loge( to get iDto thc proteinsrhe ifrhe ri$ueshad beo brought drongh absoluteerbool c -ylen od o a roult tlc hilnated naterial njut ashud. ftis is a *anple of o increasein sofmes but a de.rersein lorosity. And it ; lorositt vhich is inpo$3!t, not tecesdly rcfmes. The fo oniry diagrDs ilutrate b?othetical co itions bering on this Point.

l ' " " 1

F o o o l

1".""1F , : : : 1 .f f i l l , l oo o o l

Rc, r,

Hylotheticl

lob3ity

l;;;;l

ofprobins

rr tfu rcrnai blocL(:) represcntinglhe condition 6 fo! in 1ife,tlcrc is a relativcly free intcrolav offluids within the nat!i{. but tlE fuid is water vith di$olved o.terial in ir. Afre! Sxation dd dchydration most Droteinsshrirk ad exlibit dioi*hed porosiry o sirom on dr nghi, ad t]E fluids wlnd *e usedto perneate thee tissues h.vc ldgd nol&nla dd show much greater vlscosityrhur wate. They thereforc trke longer to get througt iie tissu6. This in tm leadrto a coDtilled hdd.oing of rhe ri$les dd thercroreto dihinished porosirv.

8

S.ttion Cafilg in MidB@pI

Itr the sofr block on dr lcft G) ! con&ridr is shom sinilar to that foud when orthocl'lorlheno1is lJed ldor to w* iniltrarion. or when a reagentsuch* glyool n ucd prior to n$ltation wirh gehrin or wilh uree-foinDldehyde.The conditior show a soft or swollen mrtr;x whidl hs exlmded ro : point at wlicb couiderable occluion olde porcs]u tako place.Again sucha con&tion leadsto long so:khg to aclieve adequte penct.atiodoflhe tistre3invoived. To all us$ of tie hicroscope, ofwhatever qpc in biology the mosr imlortet factor in the preparatior of scclos is tl,t of rie uint*of "a"q*t" po.o'ity ihe rorosny olproens is afccrcd 6rst by the frutive od tho by the dehydration Suids, ud in soft .ss &erc is little ihar the microionist @ do ro codiq the 3trcct of dre nvdve. Cert in drtenedi. or clearing agdE alpcxr to a$ist borh in keping dr tissue so& rd also in mrinr.ining rheh nr a lorou condirion, dd of tl$e cedrsood on, ethylde glycol noboethyl ethc, ud notudrlornothlhol, are Produdion dd n.iotddce of ldequte lolosity of !,:ed tissua is gencnily a matter of.ccur$c rioing, s vel a ofcorlect 6x.tion. Thu carskin [ed, dehydmled ud scaionedwitlin oneweek may le nade to produe exccller rcrioas ni lolysrer wx: dre soe skin afto keepingdoiier w€ek iD zo% alcohol nay prove di6cul! that this di$ermce n due ro contirrcd contractionof the {seep. rrj). It i suggested protein nDtrix with corresloDdingdi@nrudoDin rore $ze. wlatever the irfiltratiol rnednnnusedihc rroduction olFctiou cont iEing ldg. anomts ofprotein naLes corst timiDg of,ll the ?loceses m eseutial to rwcss, sd elpcri@ce iho* &at liis is particduly inrortut with thosepiecs of ruterial which containr high proportion ofwhite fibrou tissue.Th*e i a poiet in time rhere suh nat*ials will hiltrate ud scaion rcadily; once this point hs led pdsed imilar conditioDsnay not occu agliD. A factor of ;nponaDo itr Imnuliu md .viu work;r the toperatue in li6 of the oinal Thc fluctuariom of tmpoatwc to which tissuestaken fron qmbloodedainals arc exposedarc consid$edin the chapterotr isothelDic nelhods. The er1ot to whch oy given Diao add or combiMtion of mino aci&, or dy givd proiein conpld Day be rcvealedin *sue sectionsis goeully nore a oatter off,xation tho ofinliltration. but how long this will c@tinuc is a natter ofspeculetior. Wnh paratu wx, rdatively in*t, tLeren probably littlc or no ef,ect on srch clase of n*erial, but widr thc rcw nfltration media sucha lolyethylere glyols, polyethllene glycol monostearate4d dist€rates; potypropylene ud theit etcs; dd othes, the e*ent to which iee hydrcr"yl grofps arJ ready solubility in water win dissolveor nodifi grotcin @tedlt is e naner for future obseNatiob dd F-ars Brcaue they melt md &solve fats tLe ue ofheat md ofwx solventslrevents hor wx iniltration procesa lehg usedto display fat in tisEs except in rle cse ot polyetl'ylme g\-coL. For rhis rson cold, water+oluble oedia a.e leesdy if comllete su(es is to be ach;ved. Tlt ncthods comouly employed to produe sectiod showingfafty natcrial re thc *eezing md the gelatiqin€ltration te.bniqu6.

Catstittd^ oftheLiui f Oryann"

s .ewe d"ir pu-oosc,or,inb v. but rl"1 have$e dlad.uuge drar Ther _m"dod. o r e d F s e . d o has , Fa r r h c ym u { n a t r b c - I o u e d r c d ^ . f d . } o u l d t h l 6 k e placethe fat vould difilsc out ofirs loc.rion. The s.ne wealn$s is tomd iD dE uree-fomaldehydetechniqle. Fa!t,. hateliah shov r wide variatiotrin codpositioL dd sone wtich $e conju_ g.ted NiL\ oder chemicalgrouls, $rh a ccphalindd lecirhin, my shoq a gretter rcsistdce to solution in fat so1vd6. This lesistuce hay be jncre{ed !y thc se of appropriateregots suchas o$nic acid so rlut sone .onjugated fats nay be taker into hot rc batlu without beiry di$olved away. All meodiied csre$,howcvcr, suchasgll.ernl moDoste{are,disrcdate,rrisrear.rq palmitic .cid srers, oleats, dd nired glycryl cstcn shouldbe intttaled wirlr co1d,Mter-sotul,lc ncdia b@uc of thcir ready shlility in hot wq6. The solubilitiesof difi-erenrfitry nateriaL iequne close(iovorig.lion wiii resdd to dle cfecr of Edr@d tenpddurc of i!fltraiiotr. ror itutdc; so@ conjusated f i) ratcrLl v tuq wolld d.o .e r ,o. C - , comon mejd,g pobt ori,;frn lvd - night well .cmdn f rtr, at 38. C vh& in0Lrated wiih polysre! wd. lv,bih a r"6 a'r"( Le in LheLiqrd .obdrob ro be of 6r ro LbeirliviDgps
CruR3 ITARDNXSS OT WAXXS AND OI INFILTR-TTXD TISST.IES SlrrING, H-$trrNe

iM H.DMss oF W.a!

Wlo nolto w* is pouredinto a nould wHch lus a tcnpaaturc below dre mclting point ofdr wu it irt setsor congeals,ud theDir hafdens.At rhe samerimc it also Tlc comntcncyofthe cold rv* bloc.krenoved frorn the nould will vary *cording to the tenpenturc ofthe plue of corgellation rd hadoing; the age of the block: drc neltirg point of tk wax md thc hcat of the w*, relnive to its melling foht, wliar it vs porued.Thu wu, sudl astarafrn Na: M.P. J2, poured at a tempmture S closeto in meltjng point, s i2.J' C, vordd begin to crystafize aLnos!a i! ws 'being poucd. TLis nethod producs a block so cysa11irc nd sofull ola; incccpirgs, that it *ould liave to be r*ne1ted. It oay be uid therefor that oy w* should b - " " e d . . - p o r . , . o m " , - r o , b o , e i r " m e l r g o o i n tb e f o , ei , . " p o . . r e di f l o r The time which a block takesto corgeal or to set will vry with the surounding tehpdatue. ln a refrigdator the siting ri te nuch fater tho in a laboratory widr a tenperatue of aroud z:'C, md in goeral the fastet tle settirg the nore even,-' tire co ition of dre wax throtghort the block, od the sn:ll* dre cystal. With host \ds seftingrakB !b.e within someminDtes,de?dding on the roon tenpran u e ,b r r e h , o e u r go f : n * m r y u k e m n . h o : ^ . o , e r e nd , i r . Tfte hdddins ,p!@s to be sonething rvnich is dependat upon de htnding up of de intercrystalhr bo s as weli d tle intrtrcrystuIinetrmework, nd in sone caiesit (lmot be hnried. wiii polyesterw eo/ro (p. rz) a !1o& pourcd into a nould ei a roon renpemrlre ofzz' C ad storedat that tmperature is not suita!1e for sectionsbelow 6 r rutil dore thdr 24 hoft h"d elapsed.A siniiar block plaed ia : refigrator imediately on posins .rped io &qfte gratet hardncx, so that in 2a hous it vi give sectios at 6 p, but otr ihtwirg olr to room temler.tue it sho$ a soires gratu tho that of! blocL ke?t at room tempdature Fon rhe mondt i! v$ ponred. It i 6smed in su.h a cde tnat t]1e cold conditioro of a ftfigehtor lrevdt the esdtial htercystallioe lond movemt of the ws dd in this way slov dom the hudening proces iotead ofrceler.tins it. The aligmdt ,t'fcrystals witbnr the llock. od the building up ofiotercqntaline bond5is sonedring !,r'hic,lr ruy t Ic sone d.ys ed vlich cmot alwala be acreleratedby placing a oerv\ poucd bloc.kinto coid or freezingsurouduss. The abovepoin* re raned o aa indiotion that thereis a o.tain ninimun tinc wJrich nmt elapsebelore uy rm blocL en rcach is ae hddne$. Tli, n tut contary to the laoro lat that we plaed in a relrigeaior b€comehrder thd

Fr,. r.

Modcm C

$ridge ro.king lnurtome

Ilddnessaf tv$s Md oj Wlbdhd TBstc' the sue w*es kept at rooo tenperature (:o' C), but it n cleu rlat the Lr! 6* appJis minly to t)rue waxeswhich h:ve alreadr bccone set and h{d. The rneouenor ofrla }rdnes is goerally nade by uing a ?ieciion pmeto-. meter, but tbe folloviDs device &d ay the witer fo; t- !* i' -.* a*ay relatedto the rcquiremens ofthe niaotonist. Thc nodd.n Cmrblidge Rocking nicotome is s shonn in fgue :. Thc upper arn brings the block put rle nicotone klife ad it is pulled douwed5 by a spring. Tte spriig is prevoted fron pulting the an of,irs pivor by a cord wlich is aLo !$d for raisirg tie ao prepuatory to cuttilg uolher secrion. The diagm shom the prircipb;tiir, nor-dre det x.

!rc.3.

?.incilal fatl6

olthc roc|ins rrn

By reoovirg u\e,prirg ard ,ub\dr dng a co'Lrirrr for w$-r befiod ofmesurDg dF reJtarre to .uking of sy pieceof ri$ueor \ize of bjo.[ is obuired. Ttn alt*tion nay o y be nade od a very old mi.rotone. The new nodet shoM above

i t t Ir::;.r:)l rn

re?lr.cd by : @nbin$

oa q fti

The hodified hnrctone llaced upon a tdeworl latle my be sed ro e@im . thc resto,e olrhe blockto ru dng. ll.e nidorone !o. Li,g rm ; broughrdom ulii rle blo(k LJ6! rouchbg& loife edge.b) p-.i"s dr .ig\t ,.""";"".,r. uro dF tu. The arm i\ !he1.oDidsed ro be in a sare of blljle. rbe Dininh weight or volwe ofw.ter thea ad&d to the car sufrciot ro ot a sectio from de ftl frce of the block give o indicarion i! gIm$ of rhe hdness of thc oaterial

Sdtiot Cxtt;t1g in Mi.tolbtl

The plocedureis asfolows: r. Baiancethe rocker arn by adding sufrcimt w:ter to thc cu. 2. Fa.e otr thc Uock by hod luling ofrie.ord which supends the co. 3. Add suficiet water to the @ to bring rhe blo.t ldt rhe lnife edge- that is 4. Frod the hesulidg cylinder rlled for pouing w!te. into the o read o]f tne rnomt ofwaer used.This figtue relrcsmrs rhe hrdrress ofthe wax. J. Re?en ltep four six rime od take the nen reading. In all ca€sa stadard blocL with a fae of ro x ro hn. n ued, and every po$ible ellon is ma& to obtain r lnife vith an edgc of sinila sharpres. Vhile this latter !cqien)61 r. dfi.1, n nvy bc oo:ged ro clo.cLn-. The follodlg 6gure show rlb reighr .equired to clr a block with . 6ce of ro x io nm. withon d nrctudcdspecino.

Weighr'cqui..d ro. L. I ro I secr.on fiom : bloq q h r frc. ro / .o onr,

!rB6n wax, M.P. 5:" c Polyesb wax 9oho nt?. 33qc r,loo lolyethtlde glycol M-P. r2o C

It is olio to the edvmage olthe microtonist md hicologisr to hrdeu iniltndon 'u6, od, if posible, to huden tlen without inde.sing rieir neking poini. This objct nay be attaircd ody to a linited degre by .dding eitler huder w:xes, *hich genenlly have a high mdting point, or by addiog morphous naterial such * a resin.The addition ofdr fotuer goqally leiscsrhe belring point dd the eddirion of the latter uurlly incrcces the viscosityof rhe ni*ee In practice,drrefore, it ir no eay rutter sulstdni.ily to uise the hardm$ ofd iiift€rion me&uh without radicatlyalteriug rcme other desnalle proputy suchaswter tolerd.e, ei{osiry or The e$ect of thc additive on rhe hardngs oftlc meior comtituut demds on the physicJ.oD.riturionofrle l-dtr. s n "Loq in the iouowirg rrote: Abiil w* S b a poly&ide of strong crystallinepropertics.Thc lddirion of th; bigh Delting point, wax-likc matdial to 1ov-neltirg point wqes prodes a mi\ture of . higher neltiis point tlu the min coturitumt. It hd ben added to lorh ster w* ud polyoter wu md the icroe in the nclting point of both is cltuly sed. The c€'ector dre lldnos of thee two wes, however, is sfuht, ud ir the estcrwu figurs the hardnes is shown to be lover dh the hrd!6s of thc rlre *ter w*. Ad yet the hcdoe$ of Abril wd S is 4oJ

Hdtdnets oJwdw r t oJWlbakA T$n s ItE.Ecd

of'dditiv6

on iD6ltl1tio!

was

wcight Eq&ed to @t. !o / rc.tio! 6om a blo.k 6@ ro x rc Im,

M,?.

43

) ) ) 33

)

i ) * conlated witl rz: for esterwu. Whd Abril \E S is addedto lolyester ru littlc altcrrtion ir har&es is producedThe ditrerentelTectsof Abril wd S on dE hrdnds of &e two 1d6 cired is du. to the gsm6al alifserd.esbenveenthe two vds. Esld .IA behavs * :n morphous matenal.It n ldledly $dd!.et dd b.eaLi with a con hoidd fra.trre. The addiriob to it ofi snongly dystalline natenal Abril vd S, ba rwo efi:ects:(r) itredrG the of etrr lfu, k) ir provid4'sLp. moahob.bd,nd r. The teduior ofthe glasy property ofeto w.axmed tbar !r any ?oinr otr thc lnife edgethe dount ofGisbnce otrsed ly rhe nirdlre is l* tlar that ofered by the pm i8 b
r4

Sd.',ottCut tgtf Mi.roscot

v'hen the wax is so oorPhoui dut the a@ rcstiDgon the bevelsat rny given tim is in closerd alnost cornplcrecontactwirh the5esu;4c6. Tte latta ofti*i possibititis appliesto 6ter ws, bur vith the addirion ofa crlit ljle turerial, ws though it is harder, the aea of dr wax in cl$e coftact vith rhe beve! is redrced tlerebv IowediS lhe eidid, ud aiso tir @tritrg wejghr. The lnife dip. rhrougl 6ier. lvirh rclls'er id rhe ldddoD oi Abrd ;d s meu thar ro , b"cc atr*dy q\ulline is addeddo'Ift wa ofequrl cry,uliiry. The rc,ulr is rhar tinje ditrcroce is nr& to the cuttirg p-openis ofpolyoto w*. The mri! difro@e lio iq rle itrdee in Eeltiog point tcn?darure, It will be sem fron the two e]ldpl6 givd abover!3r rhe .ddirion of a dysrJline wu to o morphous type w od also to a crystalline rype, ha ody a hilor influoce on the hudrss of the mjor.on$itudt wlen anorphou materialsc addedto crystallim w** drey &! d cil tal adhesiv$,if thse is good cooparibiliry b*wem the two substaacesThus vhcn dre r6ia Nfli11ile is addedro ran1In lw (Cry. 194'7tlcrc i, o indeoe in the hrdiss oftlc u nixue i. comp*cd sith pan6D qd Jobe. The +tru ro whicl r-heboeaed vl.ori.y ofrhe nixtue nerits the inclsion ofde rsin in either this mkrue or in rhe eter rw mixtue (Sreednai, r9a7) k a natt* vhich requirs sone cosideratior because it mm that the dse nut le kept lorga in such ni*ue. long* tuilaation mu hudo tisues, but if the *terded innLntion ledod is mt *esii.e hole my bc gained by tougLeningaad lardaring the otutue in rh; vay, rhd is lost by a slight iuease io lardaessofde tissue The ee'e.rsofadding a variety ofsubstd.o to paa6n ws ro iturese irs h.ldrs tle nelting !oi* havebeo mined by w*eund G939). vido!ftaiing RiFERI.NCES GMY,P.,194t,U.S.Patent2,26?,rJr. s!:Mld! H. F., Qdr.J. Mt'. Sri, 88, r23. 'W^:r]l]JitJ\N, A. C., r9j9, ,srdif Tehn.,14, ss42. HrDNlss oF l\rrlr!-aro TusB , '"6om *su or orgm suchs bone, t€t!, calciEedchitir, etc-, ue tretuelly [dd. othes such* ycllow elasticf,bre io bulk, tirrrdd colloi4 albeerr, white f,hour risu. etc.. acqunehrdDe$ I a r€rulr or|ihe pro66 to wbicb dey dc subjecEd beforebcingtua1iy produed ir block fom rady for cunbs ino scci6. Such tisue may be so hard thrt rheir cutting is a sdos ploblem for the niaoronist. This chaper dealsody widr rquned ladness, aad i* nuumot. rh:r tlel I lr6b dsu6 re loo:oft to cu! ilro llD sl,c6 dd rhqefore it is md:l $ould be hrdded. ldeally dei! hudffi i! w*,hould be l-hesee $ rhr of the \&ax for they tho rquire 6e dtting clDracter;tic of$at particuh nedim. If they de $frer thd rhe he&h they drow: rodmcy m mllapse;ifthey a* !{do &ey do not re@ive srfi.ient sdlport, dd @y le ddaged dui4 ottirg o a tsrlt. The foms m&tion tuy lead to pinohion dntortion otthe sectiomi thc latter to bmel
rj

Hdtdn's oI wdfts dndof Wlrakd Tke$

is softerrhJnLhebSlmdon medim n ir beoN ir ii is{riepr-ly poerratcdrifit I brder it is dE ro cherdcalud phy'icalre"ctionswhicb hJW uno PLre fiob The asesncrt of in6ltuted *suer s being [dd o! soft is goenlly oade at the tim of enine. lho reh muchof tle hr&es ruv h:ve bm rq md d uilq 6stioo, ruy beio 'Ig"r Lh,rLnle rontoa i. feL s d.brd-don.;d cl;i"s. rhe b-G, that th. Ldfe givesa linging note 3 the bloc[-ii !dii! &l oiit my be so *roe it is sto& by the sle.imo Amate nourenem of the hardacs of inilnated tisse may be nade only who the hardaes ofthe bflnation ncdiw is alsolaom Detaik of the ne$urenet of dr hardms of n*es lave aLsdy btrn siven in the ^ Thc exted to vhich the hddocs ofthe slecinen oy *eed tlut ofthe hf,lnatior nedim n shom below in the detailsrelating to ox skir ud parafin w*. ox skin is siven asan exdple bcceuc it n difidrlt hatdal to cut

Sp*ims

vdght

The h*daes id* is a ne*ue which showstie hudms of the specirea relative to dE infiaalior @dih. The great* hardnes ofthe skin f,xed in macury-formol * conpued with the sLin 6xed id Bouh selvesto ieinfore in fgures what many derrutologisx alreadyhow by praaice. soth pie6 of skin vere cut 6om the nain pidc dd were adjac€Dt.Soth were trsted identicaly, tpd-t &on the wuhing out of lhc 6&tive. Bodr wele ci@ed in It n freqlendy said that ceitain ililtration hedia such .s Poly6t* n* vitti its low nelting point wi kep tisus in a softer conditior tho a wu sucLa puatrn wth a Hgh nelting point. This is quite fiue, dd my be redily groved but it is not alwaysso, d the 6gurs for ox skin hI1[ated h thre diftierot w:xes show. Ox skin, Bouin, pdatr! Ox skin, BouiD, estervd Ox sHn,Soui4 lolydtcr wd

IgJo gm. 2175gtu. r9mgn.

The llocb were of the stddad sie, fac ro x ro im., ajrd tle skirl stretched right rros h a[ d6. The weQLs *e so closeto one torher db! the dif,ercn B

16

Se.riotlC"uing ib Midoyopf

, ruy be rlnoa dnrrgddedbea6e rhel m j beiduflced bv r.hcsbamne.or blur_ otd,F knfe. Jr is nor nnpLied,bowe,rr, dDi,ll d1ebroclspr6iice ,cc;ors of /be$ V equ:1gu:In, In.fac rhe loysr rehbg poitr 1A produ.edLhcLd srctioB. wlx h r.tre abole bg.E6 is rb-! wiri o.diDN firtioo, dehydntioaud -halered bJltr.hob rctho& r orgu !.h $ or rki! vil !c*b , cen jr bjrdfts which n Lile inguo.rd by rhe in6lo,r'on nedlo ued. Thi, i, du" Loib conporidonof appr,oshatel)90% vhir fbrc6 ris,ue.whiJ b/ ordb,* merhod-reac]e! r ,rru4 degreeor rougn s Doturrr sbr wr h ,,{d. Itse rcmls do mi rprly ro r mrs J J{b s Ljj!5 u tu.h r"to in,o ' , ?arrEj be.orD6 hard,nd sonerines dificult, but tJ
T.s. Annococte hadnds (Bouir) !.trsts

T.S, I/itoa

hrdne$

(Boui,, dcct.i6ed,

w.r

dfu

@)

tn geher.l jt m)' bc s.id that nost tisues ud orgdu show redrced hardft$ who iDnhared wirh !o\'este. e.a:e od thar ly appropriaa mcthods den lough tissles such s nemalid skin rslohd lril !o 1ov iililrrarior tcnp...tuo. Fo. *sues ds cdtilege which re nnrally tm or lud, rhe nuch strongca harder ester 7uh r'lro shouid be $ed. parafin ws hs 1itt1eto ot{er when hddeniG ortisues ody is l1nde!coBidqttion. H,rdns Egures alone give no guide ro thc alleaEnce of de section,bnt the nd{ rie hrdnBs hd* is to r the bcnrr is &e ;;ion tiLcly to te. The fisues gilen so fd for hardrs measuenent ofwxes ad oftissres ue eiven as a result of thousods of neauencnts over a period of muy yeaa. The! ue gih _aftercareful *sssEmt of rie stafn$s of tlie InG eagc. ih.l"ner n$t bc judged with Srearcdc $ the folovins hardnss neauremen* show: Ox skin, pdafin wa Sharplaife r,J4o gm. Ayeragelnife r,7ro gm. Bl@t }aile 2,7aa gm. Such 6gur6 indicate rh.t grcs erro$ of brdrcs mesu@or Eay !e nade lllles great cde is raker to seethet all msurenents ae nade with hives ofcors!_

HdtdrelsoI wee' nd oJrnjbakd Tis,a$

ry The speed.dd*td of hife edsedeEio,adon who " roush nJ@itj (uc! $ ox skin i5 bejng ar n rhoM in rlr folowiDg ribte.

Ox sLiL

3ouin,

Polyestc. v*

Sedio6 dt at rr.. Block &.e ro x ro ]m. Specinenro x 4 nrn. Room lem!@ture ro. C

Tle 6g!le showthat in conpdison wirh r sruddd Didotone knife a razor bta& * shdpa ad lsrs loDgd. The rhiclas ofthe section- rJ a _ pernjrs a cosidehbte nMbs,ro be dr viu bolb knivei At s " oDl) reao r"rjo6 rry b" ob,rined. deuilsgr@ abde arc no' "pplic,brero orgo: s([ u r.id.!g,rd be+. .T5e rbeii FLljve iofllc$ in de bro.[. D4kesror .^i";n j,g _d . i";;. .,_r"f",,h. lnife edge. It will bc cla fron the abovedata ttat razor btadehotderssavcrhe microtoniir much lebou dd time, dd if ued with rhe right wax suchaspolyeter _ not estet_ elce1tdt secnos dav be c[t.

L]ltrJ]@4

TH! EFFECTS OF INFILTRATION

MIDIA

ON TISSUES

Th* effec* my be divided ioto two groups; thosewhich h{uercc the voloe or shapeof the tissues,od thosewlicL tu{uoce the chmical content or reactios. The ef"-cs on dsu* oflong pqiods of tioe sp@t on dehydration,cledng and infilnation andatively high tmpeetures, conpued with grady reducedtiru aad temperarues,m shom in 69. 5. Botl photograplr show a et testis@t tlansveBely. Tbe 6nt specimo ws fxd iD Souio's fluid, nd taLd itrto pd.16n w.x, M.P. J2, drough absoluteaicohol od xylme in t\e uual wry. The secondshows a tesri! i! the sinultoeou fxtion od wx infiltntion nethod 6xed by picrofdnol-wd rc2. od si/ei ?age .-hs / The 6mdd"ble shriDkageof the scminifaou tubule in t-hepra6n wa proces led to dreproduaionofhge rpae. not praot in thc othersectjon. suchspa.esarc due to 6*tion md dehy&ation anifactsaswell t to the Gacraly , eFcts of d'e hGltratior -e&rn, bot de tual bath io hot wu at a ternpenture of / /:r Jf C or so it sufilient to *aggaate gro$ly the wealoese aLedy presett In the eruples sLown abovethe di.stortiooofrelative vdme in rhe gara$r w* mtsial is quire .lear, dd a suchtepreens a condition fu from dat wlich exiss ;n liG. Neverdeles tle ituge which the histologist see tluough tie nico*ope frequeldy ows its bdghhess to srch conft&tio$. Thu any givo section clt iD pamfin wax at s p will look brighter md cleder thr the sane naterial infdrited with d aquos nedtuo such as geladn, cut a J /. slainedin the sme r:y and ,rnomed in a aqueou oouthg medrdm.TLeo,.ti.aly ud ir pmric Ge lacer la.do!,hould bc Lhebetk!. I! wil celuinly .how h* $rinlase. ad 16 drroftjn vLhu thc p*dIu wu,*tion bu:ooero" i, wil Dq loot !o brig\r. Thse wil he . djsappointinglacL ofs,\.rpn6s .!out it. This is Dot so nuch due to my dillerene of reiactive indq letwer the .qu"u momthg medim od the !"lsan or pol1xrrse mostant; it is due to the Lck of 'o"ll or l*ge ait spres in the tise. vTse contnaiou uule ti:$* no!q44r. A, rhe.se rhi.Lr6s a rrffin sectioo ,mI hrvc more.orp,cted ti5,u6p!6e!r ,hi r gcladrcs.cioL The dtrPernrirg e/ofthe den$! tisre n accenftat d by tlE d@-.!t dnpty spae, od the eult is a brigtud picture ascomlred vith the gelatinesecflon. u rcitncdqn, the $fis tice ilqrrore $e sogolbs tle nisT. rd Lhe ! ?8, lss Rrll bf dre boL[.l:ne5 \ePdatug ode r]?e oi dalcnal b rfse wil be a dis'inirhed cooEastin the sta;led section.Ir ir sbongly remiDisdf o{ e photograthic legative fron which t*o prints are nade, oc on a lnd paper,the 1 For drtsc raoo the nore otual becorc the s*tion the nore likely it is to !e rnther dull util app.opriatemcdrodsof stairitg ne prod@d

ric. J. PriFh

$rr.fi.6

on [lii

rhoLttrn..us irnlu !shr

1 wrr ndlhli!'r

ede.s sho{r on

Tte Efhb of wt%tiof

Medr| I

7:8n4

re

With elect|on niaoscole sectioascrt ir retlacrylate thcre agpeanevery aow md tben a gap or so in thc n.tsial which st ongly suggeB . niqutc o.plosion Iavirg cceed duing pobo*izatio The fore mrted by thoe exploio de no: appu to have lm msued. Certaidy ouch work hs beo done on this probbm fy Borpko (1955),dd GetB1er,M. 8., dd Ostei4 L. G9i6), .nd the chenical possibilitie }ave beer very tLoroughly e*oicd- Aa er?loiion can oDly mr howevc when the e"1lodw to!@ is gre$e! rlan the form olthe surounding naterial. lf the surroudilg @telial excrts a llsurc equal to thc proure of the pototirl explosior ttcre n ro explosion.Ifj! tact polye.iziog 6i!s re lobtq;ed uder a prcsuc equl to or gretcr tho th* ofthe ninute e"plosion lore tho this toublc would diapper. It n a uner which niglrt well tepay f"rthe. study. RIFERENCE Bonysrc,E., i9ji,J.,4/rl. Ptr., 26. r394. cerlft!, M. E., dd ON:IN, L.,19s6, Phfi.dl t chniqa in biolagbdlt sdrch. Ed, Ostr, c., md Polister,A. W.,

Ctr PTER J "RYSTAL SZt. , ,lWitHr the dncknc* of ny se.tion ofwa\ or wd-lile materialtherelio a runber ofcrystals. Thc amber will delend rpon dle sizeof tle crystal dd the thi.ln$s of tle section.Thu betweentle two surfaes of a sction cnt ar io a there wlll be liw crystllsofeste!vd 1947;moreofpmfir wa5 od mostoflolyesterw*. The 5i@ of ttre crystal, it! shale, its hrdness, od thc dcgree ofintercrptallirc adheion all have a ditect efect on the lloduclion ofgood secrioruod ribboro, ud - . * ^ . 1 , . 1 " ^ ad - . " ; ^ - . An allre.iation ottlese fa.ts ud their pnctical applicatior nay leadto a dearer of rheproblcn o cLrling3Ddrouins -d1on5. udc.udns Exaople: A roon with a tenlerarure of r8' C, and tue blocks, one ol ester wd M.P. 48, one ofpalafir wu M.P. 12,ud polyeter wd M-P. 38" C. civs that all thc blocls h.ee beo.rkelt rt th.t room temperatse for tlte or fou! da)'sto setdetruly into tieir natual crystallhe condirion, it vl11 be loud 6rt uing the sme part of thc knift ud cuttitrg scctionsat ro I lhe esterwd vill cut into xlmost 6at settions;therc will be sone crrlirg ofthc !ra$n w*, rd the polyeter vu will go into cigu-Jike cylindes. Tle knifc is a sharpone. If the sectio! thickre$ is now dropped to 3 p it will be foud alat all tlree w*es will .rt into flat sccrions, thorgh ture rill be considerablecomprssion iD loth the parafin rvs ard tLc At tle cutting cdgeofthe microtome knife displ.lclqot dd defo.n.don ofrhc vd cystals leadsto curvaturc of the sc*ioq rvith the longer radius ncxt ro thc kaili od the shortcr on thc outer fice ofdre scction.This shorter outer facc shorqs a duli surfae bccausemovorent of the crystds thic hs d$tioyed the origiml snootlnes. The nce crystaL thae ue in tie thictncs of the secdonthe grcarer the likelihood of crrsal novebent dd derelore of sectioncftverfte - unle$ 6e ' strong intcrcrystallinebohds.T]ns n .i;d' ne lomd togedEr wiii eicltiomly true cvenwith . shup lnG, thoqh a bltrlt hrG edgcrvill exaggerateit. In e liick sectionsucha in one cut .t ro p the force empJoyedto cut the sectionviU nove the c4 stalse*ily upon one mother dd a dtled sectionis rrodued. Sucha section biiniajnLsits ctrnatue becauseno conespondingforre cu be cdlio]:ed ro prc$ the crysraisba&againtuoth&pieviou.ligmdt. Duins&e.$tinrg 9f athinseclioa,/ bowever,dx iorceof gr.viry,'u]Ii ierr ro m-i<etie ruvirg
is a !!qlr. rhb L ducto fc" G,.,r. L,i"s h- "[';o; li- --. *.,,r,. -a -

ililiver condtoro r]le interctshnine adh;ion is {eaker n\d the io!;e of s.viiy. $ax sectiotr there6re, dd h compuable conaitions,it; a truism rhat I! oy / V the Gwd rhe cryrtxls thi da$erlhe rction.

Cnstdl Size I ,l In artain wdn conditioB sd hay give flattcr sectios thd rhe saoc wq in cold .onditioru, lut xt rhe sahe rld&E$. rus n bec.se thc intercl,s*lliae bonds {r rlacd. Nd t r d {,I $er"elvcs ruy "bow pra:J m-goe wit}roneuotha. tlrs producing Gver crysrals. v./ith ncdi. ofsb!11.rysrrl size,suh as 4oo polyedryleneglycol disteaatc, uy noublesne rolirg wlich mry o.c!! ar thickress od ar r rooi remlentule vhde such irouble do not occur in para$n wG, may be rmedied by cuttils thimer r.tiotu. Slch snall crystalsir hedia wiu reslond !o u;ng a knife s verti.al s po$ible dd aLo to qing a knjfe with a shal inrer+evel ogle, sayromd zeu5.. In the p6t mrdl cfiort h$ bm arpe ed to rcdue th siz oftLe pa:fir w* crystal.It is trle thrt iD som cuesirs ayralliniry ruy prove to be emlu*sing, but ils gener.i ede of ndipllatior is oainly due to its crystal sizc ud shape.

CE $rxR 6 .!'ISCOSITY

AND AGITATION

' The intcmal friaion" due to noleculr cohesion,in fluids, which inpdts to lhose i 6:id. $eu vs.o,1, is ! daHd of sore idpotutr 'o lhc djcroroni.r rd the Y bnrolosiy.be.,-.e rlc rateofmar oL€uid. .uch$ ,ir"ti\e Dd iiilrr"rioD nedrr it8uwces dre appermce oftie tissuewhetr i! is fituly studicdhdci dr nic(oscopc lh" 'arc ofperd.doo ofsu.o dud, norm.ily van"r bve""c y sidr dr" \iueof;r nobcde od the viscosity,tholgh this is subjcctto side eFecbdue ro the ch@iol costitution ofthe rcasa1t.It is a ga*a1rrJe, wir\ a nunbu olexceptiom however, that d increde in lhe teDleratutc offluids redues the viscosity. The mic(otomist who is in6lbating tisues with waxes or resir is placedin rhc Jficul oo,jrionth,r Dmd\ cae.her lo .ed,o u:cwd* "r rel"d,erl Liehteopa* / t u r e "i n o , d c rr o , e d - . e r e v i , - o s i r o y l:t e rquid$u. ftso .-r.,,;., ";d-,/ * s.ue' ud lcdur- du perm."biliry. \ rdi, iolr J,..'deni Drr .be'rfo( bc mJd; o]-rffia l)lfli;r,hf_;i-(Mioi;r,ou, n1ikr"'io" n.d," r, , olju!Lion wi!]r the e$ec* oftheir bfiltratioD tehoeraruesAt tle lower *d of 6e vncosity oer.*oots l;s pa€6n vd which witf a melting point of ahour 49-jo" C IB a viscosityoa apprcximtely rc mtistoke ar ,j' C. At dr !p!er od of dre sale ue sone of the eport, rcsin5vhich ar loon tenler.tqe (ro' C) hesurc betwed 2,@o .nd 3,ooocdriroLo. The folowing ir r list of.pproxinate cartistokevalu* of sone olthe more widety lsed iniltration hedia. Ir is approdmate becaue few ofthe tega* re chmically pure substmces,ad vuiation js alwayafomd fron one baich ro ano6er.

Pae6! vN, M.P. l2'C Die$ylde glycol direete

Polyethyld glycol r,roo UI lirure rc.2o.b ( rb or)

The above frguresshov very clerly tle advuoge *hich rra6n wf ba over other inflh,tion media we.e vncosity lhe only criterion. lsrer wd, 1960,is clos

v^casiry8d ,kit4tiof

23

to tus viscosit)',a is polyesterwu, Nhich, widr ie lower helting point hs adl"antegesover aI othef molto media The rq+z estr vax lornulation shovs 60 cen*toLe, becrue of tLe prcrcrce of et!1'l celhlose, which, though toughodng r\e mcs, is,d@Fs tlre vnrcsitv. / tn uy w* or wx-1lke midal iie presdce ofhydroaTl g.outs inlarts incresed hn.ositr asis sccnin the igures for the steric acid estersof gly@lol dd dierhyletre g1)rcol.For tlis rc6on it is u advetage in dy deliberatelonulatioa ofan bllltrat i o o n e d i . r ' ,r o - \ o d . s r w i r \ b y J r o r l l g r o u p , .A F . o n d d d n o r e i m D o r @ r ,e-ob i. -l"r -n- b-6enr of$6c SrouD.ilwaysDru .hd LIreFisJ grtuLerrmd€rcv for the wf to &solvc or dirycsc in w.ter - a featlre ri'hich nay produce gret drlficulty ritl the lLnennrg dd adhsioa ofsectiob. The prddc of trydroxyl grcups in r1rcpolltthyloe glycoL is reflectedin the relativelyhigh ignre of roo cotistokcs for polyethl'leneglycol r,5o. The lov vis.ositl of cqshlloids rs comrucd wi colloids is shown in the 1at trvo figucs quoted in tle tab1e.Poiyeihylencglycol r,joo showsa mrked increoc itr viscosityNIrd it lDs rea fonnaldelyde resir addcdto it. The conparnon indicates tnc viscosiry diliamc* bet ren ws-like rnd rcsinousmedia. This difierocc is cleafh sLo$n by the erot reslnhesunndq vhcn it is conpued with uy w*. Calcul.tions of the reladye perEtmtior ratcs of vdios i,6ltr*ion nedia bxing thosc olculaiiod on viscosityneourenents alonc would bc subjcct to a very wi& desrceofsror becnse ihc cheDicalpropcfticsofrhe ncdia alo lbve a direct buing oh the ldetration rate. Neverdrelcs thc vncosiry figuc, of lhe @rerials sdecred indicatenr a geler:1 r'ay tle relativc pectration rares. AcrranoN ofnrtcrnJ Thc intcrchmge ud cxtenul duids which is m e$entialfstue oftisle infiltratior lor Sc frodtrction of tisue sectionsis a lroce$ which is frequddy cdned o$ in r r.thci lenorcly omer. It is oftd cosidered mificiot to pJae ir a beaker or snnih containu of nroltd wd tle speciDehsfor infit ation; to pl.@ dr leaker in o ovo and to lc.ve it mtil the apprcliiate time h,s elapsed. Thc idle ebb md florv of:noltcr wx throngh a satic object seh * pieceofti$ue at the botton ofa bcakc! is r nethod ofbnftratior shich haslinle to recomend / i . l t t * e r u h ' o r g . r J , ' i . . l o u i dd d i t h * d . ! b . u 6 . No photographer of oy tcctuical uderstadng ailows his lrinrs to 1ie ia a epillary-atirocted mss at thc botton of a still-vats tdl. Yet naly histoiogists, railolognts, ud qtologisis neglet to cnlloy sftrers or asitation ncthod' at any time in the Nstory ofa liccc of tisnc from iption to the 1Bt mitute ofinfdtration. This is ar ovcnight which brings dr femlties of djfr.u1t microtony od biltration anifrcts. The time of iniltratloq !)- iny liquid, whcrher tf,ativc or nohen wu, ru)' be retuced by one quane. ro onc drird br lhe enployment orsgllc,ble-stidilg -iiie-bst stirrer lor lnstologicJ purposesis one which cmplols a vuiable reistaace wlich will give revolutionsSon alout ffty to tlree hudred per ninute. The speed nay be varied so ttut in oy siz ofve$el rhe lie.es oftisue nay be kept tuoirg dd;sins Dd faling everr ftw s(ondr.

SectionCrfilg if Mioa$o

24

The end ofdr stiting rod shouldnot luve u inpdler ofttr lropdlor type. This devic cd srike dd bftise the soe.imen.r[e end oflhe rod should havea disc or n, .botrt ole :nd a tultinchs ir dimdd (ad.) dd abont one quarter ofo hch (6 hn.) in thickms. The discshould not be lt nght dgles to t]re rod but should be rbout ro' ofi The edgeofthe disc shodd be dredi tlbt is it sholld Dot havetwo sndp rigk ogled edgd to .ut rhe movins tlssues. obj€.tion to stines ney com fion tiose vho cmot enploy then in the ove6 tiey ue. This is a v:1id objection md one wLich nay bc diElcult to overcone, but it enphadas the frobld ofw nedods vmus o1d.By using lov nelring poinr w*o such * po\tsto **, which nay le kept liquid on d opfl hot plate rt 38' C sti.dng deviceshay le radny ued. The following diagramofr suitablestirrer illus*ate the descriprionjustgiver

StiId

&

f.r specineN dudng

It nay be of glas, nd this nay be esotirl for sone 6rativ6, or of stainlesssrel, c polyetiyloe, o odrr plotic. It nay also bc sinply a 6 nm. gbss rod vili a Ji-Ll-

P"d-"J

^-,1 l""r

-,1

It is a de.ided advdtage to luve the stifiag discoo a telecopic srm so that it may be raisedout ofthe moltu wu nithout having !o tih the containd. Sucha devir nay be mdc iu tuee sages: r. Take r pi€ceofsteel rod about 6 x + in. DiP thc lottom eqd iqio solde. so that it beconc thicker. rapcr dre solderasshom in the diagram (r)

I' Frc. ?.

Telescopi. rrm of $iiier, wilh clutch devid

i

"

Vb.asirycndAgitation

25

*' ** dieie!lsd ir so,ds ! .':1;1I iil;:i*T: ,"i"T3'.fffi:"; I

'*''iltrf

i:"i#tfi:','T.n*i'"*l:T;:*'de'apeBwiu6r

sture( 1,:ed .,]!: .mtl trb tubeat ttu endof theroa, rn whichpositio! rhetwo tapqsd s di.lgh d(.n. tu deF i, pr,ft.aly uoBnb!* . *.."",t,g dJ;"

11F:T_.",",."##*'n_:*r'#;::;:x; ;;fi*:Hs-,"-ffi f m:,'llt:i"i:t; ::T"ff :;T".,ii" j:*ll:"r:#*"* rfrJ'""1r;:i:tr"Hi::;"1rffi ft !],"""ifi jilt"trTft,"1*" ,si::,i:,";P"y:J:"*tr,;f.,",i:Tf; Ht,n'x:"ff *^xT*r:;*i:ffHlir'"f,il:i:?:i:T::

CIAPTR 7

COST AND TRAINING Whe! t1lenicrotomiit raies tiis subjecttic rdninisnator or lusu regu& tin wirh a somewhatrvuy eye, ed inncdirtely ries a lno! i! tne luse striDgs. To equip a rhool i:boratory for sccrion d ring, evd of the nost simple khd, &equendyeotail the *penditure ofrnore noney thu du biolog7 ]Btei ir likely to reeive. Nerertfules it my be done very cheaplys tlt lollowing pi.$ sLow (to

orc beDchladp, i'fth ao vatt bulb { J Onc microtone and lnifc 40 Total

one hot llate, 24 x ro x 4 in. Oft nicrotone aDdkdfc

{aJ

{ts 40

Tor:1 {6J This ljll would be doubled or treued if it wete necessry to purchue a water iacketcdpara6r ovei, but by uing 1ow nelting Point wses suh o polyestern* (ur. 3r; c) " t.n'ed-dom benchlanp or a hot plate is adequtc for the pupos. nore lrofty qil N! Ior tecldcai co11ege,rcsearchi$tiiutioos, md eivdilis only be grmted but spmt, ud it sooe oes rnore will be spot thd is gtuted, blt em rc a revisedview ofreqdrenots on tlE Ins abovewould leavesone ofdre srut for ofier ugeftly neededitetu. T

NINC

Vhen a baby cu* hn 6rst tooth i! is . sigul for geneEl rejoiclng,but who a juior d@trl teclniciu c s his ftst toodr it nay le r signrl fot the st.flo rcconsidertheir trainhg mctho&. This i becase nidotomisrs are nade ctrrcientonly afto sone of expcrifl.e rd dabDq. \rr5 ' b L I Fp - r i " , b e r o a . o L c r h " h , p \ a " r d p t o c " . .r - ' o c m l l l r u - g i r c n b y the hsd rccluiciu ifhe h:d thc tine or rhe iocliDltion, ed on rcne rare dd plesdt occsions it vas evengivcn by the headof the deparrnot himdl who took a scat 'My boy, Nhd I was iD bcside&e neophltc ed shrted the ptocccdnrgsb1 sa,ving: Heidelberg . ..

Con ond Ttaifitg

27 Todry_thetrshg of br mirrorooi"r i, uderolo moreaeirttv, Les ruultv. sd uurxv by rehrj.b, qbo haveL\ftuii6 recejeedrrinirs. L l;* f"". souces ofiD$mcrion re olen ro lhejbior lcchaioaD. r. 3y evoing or &y+clcae dose. z. 3y inr.Dal clsss witlin tic etabhhnent in vhich lhe juior 3. By everyd.y instr!.tion fron the chiefor saior tectdciu

tecfnicid

oflds oM deDdF

4. 3y occasionalyacarioncohs. A11thse metho& Iave good poiDrs,though the . !rclortion of iqowledge acq&ed b my gven group tu) vary &om placeto plaa. Lro',b !-,l e w.-tne,. !h"r Lbel@- t;chjcio,r lbe tud or" da\\ qork i, Drobabl) jadcJMd wdy, ud lie dded bud6 ro hinorptrhrls rwo o;ev@ttnft evnDss a vek of ldditional efi'ort is one wlich ifu;t a6.ys be bode vidr olhusan or ad.Ddedvilh success. 1be day+eJe.ecou. i, buci bcffr in r] re'ped. bur bofi dc u\uJly devoed ro (uchsubjec6a bio,osy.o- nistotoq],4d ucrv b runoro. ') rlooe.A. a rautt of rhl tle ncllidr r Lheod ofr fujt vec of:veiins cqss:: o: day-r.etcaecldsesfias h.d onty . fes dap on miaotomy. croup 2 ''dnch is rD by sonE but not a urjve.sities ;d ..*"h i,rd;fti* .oeer,a qd.,ariFry oftooic\ from gtas btowinged Deot work to hnmtogyrd brocbeutry, but omerg4D.acelerr $o Jghrhese , ou"€sae Lleygiveodyi smaX per.frtageot bmerom,croromy6such. C-oup 3 rJe" .o mrch fron or dep"nnot Louotlq rlut ir nar reoresotin ane.ses !]le mN lruirful .ouce of n,,-mon, wherea:ir odrn ir mry be so bafm s ro be noo<xisrdt. croup 4 in muy lspecls hay be dte bst of a . At a vaerion coule rhe studenr tuy b!r'ej -ro day,ob (ojogicllrdlDiqq ofs hichnidoroby fols lfe @io, pomob ol lne qo*.. ln \Lc! r (oure a Ducb s ro ho4 p* day ory b. spor oo rcr' mllrEd recltriqrs so Lhr ir a fordgbt, - '"' --t i"g d").,'.* h-d."d lor. tu) be udl4d. J m od whilb i5eqLd ro or eve lohs;then Lbcrorathou3 spenton niaotomy io one ytu of eveningctssesor d.y reateJecl*ses. The studot or !-.bnicid d lhc v{acjon ctss i, -u,y Foo r-heas aad troubt6 otbode lile dd ofhi, bom. wdG,g liG. t@ p.nyjelo6iA -nd dr6culd6 whicho@jonal! b6et hq i! hn eRryddy so.J
28

S.dror CMng if MituscoPI

For th* rqsons dr vration come [c a grat deel in is favou, but .I four or thae groups,srrd though they bay be, de sti inad.$ate. Midotony !a: not pt bear giva t!.t status,recog'rition or building which ih. pr6s@ ofnod@ nicrc clez'ly thereshouldbe a depaitnelt ofmicotomy. Not asa branchofpbriology, anato-y, prthology. zoology, lut a sep*ate,indepadent depanmeft ofnicrotomy. of firll-titu It should be a uivmity dep&'tteft with a chair ud a.onpldtlt leturers od tcclnicd sta6 Eachnenbu of tle staf.&on the heeddomwards would havethe cuttins of sectios s his min interest,od the staFwould be divided into thor ero:pswbichecpenoreofniootomy sbom tobe unrJdivisioro. TLu' one mmir would 'pal$. o. plstic for eleoon nico
Cost4 dTftiiing

4 js an fore lilot. He ir not eamicg a h,lfpemy or a @t w}ile he beirg t!a;ncd, ad yet he i5h fact actuellybciDgpaid. With rLejulior nidotomist naay mmtbr elapse lefore he fecono io uy way o oset to his departnot, ud duiag that tim he is ofren re.eivitrg tlaiDing froo peoplo who {c lqscd by oth* problens, ad who .mot rhdforc dcvote aI the time they would doire, c aI rhe latirc rhey should mutd, to asit his l(:uing. The dswe. to ihe tEiDiEg lrobln thftfore lies in the c@tiol1ofa departmart ofnicotony, stafed by peolle who re inrdBred in crttiDg se*ioos; thc proqq sociated with thiii and who would t hrppy to pss on lieir lrcwledge Nor would sucha &panment in the lorg ro prove any dearetrlen the nethods ued er

PART 3: PRACTICAT DNTAILS CIIrTR

8

WAX NLOCK MAKING AND 3I,OCK TRIMMING BlocleMakisr Viti boke! wu. unng: r. L piccs. 2. P:pe! boats. 3. warch glsses. r. L piec6 rc L shapedpiee ofnctal which who plaad rogetherforn a snall box. The L pieceswry in size,ud are !!ed jr pairs. The imer surfre ofrhe L pir( nry be rmtued wi![ a piitjng,&ohucb s gt/csot ,o pr.vor $e

wr.dclinglo +qEFri. ^ "* 6'G;

i.? LI",ii..qui,a. rh. pi".. or

mrJ or glx ou wLic} fie r piec6 re placed.ho Jd aLo be smea,ed. prtic :tly in tle ca* of p*rFn *"x . p^-, r_hewu dclirg to de bxe. rb! r.pieg:\hgdtlg nosffed so $at w\cn rh. .; i. poued i" c}lgs L+$ ph( tlc P-oduci,gin rhe-blocLr l@Ia cysrai sa " _,d::drate rbe o\illirg Jso heJpsto preva, ile nol,eb ws Td_s'j1:j:-9g*q.y. snchrrg to rhe metal. :. PLr Lle L pieces roge$erro nale : bloc].app,opriatrro fie s'e ofri- sDccj_ ods. PIae fim on gl,5sor oD Eebl dd p;;, ;o,te! wr ilro ri.m sJd,r the L piecesare about ihre qu.neis iitt. 3, Wait @dl a thin sHn ofwd beginsro fom on tle iside ofrlE Eel:l L lieces.

rrc.3. L piec$rho$inglrys of rcnge.lingwd 4. Ifthere is a thin skir oa the surgce ofthe wd nelt n with a hot soamlaor bv rming ! blll1g Adc dom oi b u. Trke Ge ilfjtrated rpdl@ from i6 brrh of[quid w* 6irc 6ne iorcpi ud pDcf n dlomc.rry b rnf blodr by gsdy presDg n rgun rhe skb or wu

Wax BkLk Mak g ait Blo.h Tlihtbiig

3t

which hasfomed ncxt to the netal. It vili stay eectly whde pLced be@use the congealingwu vill hold it quite rca&ly. If the slecnao is small ue the w,tclsla! ndhod (?. 3j). Ifit is rnsile do not $e forceps,lut lift it gendy with a tiin spatula,od narceuvre it into placewith a hor needle. Crdrlly placedre hlock or i* pieceof glas into a shallowdishwith iced water, so thn tLe watet doesnot cover the block butjrst reachesro &e top ofthe L pieces.Ifpossibie either replacedrc water wl& frsL icccold water, or placcthc block in a rc$igedtor. Allow de block m s* ad to hnden Lefore attengting to remove it Irom ihe I- pieces.Ttis tine my vary fron twenty minuts to two or three hours, or evo ovemight depoding on tle sizeof the Llocl. h gaeral do not hury tL;s 8 , To free the block fron tle gl.$ or mctal bde dd lrom tlrc r piecerdrof it

brnkly on the bdch fron abo$ J m. .bove the bencl. Tli ca$c all the puh to sepd.tc.

sharyLaock wili

r. The irtuctions givo abovenay le lollowcd exactly whct *ing pualtu wx. Who using esterw* continue as {or paraltu wu but Lecp the top of the !1ock molten by usiry a hot starrla or by tmidg tle lrnscn rlnc do$n on to it for a fradion ofa second.ConliNe thb lrearmdr rt intetrah util ttre blo& is soLd.Foi polyster waxjut leavethe llock to cool md lurdcn on the bendt dry. Do not placeit j! wdd or the watcr wi11atrectrle moltd lollsrer wal Ior Ester w* ud polpster war an applietior of Mapr albr:rnento the L pieccsor to the bre on which the r decesrestis to be pretered. With a water soluble wx such * polyethylar glycol r,ooo or r,soo n*c thc block * hdicated, btt do not placeit in wrer becausethir wd is water soluble. 2. As molten wd cooL i! contracrs.Iftherefore oqlv . nillinct.e or two of.su i ! l . h en o L l e n r c D d i d o b L l J , b o . e , b " ! e " i n m . $ b @r b - \ d c o o l sr o f o r m a solid blocl the speinm nay be left alove the top of the wc. Thue6re always leare a goerous rurgin above dr specinen rc dut subsequentblock triming nay be eary. 3 . rhe pan ofa blo& to renain liguid tlelorget u m solidi$ the laqs lhg c-c-q!1e ta that region thereforethe dysral re largd dd often looso t}m rhe crystaLt ' at the oltside ofthe l,1ock.rhe ceDre wd oley block is more qstalline aad r' th*efore oflloora cuttins textue dr.! lie ouside. TLat is vhv rle .^ a;ccime fiodJ 6e llaced d;;:-tiic;[v ;ll; it.; i;i"; '1,;.d in d'. bb;k.

A-5lo-A'-i,i-L-pia-;-F;IJ;;;t;;;r;l,iJdani

ltf 16! --pt.tay

submergedin cold wata. Suchtrcatnot protuco a block of wu solid or the r offiide but stiil w.n dd liquid inside. Coohg ud contractioDintetuIy vil , ,' ' tho either spln drc oucdc skia ofe'd, or will !rcdn@ a partirl vaaum imide. The lomer rcaction wil r6u1t in ihc intakc of watq or air; tle latter wil produe a oas olloor rvx crystalsshot dnoushout vith:ir which lus bear drem out ofits solution in the wu. tn either .asea new block shouldbe nade.

32

SeclianCrttixg ifl Miaaso

s. tu r goq-llv u ad'utag" tr$ e\rry dpe o" wr\. e{ceDrpo.)e{(. w r. ro kee! tne .uri/e ot tte lootilg L o,t UqLida rdjc red o c"iort". rd J o ocrhnJry b Lrcp rt-eniddte o, lbF L,b.t Liq.Lid "r td , \.,lf."i r,on Ltr ,opdoq._be.rre d".oohg u* ar rlc ou ;. of ," o..J.." &"* oo $ . - c L l u d w r . o o h l e r , c t h e , h - u r tg c r l r b i , . , l o g p : . e . m d r b c i m e _ RgoD or,rbeb oct n ur provideda,r L. Ljo. fo k"ep rhe njddte \qL id Lh a r bor.5paru3 or needrca Lfk wJI i!!o !\" nLiddl,or rh" D.o.t. evcrl now od 6. With ster ws ir is ad*able to hmt the wq for tlock making - bur Dot e.ir! rhe sFcimcn in ir - to a loift sonc re,o' C above its neftins poinr befoft pouiog n into the L p;.6. T[is will prodrcc a bloctr.r"*"t, lit". t*t*" L b r . o r p { t r c d\ D , l ' r l . e$ d j L I r d c f , t r J b o . ci , \ n , e l l i i g ! o .,. - . w r L bw * < o ' u b l^. * . s ! t , . . F . , . r o o l - i e !re . - . , l , o u d .;.t-rIr."t be smeucdwidr MJy"i. rlbsm r . . give." bfl,c, b"rrt g drr gt)t'ro . ^ 8. The bigsertbe b ock Jc_no'e di.fi.l, rr s ro D-c/tur no,, or we-hhsc.. tt a Lt*e oF nde A.sdal ,o itlow rhe poii, jbt gi,s ! j,h gre"rde $hq D:@g Drodsor Largelrcs or h$ue. oy'sbould" turhcd. tu d brdfned btod Loq .{e\b ir. G whiFjI:rJslqo. v ml'r !p rd makeJ Dewb ock. Neq wd jould be r ed b,c, ae.uJ DLts nomol !e due ro pgbred " x. rhc portrr.iortuJ be dk ,o uFmed]b ooz-ing F:D$-e $e!bJ0, ,o fu,+c, idl; n o. rh; ,Leo.; ,houtdaj.o!e coroidered.

Someworles prefer rhe paperboat ncthod ofmalinq wd tlocks to the r ojece rcthod. The-papo rcntaira or box }as the admtage tiat it is oot r neid sffiitue lile th.:nera.lt piecesand asa lcsuit ii w rend h;rdr s rhe tlock c;L, rtereby dinibhing rhe Lleii@o o 'cJrc"d pn -urc r-giou wirrJ! ,hecoolnC .q b o,[. f a y $ o u g r h ep " p e rb o " r , ' o n " t e i r : e . e " q u i . k c .. o d r u wt u . L r . r . d r o p c d { " ' o l o r m" h d . s d o J - s l o l e L e r o ; . e ; c o d . n m .iart). ed. r o n k e " p r p e rb o : rl o l l or t J , eu g " o o o p . 1j, "hj.b,ho" J;.-dod ,,.d a t o n eo d o u J y .l h e o t l o e b d\ h o u l db e f o d e dn r L l e " r e r " 1 r o c o m p t e r6ec U'e '. PlJceLhe!@!. v Uch hs beo mde of u ,puoprurc @ o. r gt-".Lde. o! r rrg* pre or gras.o! a sntrr oj hebt. 2. Stickit to thc slidelvirh a &op ofvartr. j. Poui! r]t molttuvd. a. ,Addlne hlJm' cJ speo. b "bd pt .- , ; $e reqlird no.raon. r p u r h r h ep J b e b r o " r" 1 i r r d ? i J r b o i c _ . o l dv r " . . A r " l o w 5 . I ' i r t n e _d gl$ di,b or r peri dr b t ""er' 1 [o! dn purpoF.Lf,rvc,jn] Lhebloct b solid. o. To FmNe rhe bto.k gq,ty pl.n ir o". .a,he p,tr.. fli. b.,r may be ,.ed again,or rhe blocL nay be left in i! dd lelevatrtdetailsqay be .e.jft;tr on rhe pald before the block is put amy 1br storage_

filac BlotkMaktnr oftt Blork Tnknifg P.p.r bodk for

;q

33

btdcrs

!!c.9. H,Ewly sbgeofn king, prld boar j. Wdt.l GL$' Blo.ja This herhod is of Lseprincip:fly lor sn.ll or very sbdl oLjech sucha emoebae, irset lame sma[ eggs,erc. Ir h6 rhe admtage rlat the watcl glss /Preoeia, t'ery be pl*d undc . binoaia! microkpe dd lhe specimd nay be oritutaed ;,ith varn Deedles so tlat ir hay !e crrrin dy desiredllane later.

r. Sncu tbe conevc sur6(e ofrl( arbuiml_=-

warcheles wirh slvcerolor *nh Mavsi

Sttior C' nTin Midatcon

34

2. Pour in nolter wu EtiI the gL*s i nearly fu1I. 3. Placeupon the microscore stase(lov lower rino.dr nicrocope). 4. Renove the infiltrated spe.infl fron its .onr.iDer with a varm litctte, and plae the spccino la dre wu ofthe watch g1as. i . O n e n L r c* c o ' o n a u d e ' L l e b i r o c r J . . . . r r g * . r r , o u n c dn c . d ' r . .A * . 4 . * , { , . * U U - . * L . - e , d i , v { y u e ( | r ; . D p i r sb . o . , t , .. p c , a . 1 to free it, shouldsolidiflnng wu suddoiy hold it. Anage the spedmenso rhrr r h . . ' r f " r r o b c ( ,-. r l ^ . e .d o 4 N " , d , o s v $ a y d " o t u d r ' go r r h e. e q u n c -

-*t';rtf.."B.

6. Wheh the oricntation n conplete. blow on the mface of dr wd to prodlce a skin dd tlen fluge lhe whole vatch glss below tle surfa.e ofice
Wdx BlothMakiry drn Bkrk ninniir

lj .,. Quicl coolins ofa bloc[ qill produ.ei berer btocLlhd w slowrooliig. The o!1) exceprioDm tln is poi) eskr wu. Never attempto move a specinm oDceir ha bed alholr acsd wfth sotidit_ ,t. t' ing wu. This wll1ead to cystal oovchmt of rhe wa; it nay danagc tire s!6inm; it hay lad to an bdbls ddin thc vd. 4- Use cold I, pied. Nner $e w* which hasb€€nusedfor stecineD infilftarion. Atways ue &oh, l. 6 ^ :hJlow blocl(,p'rinl'lr orDrrJfi! ws, $ilt J,va)a.oot ed h,rdq to r L'ede,,exturerbd I d dcepblocl. Thi. r beJN rle irge trecsurfr. _ rbt is tle surfaceexposedto the tr - cd radily lod doMw;ds or irNdds asrlE m d d l . o l ^ + e b l o 6 . o n . r " , ' , . f r r . - b t o ; [ b o d q J c " t r p . o . c b o o kt ) . i n g mi;;I;-5trfi;;Jd mbrc a lerfea bto,r ro bc ar, "he,u rhe.m. block holder or@ only ar rhc top Jike a bool stoding up.ighr woutd lroduce a block of vet' dubioG qrality, prolably shor witt air in;epinss ;d wirh -. A helo rourd the :pemen in a blocl irdicae. an, or water.or utcmedim ortoTlron rhe .p,.1bn. Mclr tI" b ock, dd .-ir4tr;,te lie sprci-o. 8. wlletr sing L piees keepthe sura@ ofrhe l1o.k liquid ty uing : hot spatuta, rd if necesuy add a Jitde liquid vax if the contraction appos litely to be gru BlarkTtibniflr ,],.,'.tsi'l trilfird bl(k ro d,c btockholde, rvirh , 6,t heared.p"rurr. 1_1", w henthenatcd wu $ cold: r. Trib rle block b th3t vild a dbbon n llred on a nidoscole stidethe sp€cimcn wnl !e secnthe righ way u1 uder dre dcroscopc. as shom in trle;ilsrem felow. Urc a singl+edsc safeq,razu blade.

Ec. to,

Atuscmcnt

of slecimd

in bloct so rtd tle sdion be n6$capc

wil be rle .orEd

wy

D! udd

:. Tib lbe ':ds ofrh. blo.t
rig\rugis, b,i chor.' ".

*,1tall**;". *. *.oi" e"-

36

SediottC nngin Micfosco|I

a.nothanr the ribbon- Do not ovcrdo this .l',-+'hg or thc reducd ldgth of de edgcofthe bloclcwill coEdpondiryly reducethe adhdivc dea oftlat side, thelelt redeirg the nbboiry epacity of thc Du. /+. Trim the blo& so that thc *ecimc'r ir c€dEallvDlaad.

Right

Wrong Fc tt,

Blo.L tlitrming

CErEB 9 RIBSON ARRINGEMINT

/.

ON PA?ER AND ON SLIDES

A del.tritc nethod ofarargiag ibbotu on pap- d dty ue ot should rlwap be followed. The ?l.cing o{ dr fiat ribl,on nsrsr the opq-etoris rhe lot likely to lead to

IIc. 11

Rr_bboDrnogeD@i

on prper

3 . Do not lre z shiny suface papcr for this purpose.A papca which ho bm cdlodered to give a polishedsuface, is so snoodr, drat eaiom will :dhrc very qpil1 to it, p:niculirly in wde vether. The host snta6le papeilJr the or.adon h tlar rpri,JJy produc-dfor d:pliqtioe "rug-mt of **;bb.n wh-(h wix uopor la dobo!' nk&". rhl ii a sor\ rcL+isrfjlt-paps, o on um d-q *ifioui le&g o de; dhcsioo. white pape!j3 the 16! .uitrh'eol,ll. BLrL i' moestful Lotle qes od slos up drecctiou bmer. Never ot nore ibfos thd co bc nomted dut dey, becauethey may sti& ro lbe p"I'd, sd lhcy bay 6|Jectdb(. Who a blo& i! ribbodng wdl kcp on otthg x long c possible.Thm divide rle lons nblon inlo lengtli to 6t the sheetor shets ofpApcr erused !6ide

i

Always leve about six sectior in ribfon fon on the tdfe edge,b@$e they will helDrc Dromotesood ribboninewbq cutlinpi5 rdded. bid. i" pref.,eic ro,.r;shicdged bl:de,sucb* a raor 7. u",[email protected] blde. for ditns ribbon' inLoqo'rer ime6.. The <wed bLrdema.ks r deag raos r-heribboo,ud onJya sm-ll pd oftr E D coDect ot wbo t is rocl<ed ; d-i"s rh" *nilg. rhi. ;u that thereis 1es li'iir ffi ;ibbon; ;y adhsion ofthe wu to the bladeod hae thereis les likelihood of picLiag up the ribbon who renovine LE knifc.

38

Sectbi Cuttitg ifi Mimkopl

8. Ib. ribbos bay be hadled readjv bv uine 6ne forceDs. mouL.d necdl6.or d,!pc4 !4rji hd btuhs. varios optrrto; prre, "-;"6 ;;iiod..' Maadiflg Soidl Sectiotrs ofi Midat@peSlids r. Iraae one irch of a rhreeby onc inch slideclor fo labetling. a. Se that all srids hrvc bed d@ed wirh gres removiig ftrhods betore rhey dc &d b. rr7), ldes rhey lave proved to b. quite satisfaciolywithout oy 3. Nunlo lhe s1i&s with a dianond before starting to mout rhe Fcrio6, il serialsectio$ are leouiled. a. turdsc rhe slidesaahud in rotatioq readv fo, u"c. /j. smgi]l qe4 l1th Maye!'s albmei, o( flood then wi$ anylepectin sotution. _ Plcpue abour te in this wy. /6. Ilood thc albmo snered slidesdth disdlcd waler, Do not have so much LqujdJLt ft,e Jpper, .o bc a nomain of w** oi mrlGpe( M \olrdo- on lhe Jidr. HJvejur,.moor !o rt d: lubddr. .7. Cl rLe 6r* pi<e olnbbob so rhJr ir rebues abo:Lonrud, hJf irJe ir lorsth. Pla@on fte flooded slided allow &e!S!!s!__q!P!3sC, brrve'!+c Ll-$tideon " bot pLlf or on rl,. -.f.*:f b;;"' if r.,m" **.; .,", "-i i, beils ued s byjJsr having n ro spdd ar rood'Lenperaturcd po'/e,r* E. fftlE nMber ofsectios in.he tusr pie.e ofribbon prova to beju ight aiet expsion by 6 iig up aplloxi@re1y two inchesoftle length ofihe hi.roscope slide,cortinue to add eithd dorher ribbon or rwo to the sane dide, or continue with the otler ,lides. The suggered ddgenenr is shom below:

r'r[m

I;TTTTI;;I

tEl-l-L-LE

L,T*rT-r%;l !rc. r j, Rjbbons:4d@t

on slids

SonE worLe$ preGr to placeclt piees of nbbon on slideseider snedcd with alb@o ifpdafin \d or estd vd is f€ing sed, or o! dry slid6 with polyesler w*. The slideis then flooded eirLervirh wer or with &yto-lectin slurioa after the ibbor lc beo 11rcedon it. ltis is ptobably 6i- ria rhc ncthod ftst givd, but it hs ihe disadedeS. iha! dy gr6irds of the slide may bc dncovded oDly after the sectior od watcr haveleo placedupor it Ary such greasinesruy nale orderly ara,g6a@r of ribbos elmst inpossible, od

Rilban*

nfnest os Papeti d an Slides

39

nay well lead to section wdhi,g ofi in larer @ipd,tionr. Th@forc it is bener to flood tk slidebefore pLgl!a4"_-EE*fllsjf. 9. Aruge rbe .ibboro oo a .lide so rlut tlre qtioro ue in snaighrU'6 borh rjoDg md ros r.heJi&. Io. /rfthse b e) pm ofthe rpe.ihm wlic} is Doi of very grar idpora'd ftour X z sn, sirglcsctms 6on r}!. rcgionon.*p*aLe sLde3. Tb6.,iigLe se.tioB wil Deseru DFr a ururrB8ud6 ro'srll$s- puryoses.

rr. Alw,yss rh.-ffim-radr;ve. p. Ar""!s Jtrr. s-tid*r;. i roogi po"iur".-a,r $e hilhbr t oEQruc sddblc ro! rhe b.tstc.lo

6e.tln

Pdaffn w.\ M.P. J?' C s€crions Estq vq, M.P. 48' C Polygtd w*, M.?. 38' C Pobedylene glycol r,J@ M.P. j2" C

Dly at 4J' c ,, ,, 30' c ,, ,, 4J' c

r3. Never dry sax setio$ at dt meltiog poiot of tle wu bew it my car:se rle rdoo ro lotr "hape.tud beeue w* oy ger boeth rle specim@dd lad ro rle lan* wrsltg of duins subscauturopeBriob.

PARAFFIN Y,'AX PropqtiesaJPnal;n Wd

'

!

I t r d . n i - . T o . t o " h . f . d . o c r b o u ' d , g i n g. o b C : I r 6 t o C e s H 6 A s .r r , 6 L t L it ha ho .h tro h lring po;r L r g-rdr-I, .of-n. uol u o-corc , Liqud.ln a hor r o o m . b o , ( n " y b e o n - 8 - s l l e n d p r o o u c "i t i i g . e - o n J o u b ; t , c r r . 0 8 , 'when teb' r 1o.fl D !b. ao-r1 .onduo! pr '{n ** lo.* rn. owe- neitirg r',-,"T.b, er por"nob, od.\rnre loog r o rer par.tu sruduJ/ bdoD; f"i, hdder. sd ol i e-ed ml!'ng poir,. Such1* ir -o.e diftlcultro bboo. In tle no o ,o"dirior ir ,hor" , rendm..,to b.cone ondizedrd -.me a yellowish tnrge. If heatedto boiling loift ed icpt ia rlut codition for ar hou or nore n ghdu.Iy bccomcsye11owod dro brov,a, dre to i\e fo@liotr of hishe! f"rry 1ad..A: iu-I jr i" more"d,he,re.hs " o"o) f*i .bour ir: od h,s osro gle* dal ofits rornrl cnstalline cordition. It nay be foud shoNrtg rwo crystallineforns, ore Eiclbic, the otier od morc coIuon, nonoclini.. Pd,flln wd Eay be decribcd s st ongly .rystallire. p!onorocedcry"trir7 u , b]o6 i" nr*;d b) p"* -h.i"", -:'b/ i.. ,,1-h"i., oi,eclid ro .cdio". r ,fi " , .. ,r"xi"" -",;" ' i! , ligLid .o"d,;o;. +e quicker ihc coolirq rh. ".-I. r ll be rl.. .n.r- \De,drarfore block of Dd-fr wd fo! "E o,u',ioq. boul" b., oo;d ", qujcllv s po* bL. eitheru ice-"ir. or bv otacins dr blo.k in a efrigliJor. \rio.. addllio;.," p',6; **, '"- *- *;[ ;; b;tF,, y 'i;. ;;dlo rorruon b LbeEnislerlbtos. rrodur " sd,L't5,j The crystallne condition is plainly kca j! blocks wlich i4ve bes cooled vcrt slo*ly, d my be the cde in a warm bboratory, or when traco of clcaring ageot or 4remed1m de present1tr&c w*. The snell ofnohen p@6! ws may !e noriccd on drering a liboetory vherc a gret dcal of histological jnfltntion is bei4 dorc. This nearu that puticles of psltrn wd .ie noating in rle d! ed s seh r]ry wil d$cdd upon dy cld hicroscote slideswlich h3ve bca spaially prepucd to be greAGfree. Thereforc ded slilc shornj neyei le-r4,osed !o the air h a laboratoly where nolto w is

Ulk m;y ioo ell -y. lile n{trials p-a-frn ka i fouad ro .oDui! ocduded rir. wlich n"y be prc ot Lp ,o roo. whs !h; u tu molro rhis ar ; Lrselr di,pLced.bur r r6id@ ,enr.d *hich ruv Gk coD,iderrbhsdble dunlg bloi -aLi;g. l.is reidE sbouldbe drivr! ofby heatugrhe wd for , fe. -iou6 ,! \ ,o-to" C .bovcits mdtinepoinr, G ,aation to ocdrae.ir p-rasr "+ will d:sotre 7-rj% ofi6 oM \ otme of aii.at.ordilary temrcratue (carpdrer, re26).Tb$ ; also!di1y rc@ovedon heating

parufrnWa,

.

PtlEar+ secboDturqgs

4r iy1,9"e2-hl&.:llrcbic rd iB vJk rs a bfln,tion medjumfor ool) reJEed wb@ d,\u6b"ouAbt in,o itre

comgletelvdehldured.

I F.es-orwrtr n tu.primn ryixpr*qlt rcppLels_i"rj*d-*"i.h" -,".;d,iog

lorrbte ktuon cuftrs cobdiliob. I r . t o " s L a J e m b c s i voe. o y e - ! i $ ! , d . i t t r c a d i l p \ trl,.a) 6oD ?olished.urfa6 \uLha re loud U' tuy roec6.^.ed wo!n.. etc.To couru Lf,i,-ubberru1 be n corpo"(ed by leadrg uo o"i&d pre rbber ir p--,fir 1B fo! sobe d:yl l! Lso ublein a \,neN of .)d,o-rboa, chtori,r;o bydrouboro.ettrys,urual oils suchs ce
Mrciocnysrarr.hE WAxls Th6e wss diller *on retned ?arafitr wd in th& cryst l size,rhich ft sha119r, rd r_rrrcn gr.rr rcBln".s id bgbe retGg poinL. tlev r.nae iq melory Dorr .oD 4boL 62 88' C. Fo- lhi reaon rheu u* rJoneir tle pue coud$ionis intiequertir m . ro ooy. orr Lheym"r be"ddedto ddiuy p,ra6r wc to nodili i- .ry.tJ form"riondurng cooiilg. Tb"y rary D (otour fim $hj,e ro brchJr'P${frt w4, Modifudtions Joh$on' Aqhdh RubbnMj,tute ,tuphalt or gn. Pdrfinw* 99 gm. Pure rubbo, cut small r gn. Hearto loo" c for,4 a8 hor:. Deu. od kep $e uprer nLid. Ti!@ nj! ir t conrcri.J p'odu.r ol per-trx'wd Mrh 'ubbfl tp,ge roE.

99

g4

Schtlgin 99 gm. fire ue nuy otho nrcdincarions.Ther rhr6 ue given ase&mples. For iIthq fornuhe se crayi 'nre nidorohilis fornutary dd;uide,.

4

r-'"'i:jl .a | ' r " J " ' .:.'{{a.];-'.

Se.tionCt ierir Mi6oxot!

Paraffn wc r. A hard, white, auluot substrnce,verying ir Ladnss wirh its neltiig ?oi!r 4,.48 C. 2. Insolublein Ratq. 3. lbmiscible sith i'€ter. a. solublc in: a-ylene,lslzole, ligrcio, clloroforn, nethyl b@zoac, cedlrwood o4 dioxd, ethyleneglycol mon@rhyl ethtr. j. Solubil.ityiD xylere:t 6' C tu ro o%. 6. sectioDtuge (the hge of thiclnes at whici the wc nay be mt iato sectior) : 3 20 p at . room tehlcr.ture of2o" C. Melting poini otvd J2' C. z. Ribbon roge (dle rdge of tlickne$ at vhidl the sectiorowill adture to sh othd th6 fomiog a ribbon): 4 rJ l, at a rooh tehleratEe of 20' C. Melridg poitrt ofFu J2' C. 8. CoElrqdon of wd s€ctions: room temperature2oqC. Meltidg poiDt of 4oooai 3 / before d]tlojng 24%.t 6tt 9'7% ^t n tt ,, 9. Cohpr6sion aftd flattmiDg: 28.k ^t 3 p t g Y o2 t 6 4 7.5'/a^r rc p ,o. Mor wideiy urd c.lesiusascou o!-a!emd;:

,,. c,m"g *.gh, (,FGfr

."q;;;;r

€,.lgs9{Iasod"o!.

.*.*ij;r;/

'-hi.k&.- . bl".L

with r &ce ro x ro mn. ir 2Jo gm. Room remls.ture 2oo C. Wd neltidg r2. Viscosity of j2' C neltiDg poift .wax.r jJ' C i5 ro @rieroks. t3. Seairg tine of blo.k mduing ,o x ro x ro ro. i5 ro minurs ar a rooo tenper1tue of2o' C, vs neltiig poidt ,z' C. 4. Hddfling tine ofblock 20 x ro x ro mm. at a room temperatue of2o" C: for ro 4 sectioas,onc hom; for 3 a sectiou, ovemighr rJ, Elect.ificatioDofribbos tak6 pLce v6-y elily, dd is dificulr ro co@rer. 16. ?asfu wc ir non-poisonous. Wtalian with Pdafrf file Bthg [red, wohed mterial into ?o dl. ethyl alcohol,30 ni. water. Aisolute ethyl al@holGooo/").

I:19!Piry9-r*relr"

. .

6. s6e morte prrJtE wd. Iwo btu13. 7. Ma.ke. block, a bdcand o! P. .lo.

pdafrn Watt

43 ^ nde gmrtesr) i!rc pLrep:Dfr! lva tuy be Dadeb) bilg b"drsof 11lmc parahn Il ub a bad or equ! pd: lthm x)tm r, p"m6r wc / l. r: aad6aliv v pue p1.-JfiD$r. A sens orevo oore dosetysp&ed;!"d:.i"* -"y U *A i Schedute 2 Brins.f,a€d, wshed haterit into 70% ethyl atcohot, jo% valei. eoo/"alcohol. AbsoloEdhyl alcotot(roo90).rwo chuees. Pue cedawoodoit. Cedsood oil od ldatra vax, equ.l prrs. rn m ovh. ?ure pa.rfD vd. Ar lat 6ur hathsto male surea\rt all the cedxwood oil is renoved fron the srecine. CedaMood oil is non-eotarile, dd hence irs ,erwal 5om dr (ptrins isrcompltuhFdor'y b) difuiob b;o $e "*odd_ bg we seecnalreron lgiudor 7. M*e a bJock* descritedon p. 3o. 8. :rrin rhe block (p. 3J). Fof the tihihg ofdiferdt sizedstecihero durjlg inGlrrarionl€c Chapter26, p. r47. r. 2. j. +. 5. 6.

Cenqdl Nate!or Wlttutios tuithp1tufrn Wdx '-1. Keep the w* aboot z-3" aboveits heldng poinr, but no more. z. Shakedre contajncr widr e wd od !!ecj;@ jn i! at intndL to sure thar ihe wd is drtqing rhe slecinfr xld the clearing ageft is leving it. Use m agiiator or stnre! ifpo$ibte (!. 22). q. Use I volhe of Do'!,- wa rr t*.r jo-so tima Lie voime of fie sDeci@. a. Har rhreec fo..r.oooiros of ootm pr-]tu r+. B,rh L l,"tl,, b"!h l. DeLn4 rd.$ ob LhlougbslLi"! lhe rpeho i, LJe! s a roulile proe.due. (uarg Mr,r r,drbr. od a,dr€ $ r brh a. From dre l-r bad t}e:pecnlo , shouldbe reJdy fd rj,ds iD a bLocl. Wa wl i f L bfl ehojo..d for I,|.rinl]lrarioDof ,pecimo is u,rabrc for t't 6. ln6lF.!iob b,ns u, y nco, oinbto Lhe"ia md dtuiry ofthe rpe.ino. Forsome godmc od fiis [email protected]. Cuting S&kfls ofP dfrt l4/at r. Irin $e bbck s iadialed oo p. 35.ud securetifd@ n ro Lhcblo,t_holder, N[n r nar, hot sp*ub. .z. M-Le,se 6br lhe bbck-botd*. tte knife.rnek ehotder,o,l ov othq prrt oi llle m.,orohc $hich $ould IJeedjlred or !'shreqrd,ue ir gooi odc od 3. aring a\e block to tLe ldG or the bifc to the btocL, rccording to the nale of nidorooe, ad hate a 6!al check of b1o&+dse relariomhi relative !o,hc tl: kni,L dd Lie lo$a-a Lppercjges* tle btocl Fr::ie" nouo bepraxel*:.rdf" Mth e:ctro!h€r,

14

SeniokCuttiig ifl Midosipf

4, check the roon tenpemtre. js faced-o4 ti. Stln cffiing .t ro a ud continu at this thi&nes until tle blocl that n mtil thc wu sectionsarc being cut ofi-the block @n!1ete1ytrcs dd / complctelyup the faceofthe Uock. 4. Continu at ro p util lhe specimmwithio the block is rerhed or faced+tr 7. Adjut the thickncs gaugc of the microtonc to whatever thi.nes of sectionis ,,r. Cut at a specdofSo roo sccEonsler miDute. 9. M*ure the setions o they lie ou tle hrife or asthey areplacedupor paperon the l,acL If the con?re$id is high {p. 67) move tle krife rc tiat a shry* pan may be ued. ro. If thin sectionsare rcquired,say 3 p, continueto cut at tie enr steeds befo.e, Seroo scctionspcr Dinute. If theseseaiors show excessivecompre$ion a vst increae ofspeedlrorn ro*u;o secriorsper nlnute will rot improve thc situtior. the mi@toft in thn v:y will mt pro,lue s-"dionsai dncc microns ,4hmshing ' if suh frctoa asroon tempe*t".e, soiae. of .ro, ud hardne$ of sleimen, are usuitcd to tlosc requnernents. imted of the usualmicroIr. It will be foud in pr.ctlce a\at a raglllq@lder tonc knifc will mable patallm jnliltrated matqial to he cut inro fair sectiors. 12. Pl.@ thc scctiod o! ribbonss rhey are ot on ro a rcllgh-surace paper(p. 37). rl. Ior noetiig panfrn v* secrionsse bclow. Patffu I4/a, Sectofl' FlattunTM,l Mosntil'g Two methodsare comnonly emtioyed, ihe drst in which dre sectionor ribbon is placeddirccdy on to a d@ mic.oscoreslide,ed the secondin which lhe sections re placedupon the surfaccofa dish of clm, wm water.

nidoscope slide very thinly either wifi Mayeri albunar or r. Snear !.led (p. r,o). or usea slile without adhesive. adhoive Jbhd z. Flood the dide with distiiled water. 3, Add the ectior or nbbon. Do thi gotly to .void trapging air brhbles below 4. wrn the slide by placirg it or the surfaceofhot w*er or on a wrn plate. The nbbor or sectionvill eryud. j. Draih rhe exces vrrer ollthe shde,uage the sectionor ribbon in the desired Ddl'ion bv usus r camcllairbtush. i . b p F c t, o d r y o v m i q h t ,f c*t..-.', ,16.P1.,"r;*-"r",rt**.rul iflosib1e, becausethis givesrhc sccrionfimd adhesionto the glasss1ide.

r. Fili a glss dish with wtd

.t about 40 {' c, or widr dnuted albunen solution

Pdafri wd

4s

2- Pick up a pdafin nbbon with moi* cmcl han bruhes dd placetbe;hb@ d the surfaceofthe wmr water. I. cendy pull the ribbor to rmove MirH6 in i-he.d.tiotrr. 4. Rane a cled nicroscope slide fron ben@tl th! ribbor and trin ot'a seaion or requiredpart with a knife. Lift out of the rratq cohplctely, didn and &y. R,E!ERENCES C@Nes-J. A., ts2t5. J. ttut.P.ttu\."fr Thhdolo , rz, 288. Cu!.Prtn. Tfu Mido@nisBFomrlaryatulCuih. Coi'tabb.

ESTER WA)( 1960 This naterial is b*ed on diethylae glyol disteuxe, e 6t* wLich ws iovestigated by orton dd PosrGe32)ud by cuder (re3j). Dierhylde glycol aiteahre posess€r gre.t hddnes vhed cohpsd with pdafrn vd, ud nelts at a low* tempemtue (a8' C) thd the pa€fia usu.lly @ployed for sectioDwork. Alone it posese a n infitratioD hedim severalwekn*es *hich were pardy coutmd !y the additior otfour othd conitiftenis (Steedmu, 1947).Evo with theseaddirio$ fl.troing of dre seaion w* ahvai.sdiifia:lt, ud secdos lsudly sepdared&oo ech oth* dwlag this opdadon. InploveneDt of tne darrening wa! p.odu.ed by Chdtelfu dd kach Gs:6) who r@oved rvo of rhe origin:l costituents and iDcr4ed rhe diethylae glycol nonosteuate. This nodifation, hovever, h* rhe diedvst.ec thar ilr tr.rod"dI r!d,E d,.!irg fl"mnirg; thr., daogor rch - l*pof L requned d anaid to flattering, ed thar the sectioBlbd e iendencyto washoft'duing flattding. Thc new fomula put fomd here rctains most of the toughrs of the 1947 formnlatior! dd hs gready inproved de ribbonirg c,prcity od dre llattming of sectioDs.n! addition tkre is a lowqing of lhe meking point od of the viscosity. The new nituc produce e dore honogeneos blocL, duc to letter settiog of the ws ud |he s@tiomldhde duriry fl*tening. At the time 6ter war va [6t !!odu@d, 1947,it sudilqd 6on vriations ir the quaiity of thc nalufacnted products, particululy dn did$/ene glycol none stetate, bw this vas lugely a post-wu weahess, lrd was .orected by rhe nu$.. factuiersin due co$e. Today thde producrs,rhough stil subjectro somevaiatioa re gIetly inproved ed the 196lrlomulation (British Dng Houes, Poole, Dorser, Ergland) fintL aI specGetions. The fomrla is asfollows: Elkt Wdx 1r6a D;6ylen- glycold\tt:i,rc 6o g*. clydyl mddtemte 30 gD. 3oopolyerlyloe glycol dnte..are ro gm. Thc &edyloe glycol disterate usedin this fomula is a vcry hrd, rqlslu€nto trupaot mterial rvhich breals with a conchoidalfrrctue, ad is sold o digol disredde (British Drtg Holses, Poole,Dqset, lngldrd). To nuke the nixtute first nelt the diethylme g1yco1distearateud heotit util it is clcar. Thm add the glyceryl monoste*ate, ud who thar is &solved add tie 3oopolyethylmc glycol disteuate. Filar alrough a BarcLamGred eo4 ilter Fpd.

Esh, Wdx 196o

PftpeniesofEstd WB rs6o Melting poirt 4J-a7' C. S.cliob rilge r-.:o p ir a room lo1pdarureof 18.C. Ribbon ranger ',J /. Optimm ribbohiig srrrd is lo-ro p$ niluk. slock ttindry shouldbc doE wirh a siisl*dse ruor tlade dd gives tu difiIt is e advdt ge to heat the wu slighdy before pouring ir into moulds bur rhe ;ftlee shouldnot le nore the ro" C. This nak6 surclhat ant possibiliryofserding out ofthe dif,crot irctiou is comtered. It also promot$ a nore wen od homG smeos fnished blo&. r !;cces mly bc sea.ed vith glyarol, or wili Mlycri albunen" a a partirg Ali tle cotutitmts re soluble in 9J% edrrol, 5ol. rvater, ryloe, md nosr of the reagon comorly eoploled : clerhg regent" ir rnlcro.opy. The ws is ?ein€ble to boic dye soiutiom, md ribbom nay le floated on to sEchdyesdisolved in wter asmeth,vloe blue, sa{i@in, thioDin, ed bsic fuchsin. Aner flatteliog the s€.:tio$ dr dye is daiDed oft'dd tle slide is ri6ed with v,rer by adding ome from a pipene.The sectiou rc gently agitaredby rccking tie slide, aad tu ly tle slide n draineddd diied. This m€u drat a s€.rion bly bc r€a
48

Sectian&tting in Midosopl

Ttotiul Estet \ue 1960 Dierhyl@ glycot d;reamtc 60 fTffipo.ary clyceryl nonqr€&te 3o{ nehirx 'rnethylm glycol rnonostea*t ,o lp.;t 5o" c conp*ed with rhe studard nirture tropical ster wx nay be cut into good ..As ribLons a! room remperaruresbetwen 15. C od 35. C. In the ovetr rhe n;hed niture.rcbA out o..oludoDD j J day..md how" €occuL:iioD. r, n.y be, Lrillcd L y n e " r b gu d 6 k " n o F .r o m d e " b . o .( r n . * p i " ( o F u o p i . . 6 r e , u r \ rce:ved fi or rhe$p! a5, 8ntuh Dn s Ho',.e. po.t., t'""". L.n -d/,hou d b" he"tfd ir a .rrll b.-I4 dln J-,r. r"e ur .houd re Dou,eoi"; r -ri.... _d vhe it n begiming to set rh. injlrratcd slecimd shoutd bc added. The etrecton tissuesofsrer wd miarues is ro qive theh a softnes vhich embts good sdioru to le cut of difllcult nuterials. S;e investig"ri.tr of rhe -d wd madF_by Smythdd Hopnjn,tr948j: bv HerJ) " ,,. t.ost dd .l(e*, oD,i! !h. r94- tomula by Chgternun rd t4ch (roj6/. t, i5 bdie.ed rt,rr lhe ,060forbula wu Prove mosrsalsradory. Eskr Wu r. 2. 3. a. J.

Anlpaqne subsrde, ofnuh great* hrdrcs tho pararlinwx. Melting point 4JJ. C. Iruoluble in wter. WiI tolehte up !o J% ofwater in solveits sucf s erhyl aLohot. Soluue.in: 9J% edlyl .i.ohol,aqueo.trs; ethylde glycol non@thyl erhd, 'ce|jo,ol,e: " b s o l u ke d v l , t , . t r o t .h G p r e p ) Ir . " h j . b u q , , l r c t . o t :x l v o c , bcuole.fgro n monoclJonorl,'Tol. ,edjtuooo oit. .Labl phdrailere. 6. Secrionthicknes reg€: r-zo 4 :t a rcom tmpemrure ofzo" C. 7. Ribbon rdg.: r-r5 a at a."om tcnpe.aturcofzo" C. 8. Cobpresio! of w* s.ctiotu ar a rooh renp*rse ot2o. C: 4ooo3r I / be ore ca|hing ')oo^t Sp rc%^rrap ,, aher{-nmtng: t}":eftior rompterettregjn rhe b6uremtr6 9. C_omb-6sioD orrh. blork- ed if wmed roo to-s wdj $p;d b;y.; ,ho.e.r6ueh*,.. ro. K(ommodFd c errbg r8eDso, rrcnedD: 4.o; e!i1 | J.ohot. erly oe gtycol nonoc&yl erlcr, nonocHo.norhlnot Ftuql atcohot,xy1de. r. Curilg $eiglrr or Mdner (Lhcweig.r rcqued ro rur ; ro "eton.-oo e 4 r o . o r m . ) : i r l j o g b . a l , r o o m r e n p e r l , u ro" f 2 o . c viti I l e ottuediun sharrnc$. r:. Viscosityat jJ' C is zo cdrisroks. 13 s'Iu1s tlTlJ r blo-l mer,.rirs,o . ro mm. n ro minu!$,aLd loob rempela-

Btd WM 1960

+9

ra. Hddcnidg tine ota blod<:o x ro mm. at a roono tcmlcBtre

of2oe C ir:

rops4tios-rhou! l &!(uos -2hoh rJ. No electri6catiol of libbons trls plae. 16. The w* is not cdible. 17. Sectionsnd riblons oay !e {oatcd on to stain solurioro, danenedor those solutions,od rLcy will poetrate thc wx md strin thc tisu6 deelly. r8. Este! wd brcaks with a conchoidd fracue. It; therefore amorphousin it3 beLaviou, though it is omposed ofcrptak. re. It libLos with se ud sectionsdo rot seldate vhd being tunemd. zo. The secriomshouldbe ot .t 3o-Jo per minute. 2r. lste! w* c.nnot be cut wi*r rzor biade ni.rotoDe ldves, though it nay !e whm ued in tLe doubleinlltntion nefiod otwigslswold1(p. e7). 22. s€ctios may be starled t\rough the nu by doating then on to solutioro of dys sucho mcthyloe blue. 23. Adheion of sccrioroasfor puafin stioni.

Iijlttati.f

uik Fltet wax ScheduLz

into 7ool"erhylalcohol r. nring 6xedwslled tissues z. Eq"i p".t' "f zo9" "tl'yl ,tc"h;l ;d .tiyl-" gly-l nooethyl ether('clo3. a. J. o.

$lve'). Pue ethylae glycol nomethyl etlu. \ xtlylm glyol mooethyl etler aad e.ter w* 5o/5o. P1lremoltm ster wq, 3 b*hs. Makea block(p. 3r). S&ed"le2

r. Bring €-red,wxhed naterirl into 7o"l ethtol aqueous. 3. eJ% edmol dd dotroc oripthrool 95% etlEnoi 4. then

J. Pue estervs. 6. Block.

moltetr 6ter wd Fou !ar]6.

lo/:o. , lrn4o@rt 5on'l l.t.ut no'c

Monocllorisothmol. liLe cetuwood on, is a norvolatile !uid. Its reooval fron risuesis by diffuion onJy,ud thneforefou b,td "houldbe gvo o nolto oier ws to rebove it cohplelely. sririds is m advdtage (P. 22).

SectioiCnukf in Midos&n sahed'le3 Briry 6xe4 wAhcd naFrili oro eryo ethrol, aqueou. 9Joloalcoboldd 6te! w*, equ:lpm ir u ovo,t about35.4o.C. 3. 1. EsterWd BloeLMohing Heat a sm,ll mout ofester wd about ro" C aboveir, heltinq poin!. Use trcw wd whi.h h6 Dot bm prcviouly meJred. Do no, * l""g r"tr- "*. ?os the l'li ioto cold L pieces. l . q4o a t}in hyc of.onge!6f ws ho fomed lext to tle L pie6 ndr the surf&c ofrhe block wirh a hot sparulaor with a lrirk downward flick ofa lighted Pla@de specine,nin thc block by pressingir agaitut the congaled w* i! dy J . suomd &e L !iec6 with cold v.td io shi.h pie.esofice *c plaed. 6. Kep rle (urGccof 6e blod liquid by uing d bor \prul, or Bsm uiil rhc

$bole blod ir solid.lI lhis isnot doDe.ir tu, c,trD in'o Lheblock. Eskt We r. Trin the block with borl to! ud lotroh sids, .elariveto tbe miaotone bric per6ctly ?da el ro ech othe. dd quire sr..ighr The block nay be wmcd slighdy to si$ the tirming. by plachg ir in a wm ovm. Do not prolong rhn peliod or tle secrioningprolerli$ of rhe va* w;ll bc adversly efe.red :. rasten thc block ro rhe block-holder with a hot flat spatde. cuuing ir jr €ssentialto cut at tle !*hr speed,which jr 3o-Jo sectiotrslcr 3. \l!{ Estil WLt r

Takc . clcs, gre.!e-G@slidedd sbd ir rhinly with Mayeri albunerl or with adhesivealbuen (p. I,a). 2, Rood the slidewidl disriled warer. Sumcidt to cover it wirl , tllin 6ll!. 3. Lov6 thc *.tior o! ribbon on to the watu in swh a way t,rur no an bubbls de tlappcd belov the ribbon, 4 Plae the slide on a hot plate or on to thc surfae of wm v:rer in a dnh. Tte optimla tcotehnre b bctwed 4G5o' C. The w* wi loseits folds od wil becomequite dat in J-ro [email protected] specimenwill do the sm€ ft6 abour 20 secondr.It is essendlito seerhat thse is sufrciot water r:nder the whole sectior to act a d lubricet, dd ro permit movmdt ofrhe sectioD. t Di:i'r aw.y t\e ercessvate! ud adjusrdE pontion ofdrc sectios with a onel 6. Placcitr d over to dry at 4G4J' C.

Eskt lV# !96a

Jr

7. Placerhe &y sccrionsin ryloe to remove tLe waa od coDtinuewirh sidning plocedure aswitt pae6rl neL Ister wa 1960doesnot \raialle rftcr flattoiry. The materialwithin thc sc
F4ti Wax r96a Thee rc wful where hot laboratoricsor room mot !e cooled, .nd when it i couidercd suitableto bilE'lte tie slecinm wilh a 1ow neltins poidt waa btrt to block it in . high neltiog point w. Wit! 6ter iw th; procedureis 6ua11y uDe6sly becaue the staad{d nixnrre vili cut od ribbon vell up to 28' C. Shouldtie room tenperature!€ alove that iie troliol mia:tuc nay bc ued icteadMirtrtcs uith Palfskt Wdx Est* s'ax md polyeter w* hay be m;ed in snal lroportioc should ir be comideed suitabieto vary tlen lepective lropelties. xa.I will tolerate some ro,o9/o of eacLother, brt atore these6gure sone inconpatibility nay be produced. RIITRENCES CmrJ];rM\'Ww:,dd IBCE, E. H., 1956,Qr4tt.J. Mict. sci.,97, 5a3. SM.m,J. D., aDdHotrms, C. A' \948,Qun. J. Mitr. Sti.,A9,4rt. SEDI\N, H. F., r9,r7,Qrdr.J. Mio. Sci.,A8,p3. SruDM^N,H,I., 1949,Ndtas,164,ro84.

CsilrlR

12

POI,YESTER q,/AX Poly6te! wd is bscd upon aoo lolycrhylde glycol disterare. wlei n vs 6st introduceda dinfttration hedtun (Sttrdnu, r9J7)n ws bodiEed by ine.ddirion of r gn. of ctyi alcohol to ee of 4oo polycthylde glycol disteante, but frequot vriarion in the quality of lhe am polyethyloe g1yco1&teaote 1edro d increa. id the cetyl alcohol coatot * a oem of controlling the fucruriotu. A mirtue ol 9|ogin 4oo ?otyethyl@ glycol distedaread ro gm. of etyl alcohol h* o high a @tyl alcohol conle lnat it will esily cortair oy variarion ir the propertiesofthc polycthyloe glycol dntwatc. 4oo polyethylere glycol C€tyl aLoho1,reclDical

90 gn. ro gn.

is refctred to o polyesterwx 9o/ro to distingulshn fron thc qiis fomulation. It will scctionud dblon tadily et a .oom remper.rurc of rz 22' C. It nclrs at 38-10' C. It n soluu€ itr eo nrl. ethool, ro nL *ater; absoluteethmol, oethuol, ,netlrylated spirits (indEtnal, 96 n1. spint, ,{ nrl. water), eters, ethc, ketono, hydrocarbons,ccdlsood on, .edr@e,ethylde sly@l noncthyl etha (celloolve), diethylme glycol nonoethyl erier (c.rbitol), ecerone,moncblor;oth1nol, dietlyl phthallate,bechwood aeosote,od nos other cledng agentsor drdedia. Becauc of thc low mdting goior of polyeter wc ao/ro non-volatile nedia such .s ccdalwood oil od nonoclJo*orhFol should nor be usedx cle".ing agots. Th. *cosity of thcsefluids at 38' C jr so high r.lu! lmy ddges of vd wordd be rcquircd to rmove then, b@ue they are renovcd fron rhe ti5su6 by dif'uion od1 o dntina fron a fuid sucl d xylene vhichis iehoved both by dif'usior ud cvapootior. The rcomended cluhg agot or mtenediun js 96% ethyl alolol A block po1lredin L pi€cesat a wx tempcratureof 4oq C, . lood r@perature of zo' C. dd mauing J . a . 3 m, is rirdy ro cut hro'o/ rclionsi! 4 t-our. od dow to toura on soJrdrfyingovemght. Ilattening otsectio$is accomplishedat room tmp*atue (r7 ,f C) by €oning lhe ecii6 on to a microscopeslide whi.h ha bem doodedwith a z% solution of dylope.tin in water. The qces solurionir drainedofrhe dide ud rhc secrio$ tlritt fatto out at room tenperatue. k is e$dtiql thlt sufrcidr liquid shouldle presdr ro oable lle sedob b ex?sd. It ; decs$ry ro rr , soludooof rmylopectiD(Seeoru, r9J7)s e .dhe,j,e b@!.c it is foud in pmcri{ lhd Mryq\ dlbwen fa ro na.ket}ece.ecrioro adherc.Tlns i de lo tvo la.tots G) rhe low tcmpqature of inillrarion dd of

Pa\stu We

rl

sdtion dq,jng dd (,) rle :light tendarcl to w* &pesior In dE lsrafitr lvd proes the protein oftic fircd mleri.l is subje.redto r Bther high het doatuation, ,na tlir loliows tle chcnical dqorurario! which hasraLenrlacc duins 6,stioa ed dc rdr tior. Iq . ddjlion p,,fi, wd i. hydrcphob'cod whlr rcti;ns de phced or water to flatten tLcn the lrotein conren! ol the spedner is lror.cted f.om tle water by the wax. Thesetion iierelore dos nor swel to dy MLed ertet, ed L.hmiisdriedinodfla.abo.daj"C e p r o r e i nb(" c o o eh * d u d r e n t o r t o Thse facs do not apply to po1'esterwax. The inilFation temlerarre oft 8-4o" C doesnot hardor thc proteim oftle specimo to the sme enent s does!{.611 The wax itself wjll tolerate J ro% water, ad hoce the tisres never quite lose then c:o,c1 ro rJe ir * t, r. Dmg tunenhg opcr"tioropolyeLerw ea;on Lhercfore tend to inbibc watcr nor rediiy thD rlhfin se.tiou, md a they rnust be dned at about rcon tcmpe€rEe or !p to 30' C rhe proteir ofthe sction do* not becone very hrd. Nor do6 dD albhd adh6ivc. ?olye$er wd sectiodsthoefo.e require a srro{ger o! a dilferot type of:dhesive ascohpded with !.rafiln FctiosMayet'salbunar {ailsto hold polyesierlvq sectionsto glas shdes.Bakai abmen adhesive(p. r,o) shovs lcrlo holdiis proleltis, !$icula!1y whd lhe se.tiotu arc ltoated on to 5lo fonnalin, lut carbohy&atc .dhesivcssced besrofzll. A solution of eylo pedin holds the scctiotuwirhoft dific ry b. Dr. Slututage ud hardne$ ofhistological @tenal dre !o rhe heat involved in war irnhration n gr€lly reducedwiih polyesrcrwd. Thb nealI dlar in practiceDany trornally diflicult tisses becone agprccialiy enier to cut. It n.y be conbircd dth nvtives c indicated io tle chapru otr sin taneoG 6xario! dd wu irfdhation, ard it may bc usedto dcided advdrage with watubloodedui'n.ls, asind;.ated in thc chapro on isorhernic tetio4 dehy&ation ad Polyesterwd rdds to bccomesofler the longq ir i, kept in the molren con&tion, d:erefore,oriy dbt ouout whch i n€.ersry conplerely to rn6lrr.rc r pEce or pieccsoftnsue shotld be kept h !]t noltd condirion- The vd for blocL m,ling slDuld le kept solid etil required.Thc ano6t nece$aryfor dle blocksshoulddd1 be meLed dd poftd inro the L pices. In practicethis n esily arconplished by cutting sufLcidt cNps of the stock supply md nehing then in a small bealcer.Thc srocksupply is ir this *ay rever mclted, renelted, and so on.

r. 2. r. 4. J.

A white to creamsubstaqce,opaqrc, dd ofjlighr odolr. Melting poiat 38' C. Irsoluble in cold water; digersiLle in hor will toleratej rool ofwarer in solvmts suchs edrmol. Sohble in: 9J% ctLuol aqueos, edryleneglyrcl nooerhyl crher,iio-prcpy1 alcohol, but)'l alcohol, xaloe, bdol, Iigroi!, clloroform, cetooc, norocUornorh)rol, cedBE, ced.mood oil, dietiyl phthalate, beahwood creosoa.

J+

SecnoiCaukg in ^&doson

6. Scdioa thicl(lqs rdge: 4-2o 4 at a rootu tdqtrrtuc of r9' C. 7, Ribbon unge: 4-zo 4 at i ioon rempeEtur. of r9o C. 8. Comprssion ofwd sdtiols .t a loom teolerature of2oq C beforc flatrmiig: to p 2oo/o 8,,26% t,, 40% 9. Sectioruvhd nanenedon vater regaiuthe block neaslrements.nd vil expod beyod tlosc 6gdes if pehitted. ro. SectioDnattcning is exft enr. rr, Sectio6 bay te stainedin rle ribboD $ wirh €$e! \vd. rz. A polycter wx blo& takes24-48 hours !o beome hrd enoughfc secrioroof r9-2o' C. 4 & Room tdp*tse rj. Pob€ter wd my Leveits neLiig poin remperatft raisedby dle addirion of esterwd. It my be stifdea by the llse of crhyl cclulor. Thc w,r is otre which show itui6iig fll]lss thc l@gd e block is keF. Thrs it wil be fomd t!.t . blo& @ltaining blren 1 vhich Fctioad quire vell atu! z4 hous of life, wil s@tion*o lena two or ttue &y aftemrds Nor cm rhis prccers!e sp*dcd up by pl&ing the 61oct i! a rcfris@ro!. lnjl dtianwirt Poryetd Wa, r. 3!ing 6xed, warhedm.terial though gradedalcohoLro 96% ethyl alcohol sd 2. 96% alcohol md polyestd wg ro/Jq by vol:ne, or a gmter nmbcr of .,,,{,;^". if .*,1",1 3. Pm polyeirq \@. 9o/ro.Two barh, r! J8" C. BIoeFMoking r. Usc L pie.d oi papq boats,s istlucied o! pegejo. z. Do not ilmse ihe cooliDgllock in wals a rhe wlrer will aFet the hot wx. 3. L@ve or the bq1chto cool. Pialiag itr lhe reftigdrtor doesnot alwp produa good hdddiis. It n arradvutage to leavethe llock ro hardar ovenigh The loager a block is kept at a room tcmpuature of ror:nd ?,o' C or below the fimd it vill be.one, md scctios doM ro 2 p nay be cur with relariw e6e on dE third o.loonL day afts maling dr block.

r. Trim the block in the sane way s auy otier vA block (p. 31). 2. Fir to tne block lDlder vilh a hor spetula.Do nor hold rhe block in the fuges. 3. Cut at the sp6.d ofr{a6n wd - abon 8croo sections!q Dinutc. 4. E-ctlFni sectio$ rd ribbos may be et uing raar blade nicrotore ad.ptos (p. ra4.

hiG

JJ r. Plae the ribbon or sccrionon ro a solution of @ylopetin (!. r24 vhich bm spieadinto . tLin, e€r frlm on a del nicroscope slide. 2. The sectiono! ribbon vil date our widout rhe .id ofhear. 3. Watch the dattening c*ftlly in ee it goestoo far. To preveff this dein ,nost of tu dylclectin solurio!, ed wiF the es solulion rway FoE did€. 4. Dry ihe sli.le at loom tehlehrue or in e ovel at 30. C, but no highq. j. who d.-y,dissolveorrhe \fu in xylde or in alsohte alcohotad iron tho he.t d a pmffir vd sdrion.

hd

of .he

ou

aVlce the laboratory tenp*ature is in *cess ofza. C it ory !e m:dvan&ge wlo Dibg Lheblock ro u<elbr fo owiDg mi{ue .

3:!T"*'l'" TT]..o.*"" lster sd ro Cn.J Evo though dE speina hs been idltrated vith the sradld mi\tuc th. doud i! of the Hgher nelting po;nr mixtlre embla sectiorond ribbom lrsde to !e trqted much hore 6ily in wan conditiou. Should a nir1tre of hisher hear resistm@be ieqfted r ni-ture of sol2o ?a!ts Borh Ditu€s in no w ry id pri, Lbr flruding propenie..of rhe 9o/ ro mixure. ror I{XFER!NCE STEDIN, H, F., r9t7, Ndrrr, r79, r34j.

CE{rrr,R 13 .WATIR

SOLUALE WAXXS

Thce re principally polyetlyloe giycols md sone ofthei.latty acid 6ters. The higler nolec u weight pollttiylere glycols - 8oer,1m - uc so like para6n w* them ap.rt. They areconpletely in appearance, Gel andtexnrreilal it is dificult to re11 solulle in wata od rhey rage in helting points fron .roud 2J jt" C. They de rc1nbleh or m softoed by cvery liquid cxccpt nerctrry, md this propertv nak$ lie dattoiis ad adlesion ofsections so troublesone od anerdcd by so rnmy fanLuesthat few didotoFjsts h.vc dre |]tiehce to usetheh. In a w3m md huid atnosph*e they show a tendencl to bccone hygroscopic, dd lhis dctr n, ..ill fr.he' i,om Lho ongirJ e,r\ prooi c. As hfLltration nedia ihey fosss a nljdes of coustitution od a high adheion to the spccimor which ue htuitely supenorto prdln vax. Th6e propeities give polyethyleneglycols a sectioningma ribboning capeiry vhich n excellot, and dreir .henical constitutio! is suchdut infiltrated matedali softer in thm drar in most Sectiomof thcscglycols innediately dy to pie.$ when !l.ed upon -ate' or oy otherliquid, md thu it becomesalmostinpo$ible to kep rhcn on the slidc,lecaue evediftl& d;;tcgr.tion i linitcd by linitng the nea ofwater on drc nicroscope slidesuficiutrvu ; srill disolvcd to get bdoq the specidd. The secrionis tluefore rcsting on xr, not on the glas, md in dnc coune vhen tl; w* il disolved away du sa:tion con* amy fion the g1os. The nco most suuble grades{e polyethylde slycol r,ooo dd po\tlhylee glycol r.5oo. The mrnb*s re6r to the :pproxinatc molccul* weight. TLis mber n approxinate l*atse it ls alnos! ihro$ible comDerciauyto separaterue tiactions dd a a rsut ihe polyeihylde glycoh re sinilar to p&6n wd in fiat tlty arc niatu$ of variols neftiig point fractios rather &or pue subsluccs. The dpproitute mehing points of r,ooo aud r,:oo polyerhyleneglycols .re as r,ooo 38-42'C r,Joo 4J-J6oc Like tle polyetL,vloe glycol cstee t}e nelting points ofthe polycrtryioe glycols show a todocy to v{y flon batch to brtch, od the gencnl constitution and physicalpropenes show corrcspondiryflnctuatio*. For sectioningdd ribboling drc r.5oo polycth,vloe glycol nay bc considered better thu the r,ooo becauscit is hsder ud showsles conlre$iou, ud shoDldit vrl in its rm!ftclue towrds the solier sideit is sti11uable, wher6 thl jr not nd$erily trrc ofpol,vethylme glyol r,mo. The higler noleculu weight, sucho

Wdtet SolubieWdxet

5z polyethyloe giycol aooo lave rhc diedvantage rhar rhey take longe! ro leDeftate tises. AI the tolyethylene glycols have rhe very big adv&iage ovq e'ery o$d wd-like medih that they po$es ghcol groupiigs which cortu upon then the ca?acityofleeling tissuessott - or hndl soflei iid doesu infiltntion ne&m h slire ofthe'e advst ga Lowever rct ore ofthc polyetlylare giycols is worth uiDg alone- that is llmodifed. The flaaening of sectiou cnt in rhesemedia; the rerdtion ofs{tion shaF; dd dre adhesiorofsecriou sd nblos n oqly acconflished by so nuch tediou cFort, md yer vidr so litde ccnaifty ofcob!1ere suc6s dDt tLe rse of ti6e ncdia $ a stdddd herlrcd of rouline procedue is nor volth

wlne.

3y ni:ing viii laldc acid t]r polyglycols nay be modiied to . ?oint at vhich they stjfl retlin tleil lr]ncipal fotures but my !e ued like parafu or polyesterwax. Sedioru ed ribbod hay be ar with rclarive de, fattored ud moutcd without excc$ivc di6cu1ty, dd they will stick. In addidoo 6e nelring poidr oflle ni{lure is lowu thm that ofthe polyetlylare glycol med. Eren this miarure is considered suilablcody for siigle se.tion slides. The lollowing factsapply to tie mi*urc: lolyethyloe g1ro1r,5oo 7o m1. jo nl. Lauric acid Solubilitl Solublcin:o% ethyl alcohol aqucousar 3o" C or alove. Readily dispcni e in s'ater. Soiublcin retonc. crhylme glvcol nonoeth,vl ethd, erhyleneglyol nolroluryl edrer,:a1ene,bcuene, c oroforn. sedon ugc: --:o d ' room tenpcr"tureoli:o C. Ribbon range: r-rz a at a rooo temlelatue of2oo C. Melting ponrl €" C. Block lurdming: A blocL nruuring 2J x lo x :o m.; ready for cutting at 5 a in one hou aft6lreing in a refrigcntor .t J' C. At a room tempffature of:o" c such: hlock will take about two houn lcfore it is rerdy for ottingSectioncuttlngr Tbn should be doneat a sped olabour 80 ler ninute, ad tlough the best resul* :re oLtainedar a room temleratue betwftt r8-2J' C it vil stil i L b o o r d . e ( b o o r I o o m t " r r p e r - a r e . l l g b t l,yb o r e t h i r d g e .

The bjxtue is not reormoded for suial seaion work lecaue of the difiiculry ofoaipulnhg tLe ribbou oncerheyre orliquid. Serialsectiorsruy be nouted wirh succ* providbg tLe srctioro ue uoud ro 4, but at J /r the sectio$ de so softenedhy the dattennrgtuid tlat they cdrot be hoved. Serid se.tion work at

j8

SectobCattilg ifl Midavopl ro p is there6re posible, but not at uytli4 is aslov asabout f C.

lowo unlessrhe room tcmPerature

The mkturc mry !e n*d for singlcsectionvork ofa routibe or iesedch natue. Secnorodorn to z ir cn be cut ofa rilbon od mooted oo a nicroscope slide mth ede, bui once de sedioo n pload on fte slide n cmot !e noved, if . flldoing

fluidr : r. Joo/"ethyl alcohol aqreous eo hl. ro ol. Foid.ldehyde ,ro% ,. Amylolectin solntion(p. i,4 p o J , e L t l l r eg l y c o rl ' 5 m Lurc rod

r. z. 3. 4. j. 7.

t" -llv.p. 3 or u J

,e ,,. c

Briry nred wched speinm into 7o|" edr11alcohol, aqueoas. 7o nl. solvot, 3o nrl. gly.ol lauic acid. 5o nl. solvot, 5o ol. glycol huric:cid. 3o nl. solvor, 7o ml. glycollauric acid. Pure glycol l.uic rcid at a2" C. -

,, J.

Biock naking witL po\edrylene gly.ol r,5oo 7o nl. ladc acid 3o ! r. ,. r. a. :.

Sned L picceswiii M.ye!'s alblrrd. PourLJ'cnol.ff hr inro rl.. arruged t piee wait util a skin of vu hs fotued on dre iside oldr met.l L PiEe' Liquify tle ffface ofde wd vidr a hot s!,ftL Placc dre hilnated specin@ jnto dt l,]ock using hot forcePsand arange the speciod s requir€dfor later dtting. Placethe spe.imd away frcn the niddle of dre b1o&, od for prcltrefte stick it agaiqstdr sidc by rnshiry n gddy iato de coneededskin there Thir is e$dtial be.abe it is a fcatureofpo\ethyloe giyrcl. th.idey frcquo'dy show a sPlit or Gadurc io tne niddle ofthe block

6. Cool at room tempqatlte mtil tle block is soli4 but keP the suface liquid fm so@ tihe by un]g e hot spatulaor ly tuf,ng the bmo bmq dowa uroD 1t eldv

nov

md agd,

r. Lare the Uock to hndJn @np1ete1yoEmigh! d Placeit in a refrigeretor to quickcndns proce$.

Ct!ttiflS Cut sectios ar approxihately rie sme speedasprafu

vu, aboft 8o_r@sections

Flartniry abdAlh'sio,t o.fSetiof r. Snc{ a dean sliderliily virh Mry€rk albuso. 2. Hood vir! jo% erhyl .lcohot aqueou eonl.

ry_b|eq' solution5o mt., :bsoluteettyl alcoholro hl. :: y'q 3. Slakcthe slidesothat mostofrhe sotuliotrdropsof' z. Plr( " .eriondetid) m Lledrinflnoffl,,d. IG.e<.riorwilLrmcdiatelv $ho. but irfic ltud i" orJr ,Er(-3nour lni i. aJ LrLtwrl h"ppcarrtoi nlo ru,r I pr6mt rbr wa p"r or ltbe".ctjon \iI "bow " ceain cmort ot dlbregrudon-bu tle soecimo wiJt renair mdan"g.d. J. Dain the slide ofdy fi!&er rraccsoff,uid. 6. Dry in u ovm.t ebout3J-4o.C. 7. rrom thcn otr rrc.t $ a pm{fir w* sectioa or disolft dE vd 6oh the rdron, wi$, eilhtr "b$lure erhylct.ohot.m." edrytJcohoJrqreus. or -o.. cD) -r-obo'rqreou.

Ano-l-rrm6od wlLi.bwolk widr . f,i- deg,( of.u.. e- i, de,et) ro pre" Lhe q bm dowDlpon r \]rde Ltridy r +r rc uid " bmo. \o liqud : Ml. some tLe.iLkcd r OJ cn o.ete. to ue r drb -p,per -mb. - such,, .isJeue pape,.Tbe oer-hods o be or) wir rery fld sndos. p"t poltethftL\e Crycat 4ao lle purcgly.ol nry be u.d ir rhc - ,e u_y d n !trenir uc glvebJbove,b,,t Mrh rcs c\ice( o.,ucc..ful F.doa .dieion ro rLios.lr i, s,ouj rc oe tle pue glycol fc raiom showiry fai.

r. Fix narerial itr fornalin o! in Bakeis formal-calci@ (p. 8t. :. &Jir D \Jr.,rd plae d,c,pr(incn tr wrd 7\ hj, r,,o poty.thylene g )rclis gm. llrm in " rc jo niruc tlen a zs -s, r d tuJy j, ro pue *;fto gl)co r s o\c- ar "b,ur jr. C. blnldre rd o_!e r block. 3. Cut secriomat aloui ror. ?l.e rhe cut secdonson: damp 6ite. papd. This prevencso excersofwaro bustiag tien. 4. Placetle 6lto grper in a shaliowdish of war.r. The secriou will float free, and ftoln ther o they slrculd be treated* &oa sectio*, srain.d vith aplropiiate dye soluriois dd mouted id F{rdtt nediun

CE^!r!r

14

wAX SICTIONS: FAUITS, R-EASONSAND REMIDIES DURING CUTTING AND AfTIR u/r.r*u

, ,.**

,*r

r* ribbot. Ot the ribbondirid* alan! itslefllth iflto t'to or tte

Frc. 14. Tbe bbo! shoer lind, or my rylir

;4!e LdIe hx iageederleo, b'6(e{ or dored Th6e defects*y be du rc o *n1 co"ditoo-iF,h.GG "hich ws rhse bdorc *aioDins .1,ned or ir ruy h.vc uirn during sectiooiDgs a 6dt of $r in the sPecin$, chitia touching the klifi edse with a nedle c foeps, o etching tlre haia of a sablebrush betwem the edge od drc block when colleairy seaioro duing cutting. r. z. "/. +.

Hone rhe krife. nrminc tlrc blocL mder r low powe binocdar nicoscope ud if p"ssible pi& out uy piees ofgrit Rei'fdtr.tc dE slecino in r hrde w*. Ifthis faiis ue dre celloidin teclniqe or the ure-follraldehyde rein method.

'r2.rhery!!!y::

I 'l,;

Tlr edg* ofrJr blo& wLich ate prallel to the knire eJgc are no' :traisht. Tbe rop dd bonom edgesof rle bbcl re oot pr:lJel The lnife ir $ap rd blmt it Patch6. The top and botton edge of the blo& re not pam.llelwiti the knife edge. The slccimd is eccentricalyplacedin dE block.

W# Seaians:Fdalts,Reers dd RNbedies Dutif! Ctftiry andAltu

6r

E U E U E E FG. t5,

Sone oLE

of ming

nbho4

6. The wu n softer ia one 9a* due to: (a) . le! beiog too n@r rie block; (6) tlacs ofcleding ageir iemaining in the w*; (c) c.lcuing agat ooziry froh rhe slccina. ;r, z ud 5 retrin thc block. l. Move the laic to a berler position or slurpa n or rhe hoDe. a. Rotaie &e blo&+older ud the b1o.k edg6 are lalrld with the kdfe edsc. rnove rhc le!, 0) isblock h ptrc vd, G) reinnhate. ,6. k) /3.Ilales Appw h theSttian

Fr. :6.

r. :. 3. a.

IIoG3 i[ t[e s!€inrn

Theren u dr bEbl,lcir dlc slccimd. a p;"* .fFiiilJ-t".iJ eithu io the specirno or in the wx. A sofl latch of wd due to tlaes ofcleding agdl A so& liece of slecinei which n tcaring ifftead of cuning (incggplgqer@q;"q)' J. Watd suckedinto the tlock dciry ooling.

62

Sati.n

uinfi,l, Mdoleofl

r. Cdritue drting ifdr tisus do not appeardistoned. If rLey do r-block. z. Renove sud or grit from the specirEl udd a iow_fowd binocul.r niao_ 'copeif po.ible. Usea rcry 6nc needte.If thi ,,mor be ddr rr-inflhre rbc block in . harder nedim. 3. Re-block. 4. Reiniltlate. 5. Rcblock. The Sdtiors Shau Gtula Pdnll ith tte E.lScol theKn(e

F!c, rt.

!'

Cnck acrc$ the sction

r. A bbor kDife. :. t flnifr." -edl"'" too hard for the rhicknessof thc section. 3. The hife tilt ls too small. a. specimiritiffid. mgle too_grcar. 5. rnifiL&il The cdge of a nicrctonc kdfe, rherher of glss or of nctal is wedge+haped. wheD srch a vedgc ii pushedinto either resin or into ws the rodercy {or rhe thiDer sliceis to split awry frod rhe hanl block. If rhe sectionis a thtn one rclative to the hddnes of the blocl
r. ,. 3. a.

Hone rhe lniG. wro lhe bloc! Jisbdv. or (r Lhinacrrftjod. In6ee ihe rilt dglc. sring up dorhd specino using a &Screut Sxtive, dtemcdium, erc., or use eot\er illriadon nediun. 5. Use a hG with a maller bcvel ugle.

Wdx Sedian!: Fa"Ls, Fr6an! and Renedks Dttih! llhik

Crti,g

and.!kl

6j

Ma*s or Coftugatio* Ado$ the Ribbafl

ftc. 13. Codusatedscti ons

:. A vibrrbrq lnife edee. t, ,

ljLock roose on holder. H^]J.,l^^.. ^" -,;,^,^--

J. KniG loosein clanps. r. Move thc lnife to a shrper rcgion o! hone it. 2. Ue a thickd, str@gei knifq or ifthis cmot be dooeusea larger, more powerful miaotone, whnh vi hdvea thicLea snongerhifa :. Rmove thc blo& od stick it oo to the b1o&-holder again,taking carenot to trap an bubllcs letwmn the llock md the ho1do. md + s. Tightcn block{older and lnife c1mps. C,rred o 'l Ralliry Sstians A sidevicw of tlc edgcof a nmber of lnives staniq or tie le6 with one which hrs a shary edgc od pasing through r serieswhich becones progresively morc blmt, is shom below. The eFectof a blut edgeor1tlrc scctionis to indee a cwe which lconcs nore plonouced a &e hile becomo norc blut. With very blut knives, wkre the *e. ,l e secdonrI L. torc.d bto a rou s r is bebg or. edei h- 6;-.r, is. -r'. '.--.0 by rle rcr (cc "i l" .i"ud tbe kofe edsebccomcs nauover a dre hife become more blut, md the presute ofthe wx ir that region lorce rhe sectiot nrto a curve. r. The IaG is blut. 2. The vd crystd sia ; too sna11relative to the thicl*ss oftle sectioq od tle prevailirg @nditios. 3. The lniG tilt is mong. It nay !e too horizontal. 4. The lnife bevel ogle ruy be too sr€t lor that pdicular infiltration ncdiun !t dlc givcDthicknes dd rooh tenperaftre.

W W W w"* W Sedior Crtting h Mnfts@ll

f,,

/ / / /

r. Hone the lnife thorolghly. 2. Cut fiimer sectios. If rhe crystalsizeofthe ncdiun ls so sn,11dut it is cau.ing trouble- thimer samom will be fl:ner. 3. adpt fi. ffie soo\ar it n --. pe,podrcul*. & Try e lni6 with a smlc! bflel egle. Thi.k dkd Thin Sedio$ Fo1ar edchOt et

Frc. 20,

Vriatio,

in thiclnc$ of sectiod

Wdx Sstians:Fauks,Ree$ dnd Retr.dhsDuting C'uiig 4id A.ftu

6J

r. The ws is too soft lor that pmicular thimes of tle srction. ,. The block i. too luCerdadv- ro Lne,rength ol r.heknitc. whicl is forcedout ofot digs irto the bLock. 3. The hifc is loose. 4. The laifc i !1unt. j. The block is looseon irs holder. r. Pl"ce the llock ud hicrotomc, wnh hG, in a rdrig*ator, or rehiltratc the specino wirh a lrdo vu. 2. Rcd@ethe sizeoftlc blo.k ifpo$ible, or cu*he block on a srong* nicotone, or 6t a strongo lnife. 3 ad 5. Check all pans for loosoes. 4. Horcthc k fe. TtreSdiots R.f"se b llbban

O q O:::r 3 Flc-2r. somea6es ofloor nb6orins

r. z. l. a. 5. o.

The to! ud bottom edg* ofthe tlock arenot araight. TLe top ad botton edgo m not paollel. Tbe bb:!,qqmas havcb€ roidrd ofbr,Jele. bidig. The Lrife is blut prodrcingo lirg ro rolhg rrcor. The biG tilt is wong. the knife isgredf, Lbu !'rvmling e eJ.y.IdiiS rovemd( of tle ribooo d.* i|s l,ii7. The relation$ip bewem mon tcmpcntuc, melting point or hudaes ofwu ard sectionthickfts tuy be wong. 8. The wu my be contminated ddl edryood oil, etc. v'o. The tuftine sDced mrv be wro@. Too s1ow. o. I h{e Eay t€ r b.ilF p blo.L of \m betud lhe tflIe.

Stti

Csuin! in Midoyopl

r, : rd j. Rrnin dle blo(k so !h$ rhe top od bonofl rdg6 de perattelwiih €ci orirr ud Lie edseofdr njdoroDe Lrife-Md arest;shr. 4. Move thc ld6 mtil : bcner larr i, foud, or hone ir. J. Move the knife inro a norc vdtic,l positioD. 6. cle dr- lnife ylh xfloe. 7. The h{dd tle wd tLe uorc .lifrcdr ii is to obrain , rilboL Widr Nch hdd wus the thimer the sectioo the bener thc prospets of oblaining a ribbon. Thee 6ctoa are gmtty iniuoced !y the r;n-redp-"t"re. Tfiiefde if. hdd vE n beiis &d cur lhe secriotuiE . vatue! rooh, o! rry @lting rhm thimer. An clectric lmp fai+ ned t,\e oieotome nay lmdtr@ d; risht nbbonins tdFafi;The bl;& my "1" be dip?cd in;o-notten w* o? a Iow* nelting point - sayaj' C. This vil lrovide e outsideslin wlich vilt halc bett€r adheivc lroperties at thc giver tenperanrrc ofthe roon thn will the hdds iside w*. 8. Reblo& usins pure wd. /9. A sm t imlact betweo the sctioa leing ot a:rd rle oDc lyiis on rie lnife edgei5 dsdfial to lrobore good adhsion betwed rlrc scctios. Wirh !@fi! ea M.P. J2' C a dtriDg rate ofahour roo sectioropc minutc is rbort right at a roon temperatureof 17 uo' C. Wirh ests wd, wNch should be ot at about 3eJo pc njntq iie begiming ofthe cut should be m.de very qui.kly ed thm the rct of the strole should be very s1ow.This initi.l speedproducesrhc fora ofimpaa de$ary fo. good nbboniDg. Io. Sone wses, !!nicul.!iy polyelhyl@e glycol etcn, show nore adhesive prolerties thu othes, ud lhey my shoq a tend@cy to stick to the tack of tle Lnirc.pm, 'rjrl' q o clc b o"l L " :-ge ooeod r\e kdlc is €cror.. tr &er cir.ud.tucB lhe kDfe L bebr,"") .ron Lhcblocl dubg cle domwrd cut, bft ir sFiDgsb.ck slighrly vhen dreblock ispsr. A . resultthe bloc.ktouches de backofthe hnrc on rhe w:y up, ud leavcsa dcposit.This depositis ind@$d at eachcut ad fiml1y it aliecrstle sufa@ of !]rc upward noving block dd prcvats good ribboning. Cled the knife edgewirh xyldc.

r tus!:.: :tt,*t:gtv* :"' -9:v!!:.":!Pt:r,: r. ldperfed i JFrdol of rhe obyc $lLiJ L bot .utpoEed adeqrrely. md rh";J6;" .i;-h-i: ,. Spe.imfl ovdh""Fd d.dne *- inflEJriob.bd ir i, -terefoleloo hrd ud br*L,

"p,-'i+;;Jd*L:F.*

3. Kdfe blut, dd tuy bejagged. tulI of giinf tut{i,l vhch prodlcs No. 3, a. Sldno J. Spdifren surouded with a shiny polnhed covoing to which the rw adhere,e.g. mp 3bdomd,.4J.d/6, erc.

cmot

W'6 Sedians:Fdrlts, RedansdfldRenediesD"tibg Crtting drd AJtu

67

'. Reinilturc thespeciaeD if! rbper m--lv,o bc due ro Br6dd( ltuglb of h r e n m o l t o u " r . l i l h es p e t r , t r l p p r d ! . o J v cr " . ^ o t s " r c i r i , , n e h the l1oct, rakethe slecihd into rylme, ud then bacLinto alsolute alcoholE somehouts belbre bringing up ,sain. 2. Thiov lhe specinor away and st{r .6esh. If rhn is nor posiblc, as wo!1d bc rhe 6e Nlh a mre specine, takeir bacl into xyleDc,absolLlre .lcohot, ed eo% .1coho1.reeae it in tllis lor sever.l houn to softcn dd britg up €ajn into absolutealcohol, r/enc, ud noften vax. 3. Hone knift. 4. Try ro renore ile gdtty naterial with r tery nle nccdlc undd r lov power :. Littlc cm be done sith tlc sptcintn iu dre w*. It will pay to taLe uotier fixed srccino inro 7o9! rlcohol. rho sody brush rhe su.6ce wirh e sruU " o n " o o l m o i . q e d $ i f i | o , " q u c o uK . o r l . R b e i ! r o o oJ . o o l , ,/l;.e. od Gm bru\ rle u,4c" .brin wib roFon . ool n o.renedwidr voog onic acid. Rirue wcl in 70% dcohol, taLe up ud iiilrrate. Thn tcatmmt sho d roughen thc sufrce uthout d.rnging the inr*ior. The Sstia,! Ca\qle or ShatuExksi,e Chryrsjo

l-o7o'""n compressed sect ions F c. 22,

Scctior slorning cx.esire

comFrssion

r. I{the slccinen .oihlses bur oot rle wax rhc stecihd showsinadequatebf,ttra,. iith. r'* -d ,p""i'.* collapserie wu is too soft to bc cut at the prevailing ,ooD emp(ru,-. o, n cobaiB.olming re/g"". .u.\ "' c,d,Nood oJ. eih)lne gllcol nono ed,ylcdrr, *c.

3. Thelnife is vcry l1mt. + The sectionsalc too rhin ftr t]reprcvailing coqdirionsmd the comaircd materirl. r. R+inilftte dr spccino. 2. R*block in hrder or in rufi

wrx.

68

S..tionCuuingin Miooiopl

4. Cuttb,Le-scrtiou.or h*dm thcblochbyplchg iL@" re( se'ror. rhtbG rl', dat-, "* i,1ta "n *, r,iJe h! theBlark a'1iL'way u!, thusPa!.ntin! Ribboiitry

:

i ti

Frc,23. So,ne elsesotsecdod[f@s otrthekniG

rt'" Ur.; g.*'y o. oi\ ard tie cn sectioncmot slidedom ir completely. 7i, ' At iie end ofrie cut &e sectionspringsup the lniG ad projecrsslighdy beyond the edge.It is thrs *ught by the llock when the block psscs the kniL cdgeon is tpwrd jomey. z. The blo& is looseor the block holder may fron the bloc.ko the seaion is bcing cut ud theunovo 3. rhe llEfrovi towads the block whu the cut is completed.The block tL*efore totches the edle ofthe lnife on the way q md lifrs the sectionofthe hrife. JigtLly a. t Le w* 6 roo .oft or cr) ulirc od dre top yine of r,helb,Jr l e,.t<. lot*Jo-fbiir'g .i,t ; , clro :rocr. TlE b;okrn wr "dlee. o rte.enioo ed ovcrhugs t.hcknifc cdgc.The overhdging lortion is caqht by the retuding blocl ud the sectior n lfred oFiie !nic. 5. The block ls mtlu iorg dd bods a it i5 being .ur. The sectioncut fron tlc blocL ir therefore wedgeshaped,ard it La"es tie qpa sdace of dic llocL longo tho tle lowu *rfrce. so much 1orya tLat the rpper ed lies bctwec! the venical line ofaavel ad the knili edge.The end rh*eforc prese agaiut the kdfe edgewho the block is on ih upw.rd jourcy od |he strtioll on the bife is licked ofi:

War Sa/Jian':Forltt, R?^tutud d Renedies Datug Cstting ad Aftet

6s

6. Tle cl*me oglc letwco thc block od the rear facetof dre knife is too snall. This preventsthe bloc.kclening the knifb on is wy up. z. The lnife is blnt md the l1o& ls conprsei * it pxe the lnife edge.Below tie ldfe the b1o& *pmds, to{ch6 the lnG edgeon rhe way up md 1;frsthe seoon ollthe tlrfe vrr6e8. Built-ur wd delo-siE on the brck of the hife produe a rclgh ud uevo surfaccon the block. A: drc llock riscspct thc lnife efuc the wd stickiq therc and rlt rorgh surftcc of thc blocL tcnd to producc rolh of v* wlnh roll rood t1r hnfe cdgc ud nakc drc scction stick to dre |lock. 9. StaticclcctticiJy gocntcd by the posage of a parafin ws blockfalt tl',s knlfe edgunakcs tlc scctionadlcrc nlnly to n\c knifc before dr wtio! n conllet+ cut ofi'drc blo&. TLe tnd ot prodnco a we in dr sectionwhich ace like : spring md neka tlle sectionprotrude over tnc hife edge,A ir emple r.

z. Rc(rsto dr block to its holder md make sre that all pms of the nicrotone

4. J. 6. 7. 8. e.

R*block in lrcsh, clon wu. Shodcn thc block or cut nr a cooler rooo. Adjut the clqrdEe ogle. HoE dle lnife. Clem the back ofthe l
/ At times .he cbargeofst tic electricily may be so grot that sectioncutring hasto \,6e aludoned for ihe day. should rhis troublc occu frequody ud provc to bc a souce of delay ad btoko working hos a chugc should bc marlc to csterjvlx. Witi thi5 iniltratior Inedih therc is no staticeleariciw problm. 'fhe

SediansShaweitherBdtrl Diltottkn ot Pinushion Distortiul

Frc,2!.

xft'ecb ofmeou.l hrdr$s

oavax md rDeciDm

S{tion C"ttiry ir Mntuya?y r. In brei distortion the sp{iom rei.fofccs the wd. The w* is too soft for the prevdling conditiod ed collapses!r dr o!6ide of rhe s{tio!, blt do6 no! do soto dre samecxlenr in r.hemiddle due to tie suppon oftte spccirtl. Thc rvax r u 1 b c r c o . o r r -b _ -.. e t i , ' e - t c r r _ r e o f d c , o o r ioo hiJh, b..,.. r n d e 4 ' fr - , - , o i ' . f f s r . . n . p . c , f l ! : o . b { \ e ! t r , | n i r eL r o o L . u r r o on sectionsoftn* drimas in dle givetrcondiridN. 2. Pincuhior di\ro.rion i due to colhpseof the specino, whicf n softerthd iie suriouding s.d. Ir rnal.be in rhis coDdirionbecnse ir tDs not bee! rutrcieni]v D r r i , e d M l h \ , d . l f e , e r n " ! r , . e . e c ob?- q , . * . , t , o r o r . . e , i r g r g . r o l lir lresent in the spccimeD. r. Re-block tle sr.ind lsidg nq', lure vax, of. trardd chanclcr o. ofa highcr nelring poini ALtcdari!-ty cut rhe secrionsin a cool roo 2. Reirfiitftte md re-block db specincn. F . . . 1 t , . , , u . c . .r e m e d i e a . r r e rc c . . i r g .

L The sectim_re dmtbd ord fail to flnto when pbced or a nidoscope stidc with distiuedFarq. Waming a! $our 4J. C dlttm dr wx but rhe specinen re:liq a lirge Nnbq of wiinlres. when rtc flaildr,,s ivater [* ben :dn dnjned ofr dd ihc slidedded, rie wrinHe, arc sdlt f.esent ; lhe sre.inen. Thc specnno Lrs bem incoollctely innttr.ted, ard a vfd $luble dtencdiun oi dexiag ager h6 bed !sid, hacesofrvhich stil renain h dr srecimen.This p q n - r , $ J r , . o g e rD , o d , e\ t n r n . d . a r r J j o $ j g i . r o x ; U . L i g h r l lL. . M o I h r J d et . ^ " 1 b o r b d . i q . r t L , h . r c { " , g ' ) l r d r o p h o b i oc J, duelore it beconesor ienains siighity wiiHed. The specino cont:iN tis$ese Hch areslrongl).resislui ro iie iiflrarion of,.d cvo r.hor mrydn:g hc leeu doe to osure thr. Suchrissues, cvens.heu rhey hde pafd dno"g!{"ter-reistot fluids sucho cedrwood oil, rc srlt hy&o'phylic, atd still s$ell slighdy rvhen iu contactwittr wner 3 , The w* is to.oft ud_ tle speciher idtead of receiving 3d€qratesupporr r c . i r . _ o l i n l . h a i , o l l p r . r . d F d i . - i . p " . . n ; t h o . g r , s . 1 1 " .- . . r . , d r : r c "o r \ . i n l l n s " { c h D - - y - . . . . " * . . , . , ; , l - , , - ( : t r o . t r r e . n d c J . 4. 5. of the stecine, vlicL dunltcs as rhc tnife pdses Td.fyt"_:lq'i!'." 6,

srcli:tu ere roo iiill i, vie!. of dre prevailing lrysical conditions. :le rne speedor ruftDs b \vrotre, 'c j.". . ; Lr: .ti.i ;, ,o. ..-rr ,o ,tro, ,r,. .((.or to errrno. I )v,-, Tle )cdi . i -ouo's 'l-e edgeof rh Lidc.

Wox Settiok\:Fdrks, Redsan! ahdRe edie\D,tiflg C,fi'g dh,lA.ftet

j7

r. R+inilthte tle specidelr. 2. Litde can bc done to @!e dns .ondirion. New rieccs of djsud iniltrared with the uea-fomDldeh]de tcclnique or Nirh celloidin would give better rcsults. , . r a lr n p r r d l l i e - .i r g r J . eb o . i b ) p i , . , i g r i n " " c f r i " - " J , t 1.,, - t o,-. .r d ^ . . e g r e r c r . . p o o a, o J r c p e c i n , e " . ]h! .,ir-,'Lrot. .r {rl rerddrBre $ rlh e drdq $,8, J. R+infltut€ tle slecimd. 6. Cut thi&er sectios or clill tlic block !s in 3 G). .. \ary&etul'ilsf 4 r i o m b o u r r e r . o p e r m . r e n r i l e , p e ei d. r o s J r wrxctrde rcdoDssnowleal vrokxng. 8. Sccthai s!ficiot uc. ofwarer n available. ;rid;u; J*"i. a.y "*" ",t * a. ",rrace oi thc slideis floodcd e. The4$tl6; w i \ w r 1 . l f - . e r i o nn r o u c; r g r l e e d g ei r - d I . ' - r o r h . d r y . o a . . " n 1 pr*eur ,6r otLhc ribbo e.p nJing. Sccrjon'"rould d,sefor be kclt -.Ie clcar of the extroe edgesofa s1ide. IL

The sediotu or riblos fail ro sdck ro rhe stdc during rleir teament in variom

t . 1 ) c m i c . o , c o p' e %e. o J - b e b r c* e , l b . a e r d R r f t c n j n g nuc atie PDcedupon cD. 2. The siid$ had origielly bed lvahed in a rcxp or x dete.gcn!solurionto deen thcm. Minutg tres of dN rcagentsu. .."cici.rt r. " o- *"t". to ..*p bctwo the srnon ud the glas slidc. 3. Thc sectiou Lad air patchesbetweendren ard the g1as. 4. Alkrline sohtions h.vc leo L6ed in the srailins procsses. Ttcse solurions rcrg_rvo6..n ri di ,olv, bo t. . . tb ,1n t,otdf. rhc sr,. .r !o the ,de. I o n s a d ! - e r o I e p o l i l . . o. r r r o r , h " d . . 5. During ffatteningtle watc on &e slidc hecaneovchexred.nd perDi$ed trrce of oeltcd wax to get betwco rhe sctioD ed tle slide,th$ a owhe the r.rion to float :way rvhen the wu on whidi it resred\as dissotvedin slcne. o . T h e n , ( r i J . . J , . . h i d n . r : J u e I . u r i J e.., . . r r o o . r d t o e d * o n r r e slide.P&rs ofit duelore arelloating fiedy in liguid c sooqasdr wu ha been dis$lved_away in !l'lde. F.cqucnt liquid novenmn tkough tirc various ftagdtsloso rle rans which wcrc totchhg dr stide,od e,;u"q, tins bd, to completeor gartial rvashing:way ofthe section. 7. 1oo mu"l M-) ei" Jbum6 !- -ber J",1. r-L lurd .o-.n* o . eocgelbbo - \ . e i v eo . r , : r : s r r e p l , e d u p o n , . I J e r d g r ) r e r o .r q r p J r s .r d de g1y@rolienaib there evcn afrer rle rcrion l$ been €.trened-nd the slide dr-ined.Gllcerot , ".":d.yTc L_1., d d- "ben t.F,e L cxa.. pie "ur ,Lc : {pe,ho neve!re_uvroucte. rhc g -\ ,ridebu! I B on . 6tn of Bt).c,o . A.

12

Sation Cttting ifl Midatull

soou as t1rcsir'deir htoght tuough ryla1e ed into ,bsoluie alcohol od 9o7" alcoholtle glycetol legis to dissolve,aid by the tihe rhe slidehr ben ltought r o w " r . rm o t o f r l r : c c i o u r - 6 0 d l g l o o . e . 8. The .dhesiveis old od hd losr its pro?enies. q, Thc sectioaor slidc hd aor lee! dricd long e'ough. There is litde vhio\ d be done to curc the conditioro oudired alove. Methods ained at preventiouareindicated. Sl orrldth. tur '.-'r'e or - D'rJ olinroftut J de. "l-o. Llcscdefe.Bir rill be vor-h rliJe Llirg r1c rcnud* rJ ough the fo ovirg: r. Xylcle to remove rtrc wd. :. Absolutealcohol. r. Absolute rlcohol so ol., eth* ae n1., allulose ninate r gn. a. Lifi tle slide&oh soltrtion 3 od hold it in the air for a Gv sdondr eril it is jd begming ro dt. P'rc jtrediJ.- ) in,o 8oooJrohor, :ooo 1 .rcr.Do bor l e L l - f . ! d . d , . T t e 8 o " " x l . o f o t ^ o n g c , tt,t r e u u o . e u u l o , co d b o l d sL h e se. ioo. to rL..Lide.\-oElorirc,a"-'.r*.d"*. 5. to.a alc;ihol,30% Jcohol,dc. With *dple z. where ex.6r,Ma1.d's alblIm bas b*n s€d oo dethod will nDk-e seh se;od sticL;-o i6iiia". r"" r,l"v*, al-.* shouid bc ued or naker ;d];;ddis' adlesire rlr*n (p. Do). Farlb ali.r sectiooiig: Ister wx. L The sedionsare clulled scopeslide:nd wfncd.

ud fa11toflrtten when placedon w,rer or a nicro-

As for paraEnw,,! vith tie folowing additioir: r. The scctios havebeer cut too soonafter rie block ha bco nDde. \ 2. The sccriotuhavebeer cutoo quic!1y. 3. Too nuch heaths beenucd in d cfi-orrro nule rie s€rioru flattm.

r. lt tuI pay to Lcepe 6ter lvd bloc! 2 Lous beforc attmpting to cut secriou. This oables it to achicvca honogdeous f!d!c$. 2. Cut et 3GJo !e. rinrte. 3. Do not edploy hcatoverJooCtr. The sectio$ or ribbos fril to stick to the dide duing their rr€tnmt

-Asfor pmltu rx. As for prafrn w*.

wirh

!E{}rsr

15

LOT''\?'ISCOSITY CILLIJT,OSE NITRATb Thi reagot is al$ krcM s celloidin, collodioq prlodion, etc. sinceits introdlc_ uon ir Ge Jrvr p'rt o. r}c ru..e-nti c-orury1o,,u,r.-8701i. ir beq ttrc mo\L $idell bed Dof-ribbodingiDf,lF,rionbedffi. ,r" pccjJ farue is lhr no trer L lscd dul'g idlhrdoD. Fo, .ad) ,e a rL sr. ,c.! sahe,. Lr n!j]e heb vi,cosjcv or its sol.,dorb.PneuJrob ilro ri< r, ws ucrcfore , tons p,.cs,. i,, -.dem m(!Iod, oftujLf"fturc mbh lor vLcosiry.eUuto"e ni 1rc ro b- produ.ed.!o il,:! innl?don dF6 tod"y de rrcl lcs .ha tber qerc r( tnrertu;d r€acn.ro"al Ceilulor ni'me L,oJ.ble ir _brc'-recti1,,tcohor rd etlti".d. s-.;ii 5o 5o; ir clovc oiJ.o I *tu,e. m"d,t bdorc dd .oy oLIo ern.ir : not \oluble D Lhlo.otorr or ir ,l.ohol wiJr wkr p,ernr, rd btob uc dere,bre gorally trdoed n cbJoofom "irr.rr 6r *po,. * r," Lqjd. dd sro,edjn ,e-8o% alcohol ud warer. Cellu1osenitrate inf,lrarion is conducted xt rooh tcmp*atu.e, ud therefore ri$us $o$ Do he",rhtugc or r", ,rqf,c... ud "r ir gfterJ "o n r i, " ceUoidin blolt Lhn in otr orI&fir wd. r d o ng ,o l| c.,c ,nd co-p-t uru- tud u s u . . ! ( h 6 d n J c i t r . dr e e ( hr , c e i v e- , o r e p t o n , h e j , p o ( r b ; w i l r c r r . J L i n e To the hislo1ogistture is a snoodl clDrn abou! a celoidin secrionwhich ij nr.tv or oer*.m ir a pra6r wa \eftion.rd nore worl woutd be done u.ue tts bediM Mre,ir ho! for b onge hf_Jr.,ror rire. rhi. rrv br dFhbg ao; to ro tu4 does ffDt ofd ior wudhtrntioh Ab,otuee$, I Jrcbot rd ed* .rc g4rJy urd o tbe sotrenrfor to* vi,cosiry bFot'ur'lo\e. bu, )onF work* prLr cloveoil. ,m1i,ar,te, e(. n h!,cvrr ,olvmr is usedall the fatty natenai ofn\e dssus is disolved uu,. Ar dmesde s1om6s olinnltratioa hd 1edto variou nethods v]trcby inflrratio, nay be aaelrated by hering the celluloseoihate rclution, rhm driving off rLe rclarile rclvdJ rhercb thickoing tlc sohtion ed reducins rte rine wiich tln ?rocesswouid no@a y take. Thse is litde to be seid for ths q,pe ofapproach. If rctids arc rcaiiy nedcd ar rlat sped it wodd be berrerto ue a !n"m o.tma .r sPccd,slcr aslrc(fug. 3@dr tlc nia'dellDlse is Lepr bodr in dd aro6d iie srecin€n ud b@use it . 1". r sffobgrendoq Lorc.cpr\..ib. {ben Jrc.etuoD.lr" D 1 dtrousb(Lninc brr}5. ,t L o,io b-a- -o {r, rhcd rd wled,nnctut n Lu]I ,",t..,t-i-*.a.i r--. srrir' $'L a. \ enJ,.r. cde.tu btuerc1y, roso,,sd bdd arrue, re largety ftvoucd ro|i,. wo,", be.ru.efi.v tuy bc s(d pros,e.,N.l)_ +{ r d"y do"D; hied diflrolldon lr!-' {.idilg - rd be,6c tie. 1r,611"^r to.emo!€t by .Lohol $hdob. 5houldoteltrmc in butl "ccu, I i. o e*y *tt< to rmo,c

74

Sution Ctttitg ir Mictottopy

A wokns ofthe nitocelluiose tr.hnique ii thar sectiod cut iD the lrsual1^,eyare gmeraliy thick, sone rs-+o a, but thn *y "1sobe usedto mrled ad@tage foi low pos'u binocuk work, wJ'erethe trmpuocy ard g'eat de?th oftlick setiotu arood 4erm p revcal a spatialuugenent of orgus od tisses which thisrer sa.too cuot posibly sugg.t. Celloidin block my be ot who b two conditios: r. I! the $et statewhd they arc leboved f,oD 80% .l@ho], 20% wter dd cut dnecdy oo d midorome. Eachseaion is placedimediately into 70% alcohol ad the block is noistoed vith this fluid a eh sccuonx cur 2. In the so*aled dry st.te who a celloidin block n taker *ori its stonge nuld j*o cllorofoo od absolutealcohol 5oi5o, tho pure cllorofom, then ceduwood oil. Two or tuec batlx of ch regat sh..ld be ued, md ihe Plocer @y ta.kcsoe wceks fo! a ltge !1ock. Eventmlly t1t blocl is thorotghty ioprcgated wit! oi1 aad is ot in tlar .onditior Ir is much luds, * a rule thu thc block ot ia the wet coo&tioa ud asa re.ult drimr sctions nay le obaiaed. The proes is long md te&ou. of the prutiol data tlat ry .e[oidin block It wi11be cled Son d ddistion !o a ontent ofro nor. tha perhapsaoo/"solid (cellulosenitrlte) Dd 60% liquid to the required hrdms. This is a condition wNch alir it !n ler dried ot ge11ed brings vith it sectiotrcuttitrg advmtags foudin ro oth* nediun. It also nem that srch setiors ernot be dried, beousethey will losethe 6o7oliquid bdk. Wha thn fappos the sectionsd,"y, cud ofi'the slide ud becone distoned to t loint a! rvhich they are useles. The esy sfu of the lnife throqh a celloidln block n due to the fact tlar in dy ?ositioo the eoEt ofsolid celloidin beiog ot is ody 4o% ofwhat it rp!€rs to be. The 6olq .dded expsion due to wlatever liquid is prsent in tlE block gi\€s a lutrication which pronotes an elty snooth, sliding action of the microtorne laife. This is rot the sane conditiou asthat whi& 6ists ih a resitrsucha @thyt neth& .rylate which la b,,n platicized vi& &butyl phdblde. Suche additive prodes a softe blocL tto ore of pue netlyl netiao/ate, but such a block cmot hrvc the butyl !h6"1atc repla@dby !1achg it sry in 8oo/"dlcohol.A cclloidin block taLo fron 8o% alcohol aad wat*, md plaed in dnoro6rm .em.i$ the sahe sie, but the alcohol is (@oved by tle cllorofom, rvhich in tm nay le rmoved by cdar wood oil. Tln suggesrs" ge1-likecon&tion or a rt-like structue ofsuch porosity that tlere Day be a fee intercJuge ofv*ious liquidr without ct4ge in fie volme or condition of t-heblock. Nitrocelulose in the dry .ondition is *plosive. it nut drereforealwys be kcpt doped vith rbutyl alcohol.When weighing it thae js no ned to takeifto cdculation &e weQlrt of alcoholpreet. Tmes of noistue (water) in a solurion of nitroellulose lead to a slight t'$drs in the foished blo&. Thn is oot a seriousde6ct, but sone workus prelcr to weigh our the req&cd @ost of ellulose titrate in the dmp conditioa dry ir oD a filtd paperovenQht rd then add it to the tequiredmoot of.bsolute alcohol to vhich I tho addeda sinnar qmtity ofether The rqult is a block otgreatd cldity.

Iau l4,bsitI Ceu,bs;,Nitro?

7s

It m,1 be ued D coDjdcliob wjth p+"flir r* ir +c dolLte tuheddi!! or utrtnboD doe .. n whicblhe.pdrDd is 6,n in6j._"red rh ,e utosr ni$ar;d rxe t n pd.Ej wu. Two nerlodr ne a,-nJble:obe ir which fie rpeonft n Iisbd' i!ljF*'d (Pfterc ro-') lMc a ,o;5olu,ior of cerl,to.e" n-1, od Lhc o u e r . b s r l c L ,e . q n J b o c k i , n J d - u d l h o ! r . e r e d , o m o t r o p , r . q i r .a. .rnerolmcr i\ gre"!) ro be pretereoro rlc lrr v. a d is ssLed in the cbapreron 'Dooble bf,ltratior'hj,ration uith Cell"loseNitrdk Satntiaf , . B r i n s , } e6 & J , w i i e d , p - i n e n J r o u g , g r u J e"d, o t r o l s_ J o o oj.o o o _ o o o . eu-o.o soso(rreeflryl:lcohol, z. Alsolute etl]1 alcoholud erhvtcttrercquatoara. " 3. rho to absoluteedryl alcohol 47 njt low viscosiryceluloseoitrare 6 gn. a. ro% LVCN iD tle seoe solmr J.2o%LVCN. Tbc tine ofmfltr-tioo ir rhe.c|1udr wiX vry " ,ordiig ro rbe ia otlfe d*.re. ^, spefutr rbo. ( 20 nm. rt n, rhi,u+r pn wil rale ,pproxim:reJy -hreero four daysin erdr celloloscsotution. Thi.Lenirgthe Celklok Niltat! Sal!'iol BloekMakinr Beroreir L h"rd.bedbro J bto.l .,ri_btero. cuRirs d c ,oo; .oturioo.hoLld be , u . K e r c d b y " t o @ g ! ! - . o t v o o e ,e p o f u tac L i r' c . I t L b d o l c b y p o , i n e i n r o r g a , d ! h r b o r s o i n c l r s J * p r 1 1p I y o f . : o o . e l t u J o . e " o l u . . o n o d ; e . p c . i n d . p o u r b o u ' o b " C , n do f r h e r o @ t o f . e l j J o . es o t u o o oC. o \ t r boln.rqxs $.ur u Lplhcd beake,or beltj.r. tu li" rctve! e,Jpoh,e.L\$olurio! $r,u Bet !-rf:,: wl el fic .o[.don is jlr por,bte plJ.e !h. Lh wr_r d,e .mal] @,d,e or!exdoJe,rr_te b i ' u r d c ra . c c o r db eI j u $ t , d e r i e r ej c ! r D - I d ; t r o i d o r c r o , r . r h e i d d o r d h v _ p o u r s m " k ed , ee l t u l o < . o u r i o ng e J . ud wt_o thb x"s happsrd pou, on !o ir lhe trr rotLuonvirl rte spemen in r.

Ho.dening @ltqidin bt@k Frc 2J. H,.den g coloidin bio.! in cblomforn vapou

?6

Sttion C|tinfin

Mictoeoff

Aroee dE specimenon the surbce of the gelled cellulose,ard if ncasary levc tff dish-uder th. bell i". {ithout the c}loro6tn to harden a little norc When rhi, h* 'echcd . pout at .ti"l +e clora; f*1" ]llkee pieceof'ot rubbc' pke che.bi", ofor- d.h udca. Lh rbe bcrljr do corp etelbc br dains rle 6@1 h,rdtuq mryhI.e fioF:4 a8doub o-loogerdrPsdtg oD r" iizeof fie sPcci@' ct tli specinmour of fi. ai.L wiLI r slup 'JpeJ. od le'vc ' rugr or 'bout s-s !m. arc!trId ir. placetle block in 75o/"alcolol otil needcd.

Sone worLers prcfcr to ne anylacetates dre solvcnt for 1ow vircositv ccllulosc dtrate iutead ofihe alcoh"l-erhermixture. A methodput fuw'rd bl Bedeti Ggao) r. Sdng f,-<ed,wshed hlterial into aL."lute ethool th'ngh

the lsud gradcd

2, Amyl rcetale. 3. roo/" @[oidin in mylacetate. 4. 2o7ocelloidin, ihen 3oo/o,ad lastly 4o%A speciDtnof"bo n zo nm :ho rld be en for fo*v+ ght oou' ir *'h rc'urioo The il.cl L Lidfl'ed t cllo-oforn ligrd or ir 'hJorororo v P r a rrrqdv &.cibcd mc 'rorcJ n 7,"" J.ohol urre'luned l r Pd'dr€' of ellulse r rare u d-e L.. mirnr- s ,o JLigbJ',r Une "JdrdooJ \rddiry olGe block is neh Jy. TDe.Li D"d" for di' m*hod i' Li* b) u'ing am,lretJte s tlerolvdt mo,e low vl(olry trllulo'e n'-,re m:1 be dl'ohed: thrt $" mefiod s quicl* tlm the bsic tedniqte; od that it is chealer.

Ir[ethod3 Cellulosedtrate in the drv, chip cor&tion is a hrd md brittle naterial Like nost olrtio i' rur be 'oftocd o rllrictrd bv Lhr -dlitio" of"uir'bre reg@Ls hGe i. addedir dd r-'rch (roao)dio-ryl phJuJJate i"u"*i,," .i+.a bv cl.{anr gel tulshed of the propenies cu*ing snal qu;dry to i"'prcve ii. f,ow, ud r. Bring tr{ed, w6hed spccim@ into absoluteetneor' 2. Absolnteedrdol dd cthef, equalPds. 'ther' 3. 5o/olow ajscosiq celuloscnitnte in.lsolutc $d r. o. r. ;.

:ro:5o ml 20ol"LVCN h absolutr/edrer .ro." rvCr i" absol'r: "h,noL:ro. etl,.r zto &buryl phthallae5 o b" Jc'Jo*lv ir, &.:caror' ub-l rt becom 5 r' AUo" Lhcl,r lffic Pl,ce in 7r% "lcohol t. harder' chuge the alcohol ovet a period of o!' to

e. Trin th; hlock,ud rdove thehardouterrin oflvcN fastoed to the block holder.

Itnthmrcadvtobc

Lau Vk1litl CelkloseNitrdk Fshniry a CeltoidhBIa* ta theMitutofue rwo methodsru1 be u,d. deperdingoo the rype ' ormcdomc. th* ued witr de hrd or b-d *i-*-..

77 lhe .impte{ i\

r. lrin rle blocqa..oq belo$. Thc poiled prr .rould bc tle f$r ?M ro b. dr by dr mi(rolomekulF. tlr nim .bourdbe madeb "uh r u.-t Lhlbc b^e of Lbeblocl ii hger dro $e upocrface - Lke r ruJrcd r)r"nd. T\i. helpsro bold !h- blo.anrnl o , - ". ' or ,l," *, ,o,o.".

F c, 26.

Slape of hinDcd

ccloidin bloc!

2. Dry the block with a lher paperrd placeit on the block holder ofthe micotome. lt doesoot needto be gluedthere. :. lill the well oftLe nicrotohe witi molten puatrn ws, so tlat the wu cover the b1ock.kave to ha.d@. The wu holds drc celluloscblock very fioty, rd the dm of the llock rrcv@ts novenoi rhen it i5 bciig o,t. Sledgeat Ratatl Midatobes Wfth rqeseinstl'heirs rhe .elloidin block nut bc firnly glued to a woodm or pla*;c block, wLich n aierw*ds fitted into tle rnicrotone aid saft1y clanped in r. Take z dry woodo block whidl wiU fir into rhe iloldr deigud for ir, ad rougho tle surfre by soatchingit. z. On dre surfaceoI thc rvood pl.ce a dror of ro% celoidin. 3. on to dr &op placc the celloidin block. a. flace a sn.ll veigh! on drc cclluloscblocl ro pressit to the wooden block or plae u elutic bud aromd then loth. 5. leve to dry ud hardo io air for abour one to rwo hou.s dd rl@ placeinto z:% alcohol to hudo. The block ; readyto dt, afrer u hou o! nore, Csuiflg Celhlos Nitrd? Sections ]!e wct methodn tlat in dnch lhe ce11ulose nilrate block is t.ken flom iri rtolage fuid ofz:9i alcohol, fctocd to the nifiotooe asaireadyoudiDed,and is cut virh a niootonc lnife wHch is floodedwith 70% aLoho15 eny sectionn cut. r. with the weil nifiotobe cn avay n\e pra6n vd imediately in from of tle ooed celloidin blo&, so lhat the tust thing dE knift touchesis the pointed od of the ctlloidin block.

78

S..lid Ctttkg in Mhotun

Ftc,27, C.loidin blockin thewel of. toch Eicrotoderhoeing!di:l

Emoul of ld.lfin

2. Arrdgc a letri disL 6led wi$ 70% al&hol hddy by the midotone, and lavc a so4, fanly lugc (Del hai! bruh restirg with its headh the alcohol. 3.3r!shthcmiclotonelnifewiththewetbrushsothattheknifesurfseisli!.rally suDtliedvith guid. a. af,o b.ush the surf.ce ofthe block. J. Raise the blo& h iie midotooe by the apllopriate net}od ud nake a cut with the knG. The rut shodd be a slow one, od should bc nade with both a fomud ud sidewaysmotion ofthe ldfe. 6. Thc sctior my be lllied ofthe lnife eithe! wirh tle brush or with the 6ngcr, md rlaed in a &h of zo% ncohol. Fron tho on the scctioB rc lretcd in snall dishcso loosespecinen and rc nevc allowed to dry. The sde dtting tec}riq@ i5 u5edvit! 3I othd nicrotoM, slight adjutnenx nay be nece$aryfo! any given instnbdt,

nakitg whrtever

Dry Meihod Tle block is tako tlroqh the folowidg sbges. r. Fron its stonge duid ofTo% al@hol bdng dr ce[ulosedtratc Uock into 90% .lcoirol llus j% ctloro6m. eo% al@hol and cHolofotu, equalp.Its. l. cllqoforn ud cedarwoodoil equalpans. olithe cotainer ud add norc cedcwood oil cachdry ultit Leave the stopper J. tLe nirtue i atout 9j % d-d.Mood oil. Renove tie block fion the on md r1low it to d,7 in an. wipe dre block. 7 To fx tlrc blocL to the woodo blocl holder plae e drop of ro% LVCN on the dry wooder blo&. lrs the celloidin block firdy on to the vood. Hold it iE position vit! a lubbd bdd ud aliow it to hrda in cl oro6m v.!ou lbr 8 . Cut on a slididg micotome, od noisto thc blo& wth redamood oil during Placetie sectior * cut in ce&wood oil. Tntfer tho m xylere, + drm clnorofom rd absolua etbaol equelpa* > 90% erhdol vith J% cl oroforo, zoTo od so o dom to wter, The ccd{rood oil iifitration procas a oud;nedabovemay tale an}tling floo 3-3o dap dependingor thc size ol tnc speciner. 9

Low Vis@iiryCe rlrse Nibdte

79

Cdlrlak Nihate IfseriJ smiou u. needed it ir;rvrirbly erie"dd quicqerto s Lw*h6lnatioo proce(, brr o.rionJlly i( nay b. e, o'i:J ro obdn Lenes of."UoidiE secrioB. Muy nerhodr have been lur lorwdd to stick .elloidin scctios to slidq in sdal fornation. Their adhesion"however, ts accompded !y mceltainry, ad rvt rever mrlhcd h Lredn qill p,o\e Ledioui"nd rimHo6urug.

r. Tale r rmb* o'petric d;rh.. drd irlo ore Dl,ce$Jee 6jF'p"peb. 2. Cov6 the pap6 vith ?o% alcohol, aod 611rie othen. 3. As eachcelloidin sectionis cut prick rlle cclloidin with a nosDred needleso ttDr the fist sectionh$ one !!ick, the secondtwo, md so on. The pricL should be onside tlt specine4 and the preoce of the 6lter papcrsmablestte needtcto go throush the celloidin suficiotly to lave a vi.ihle nuk. 4. Tra$fe. the tust rm to the tust dish, the rcond to to rhc nexr ad so on. 5. Froo then on aeat erch dish virh iB ten se.rios nl the u!.1wry, rd whd the sectiou arebrouglrt up into thin r11oe balsm t}le ph prick vil te visibte edd a bimcul,E nitrdope arrdthc sediotu hay be {roged it order c they

Tlis meihod (Riclar&oa r9J2) consistsc$mtialy of presing @ oidin section5 rery tumly againsta slide thidy coaredvith aibme!. The slideis bakedar jj' C, .!d the albmen thel Loldsthe sectioDso s!.ongly d,ar tle @lioidin nay be disolved o6 r. sne.! a slidewith Maye!'s albutu or wirh t.ker's adhdive altlbct (p. pd. z. Placea drop of pue turp$riDe on rhe slide wheie e.ch rction witl b+laced. 3. Take the sectiono cut ard storedin 70% sLohol, dd placeir upon de tu.pd!ioe. Na!.le'Jr rh* r,.h .(do1 i. der."bd "q r.h {ilh rupft !iDe. a. Lowe! gdtly fton ole end of rhe dide a pieceof ceUorhdc wtuch La lem soakediD w.ter, so thar it covcs the se.iions like a covmlip. The Gllolhde shouldbe thc see sizea rhe slidc. j. Hold the dd ofihe rlide and thc piee ofcellophue rigHy fet've@ finger ed tLd! ud snooih it in luy dtcctiotu vitl . finsJor ir6h. 6. As $e sectios L@ne flan{ iacreae dre pr6sure. PoLi (le ceUop[de qtfi r du.rer.od ue cou]de,abe pr6ure. 8 . Pb.e a !ie{e of pald t]le sizeof the slide,dd nojJt€lEd with r!ryatide, over 9. Tho add a slide and bhd the whole lot tighdy rogetherwith thin string. Hea! in e incubator at JJ" C for le6o hinur6. Duri4 rhis dnE tu .llumen wi[ set,but the sectiolswil Dot dry out.

80

Seaiaf Cqttiflg in Middca?y

rr. Cuefully tcmovc rhe top 5lide,bur @intaid rhe celophd. iD.onact ulril it cu le rude wet again.To do ths pluge thc prepration inro a dilh ofvam 12. Rdove the slide froh the wum water, dilin ofi' *ce$ w.t€r, pal ofi. the elophoe, dd plnse the slidcinto ajd of r,lde 13. Trmfcr thc slideto retone, seve.alchuges, edl lhe ce,lloidinisdi$olved .vay, 14. Absoluteethmol, dd from tha on trcat a pe6r wd 3a.tiod.

CelloidntSectiafls r. Ncvc! hury cclloidin s*tiom thoush tlc ligher alcohol ad olloroform mixtues bcc.uc the rather thicL, d@secelldosc nit.ate holdr oo to the wata o{tle erlier fluids vcry vigoroudy, md it is aa uy rnatto to od u1 wnh such sectionslooking dnte or mistv vh& in bat@ or ralenc bccausethey still letain trac6 of watc!, 2. Should the stained cclloidin sectionswhei finally cl*cd md taken ilto a solution olbalsan io r11o"" in a d$h, shov r deerecof curliog which wodd mrtc then nouting dificult or eva impossiLle,ticy nay bc inducedo tuno out by llacing two dropsofclove oil to cachro E . otthin balsm solutioa The clove oi1 should not be addedin greter q@tity or it vil hake the celloidin so soft tlat nuipulation oflhe Fcdon Dy bsonc diffifth. 3. stainede oidin sectios shouldnot le le6 very long h tlE nixt!res ofal.ohol od cllorofon becausethesereagentsvill reducedre ciarity dd lrighrnes of 4. C.llulosc nitfate is not d adheive, od teclniqua in whic.h it is ued * such cdo! bc lcgardedd coh!1ete1yrc1i.Ue. s. rd4o elloidir sectior de requircd for serid vork they nay le sroredin r jr of 7r % alcoholif it ii in onveniot to mout rhemimediately. Eachsection should !c scprated froo the next by: tlin pi6c ofraper, such* a cigarene papcr.Thcy may bc storedindetuitell 6. Wha they are stained eloid;n sectionsshou]d bc t&m into zo% alcohol, eo%, eo% od cNoroforn equn paIrs, pure chloroforn, pue chloroforn md rytare equalpara, ud tho iDlo !!re at-lfle They should tually be placedir a dish of thin xylen+balsm, ad dry nay thm be momted io Co:da balsan. REFERINCES CBI@N, W., ad IBG,E. H., 1s49,Qua .J. Mio- s.i-,20. 43Er!]\m, H, S,,r94o,,4flar. Re..,76,233. DlJv^1, 1879,Jawn. del'atut., r8s. PBEdB,421, ZeikhnJrJw ui'y"yha.tlnha Mikto'katk ,nlJ* nida:ka?k hc Te.hnik., 34,342, k(::t:Ijmso\ K. C.. r9j2, Qudn.J.hid. sti.,93, 3jrGA^t, P., HarnbaokaJbasnsntohthhi,lre. Coilrable.

CErrrR 16 FROZEN SECTIONS AND GELATIN SECTIONS T;su6 nay be so hardoed by freezing dcn that sectiotunay be @r wirhout rhe sudl iifdimtion nethodj beins reqnired.The turd;l tuy l,e frcsl, th:t is rni*d or [aed. Fomo]-slirc is foud to be a verl suitabletrutive. The advrtage conGncd by this nethod is ns speed.Ir alsopreseres faty tisus, ed neNe nbres, nere odings dd end plates.The nost $e6sful rcnc sraining rechliqu4 de geeral]y 3$ociatcd vith frozo scctions,in which no iniltrarion oediun hasbeo used. Very thick bloclc of tissuecmor be ft.essfully frozo ai1tuough bccaue the rale ofcondudion is slow dd there comesa poiot where the wrnth ofthe roon prevots fu.lher advuce of thc frozen regions. This point i about r.oo ljo cn. fron lhe top of tie block holdcr. wnl gelatnreinf,inated rissuesir n lowcr, ud n The cormonest mcthod offr*zhg risrs n ly the useof conpresed CO,lion a cylinder. This is led by a tube to the nicrotone block-holder dd froh rhe iside dd rop ot &e holdq ir is lemitted ro Uow our &rough a sdis of ho16. Coning oLltat a Fe$ue orlroud r,aoolb. to thc squareinch rle liquid CO, npidly erlards dd so loms thc temldature oliie blocL-holder llar ey nat$ial placedupon it The lreezingmethod is enplo).ed for fairly thick se.rionssucbs rc2o I. ThituF sectionsnay be ot, but if reqdrcd ft n beller to infiltrate the tisnes with either gebtine G. E4)or s,irh gun rolutions. The nethod n aho useftl bohnically \!1rere frozen scctionsof pldr stemsand roots on!1e nole evcn dd rhimd sectiotr !o bc crr thu are ottained usire a hod n i . o . o m e , o ! ( i r y p i d i .c a , , o ,e. r . . .- i o . r b s r r d i , a . The adv&tlges offrozcn sediotu md rcrioning havebeengiven, but rch sectioas rvil rct conpue for thime$, clariry of dcoil ed esines of trudliry i,ith w sections,dd in practiceir is foud rhat frezing teolaiqucs re sed only vhcrc wd i'IlEatio! is quitc usuirable. The uc of freezing nedrcds witho$ iintt..rion n rpllicble to solid or honogdeos blocks of tisue. Thu . contlere secrionof a nevt rmvere to the body n the abdominal regiotu voutd be ueles be,:Eusc thc vanous orgs wodd all serdate 6on exch odrcr rhetr the scction ws placedir q l F r . A s e l l i o Do f a , o l d o r g u e J . L v - , m u . . ; & d n " ) , L i g r o l b trutue, .tc., is most suitablebecausedcsc re closeldt mtcnaL and do not f![ apari when noating loosely s sectio$ in sn.11.lishes. The prepuation ofstained, nouled sectionshsidc tcn mimres from rLe receipt ofLoi m,t*iJ n of$ p- horogi.Jv. ad , rcrlod oFr odutus su.\ y(doL r. given in dehn by claydd Ge6o).

82

SectiorCauin! in Midasopf

Ikdif.^ti4s

ofthe Freeifs Method TLesecouist pdncipally of $e ftilizarioD of variols glms d iDf,ltlarion hedia. Tbeir !!se@ in the ri$sc idllrrs ro tle 6ozo section a greard f,rhnes od snootlDe$ rd if sptrd is not esential bctter secrior will be obtainedby ore ofrhe gu nethods. This applics larricolarly to secrios vhere fafty marerial de ro !c shom; wlue hot-rm hardding nuls . tnsueuuilable fot ws inilttation; or rvhere shri*age nay !e excssive in secrioniis ftthods which ineolve .ohllere dehrd.atio! of dr slecimcu h doesnot applt to scrioning nedrcas whi.h de dcvisedlor the stainingof ncre ddings, i! vhi.h 6e dre lrceIre ofgm in the tissuqlcadl to unevei delosis ofsllvd.Il drerefore,silaer stainingncthods are to be nscda srtght neezing ofthc tissuesvi out inilration shodd be foloved. . run.coa 3 0I . n '4 wJrcr - lrhr Add thc aboveto

C,u-yr,p (Cole..88a1 wrll Je "oac dl/, ro di$'\e

Brhg tle nat*ial to bc iniltrated into water, md w6L we[, ifne.essaryovdnight to rcmove all tracesof pramative sucha alcohol,K.nertig, erc. D ex t ic Srtu! (Af do! ob, as2e) S]"up 80% alcoho! ethyl Wlitc d*trin

Jo El. 50 nl. rJ gm.

The stock sug:r qmp solution ii nude !y &sohiig j 16.of canesrgd id 2 pints ofvzter. Add dle der.njr ro the syrug, boil util dnsoked md ther slowly add the alcohol stiriry whilc doiry so. This nediun i, usefll both for inilhrion and fo! adding to a specinar which h* not len innhalcd a it is beingfrod on d1eblock-holdei ofthe fiazing nictotome. ro this way it givc suppon to tle tlsues. BlockMakrgfot Ffo2esSettirts Apzn &oo the gclatin techaiqueblocks arerot hade lor frozen sectioff. Ctttiig Ftuzer Sectio$ r. Tcsi dr CO, qlinder to seedut it ho a good presurc of cubon dioxide !y comecting it !p to rie nicrotore. Gotly opo thc ncedlc va1ve,dd wheh ir n chd tlDt the Co, is nming we11ad rhat a1ldrc comcctioro *e tight dme tlc valve. The cylinder coou;c liquid COq md it is 6sd1ttul tLat dis iiquid, not tie gs, cones ftough the tube comected to the nidotohe. Somc cO,

Ftuzd Sectian!obdCeldtin Sectia,s

:. 3. a. 5. 6. -. 8. 9.

83

cylinders have inside them r Iong tube vhich m foh rie botton of the qlinder i*o the cock or valve, so thd vhen the cl'linder is ulright liquid CO, is forced into the imer sighondd out !s a liquid th.ough thc tube to tLe micrG tonc. Such a c?linder is dtrefore ued upright, but othcr cylinders have no siphon tubc rvnhir ard they nut be ued upsidedowl. Should cylind*s be lsed in the wrong rosirio! ody coolrc$ed Co, gd ujll be forad out when the valve i opmed, od this vill be imufiicior to freeze thc natdid on the nirotone llo&-holder. lf a Iair anout of frozn seaion work is vnu.lrd it ; e*enti1 to haveat le6t trlo c.rbol diondc cylinden, both fastded ro the cuttiug table or bocL i! sucha way rhar who one ros out of CO, the tule nay readily be cornectedro the otler cylhdet. Placca drop or two ofwater or thc miootornc block-io1dcr. Phce the specime to be ctrt on rhe top ofthe holdc. widr dr varer bdeath n. Avoid t.rpphg air brbbles under the slecnne! when doing s. Tm on die CO, by $ing the n*dle valve, rud kcep thc spccinm firnny pr€sed to dr llock holder for a Gw sccondjNril tlc liqlnd begid to lreea withdnw dre iryer or sratda od continu to Leezetle specimen. Ifit is recdsary to do iithoui m]. surport for &c risre continuedt ieezing fttii dte lrosr line cleels of to the top of lhe spedno. In er $e L rorobt tnife inrc ir" hold*. oght.n r ir po.ioorr. Adjlst the positioa ofdre rp.cincn uftn dE knG i5just cledrg it. Make a ttial cut or two mtil tlt specind is facedo1l The $a$s ofthe otting is depmdot io tlc 6nt pJce on tne shqle3s of the microtoDe lniG, bur n.inly or thc desreeof freczing. This i sonethi[g which the op.r"tor nut watch very ctosely.If the tissle is frozen too ha.d it d[ chip, and will aLo damagethe lnife edgc.ifit is i6uiicidtly fro,eD tle secrior wll Dcrely ap?q asa liDe ofslurh on thc loifc edge-T]le condition ofthe tisue at which secioning is !t tu ortimu is sonedfng which can onl/ be decidedby lhe !!6oromist at

ro. A5 the s{rioB are cut dd lie on tie ldfe, pi.k thcn of eitier by presing a firyer dom on to them nd tho dippnrg tlc tngu wirh thc adhcriog section into wat*, ot ue a soft bru:h iu the sue way. rr. Frcn thd od Ecat the sectioE d loosespNimcs ed stain rs requned. r:. After stainingnoum the sectionsin grycerin-jelly, o! h rudt: n€diun. If lhe nateriJ ha becniniltlat d with gumilrur trc.t s a\e lorcgohg but stick 6c specinento dre bloclc-hol&r widl a dro! o! two ofthe go*yrup nedium, od in additiou dtoo sone on to the soccimenso that it freezs asit rm dom dre sides. ID thir 1v!y thc specimfl is so.o*a.a -itl " -U oftozo gnn-syrup which gives ir b.ftcr.uppon aud 'beeIoR belp,ro p,oducebr!er \ccrios. Shouu n be necesry to trear dreses{tiors oD nncros.opeslideso serirl scctions, plae eachsedion d it is ot on a deaa greasefrecslide Soodedwith r% liquid gelatid. L.ave thc slide @til the gelatir geL od nedy drier, dd then pbcc it jn ro% formalin. Tnn wi hardd dr gelaline dd fio!! ifen or the sectionsoay be

84

Sdon Ctttibgin Mitut@p!

ceiatin is a protein vhich my !e dissolved;n wter, and o suchit LecpslJste logical d$6 hrch softs the doeslaratro vd or @y other prccs which involm coaplete &hydntior. Ar a wrter solublc natcial ft drerefore r*aim viftin dr tirsu* to be ot fas or f:tty conpodds vlich woutd be disolved out by ay hot w* ioilE:tion method or by the low viscositycellnlosenitrate leclniqE It is for revelatior of f:tty substmce alrr tLis netlod n aotnally enployed, though it roy alsobe ued for setiooing puryoseswhere rissus may lecone exce$ivelyhrd $ing otler inEltation media,d for neuologi@l work, or fq the quickst possiblcmethod of sectioningnext to &esing Niihoft,ny prflious rreatndtAr d ilcft.ltioo me&un it ir lsua y @ployed i! wo st.ogtts, thin gelatir which is 12 J % io wats, ud thi& gelatin which n 2j 96. Sonc workcn prefer to ue solutio$ of ro%, 20% dd 2J%. Any gelatiDsolution, howcver. is prone to brcakdom if kept for my lagth of tinc in the liquid cordirion. If overhcatedit brq.ks doM to mctlgcl.tid, in wLich coodition it will rot set, or which wil rrodu@ a softer gel if preat in my qmtity; ad alrc if kept evar at ! low tempentm jut sdfcient to keep it liquid, it is subje.t to bacte.;1 acrio{ which vil evdluly lqdry it. celatin solutios the.efore musr never be ovcrhcated,sd they sholld 'Phenoxetol' s advisedby Gough ud wotirclude . h{tenocid.l agdt such a wor& (reae)Fo! shal specihen! say 3 x 2 x r cm., thc time of?oeaadon ruy be so shon that problcms ofliqdf.cti.n and brerldonn of gelatin do not occu, but viti iarge spccinds srch s a whole huqd l@s (coush ud Wotwonh, reae) where a total infiItlltion tine of four days may be equired, Jiquifacrion,dnudon ud de mtuation of rhe tudltration nediru ce coutcred ody ly cueful attention to the detaitsgivm b the previou pugraph, md by dre introduction of frelh ban\s of newlv disolvcd selatir ceLA reaim uc r:surlly trcatcd u loosespecinm, md their stainilg md Snal nouting I accoopJnhcd by uiog aquous or weally alcoholic solutiom. To delydnte suchscaioro fulll ud to mout in a nediun fth 6 lahm iq xylene rvould be to dnsolveaway uy f.ts or snnihr sulstdces. The quJty ad setting lrop*ty of gelatin vri* coniddbly. Sohe slight differenm my thccfore lc mticcd in thc cuttiog proletio ofblocks nade in dre s@c way lut vith ditrc.drt batclts ofgelatin. Suchdiferoces iie well \ithh the ac@p!able rmge, but thcy may !e euggcratcd by aeles ffetnot * indicatedin earlier pd:grads. It is o advutagc to cool biocLs of gclatin in a refrigmtor s soon d the biock is nade, but blocks or solutiou ofgelatin shouldnot le 6oa. CelatihIbjbtatid Madifdtians The nost wid€ly {sed nodidc"tion ofthe netiod asput fomatd by SoI$ in 1884 is tlat ofBake (r9aa). The prnlcipal Gature dsociatedwith this ncthod is tlrc usc of fomal-calcim-cadntun s a pres*ing fluid ftd trqtiotr wit! formal-cdciu. The nethod $A dokned for iovestigetionof the golgi elendr, but it i5 [email protected] a fat or lipoid presenation techdqc.

han

Seenobdra CelannSe&ior!

Ej

The fuli nethod by Bak* ;squot€dhere.

Iodaldehyde aool" ro bl. celci@ clbndc, anh ro% aq. robl. 80 r . Disliled wter Precipitatedchalk should lc added to this solution to neutr.lix ey folnic acid which my be produadAfier fstior fo! 24-a8 hous or longer t@sfer to: lornal+alcitq
ro nn. ioml. 1o rJ. 70 ml.

!i*ed tiwes nay be ptsesed in dris fluid mtil required for sectioniig. The @dbim chlolide act5on certain lipoid mabnals and foms isoluble conPoedr, md thus prevots their gndul los or breakdom in storege.

r. Sring ited, w*hed matcrial dom to water, ud wash in rming abouriorrr ho$ depmdingon Lhe.Le ofthe.peciom.

vater for

ri sn.l celatin w.trr 8i bl. fKreP thi' liqdd in s oveDar l7-4ooC r .j. J Phooxe,oL for about 16 hotr$. 3. Tho to ,J Cm.l cel,iin wra 7l nJ. fKcep rhn Uqlid n s o\4 Jt l7-4o" C , ml. I Phenoxerol ror a nj-ba 8-,6 hour, dfoendhg on $e .;e of thc specilr@. tlehth BIa& Mdkna r. ?our iDto a sna11glas dish a layer offrolly mde z5l, gelatio. A1low to gel. 2. Add more liquid gel.ti!, md du t-heinilnated specinen.Ajfuge the s?ecimen witi a nouted needlc. md leave the Ims to gel. Th;s nay be h*tmed by placing the dish in e refriguator. 3. Cut ort a blo&.onttniDg tk tnne with a 6t scalpelrod lLce it in the foroalsolutior to hrdd for 24 hous, or nore. calcim-odnim iq wdh the block ruding wter fo! 3 a hours lefore placing on the freezing 4

86

SeaionCruinfin lrfr.toyopl

Wlo 'maii,oieceofrisue re beingused,suLhs nr lirujaD gtdd. joo dd ,ooo rcrubotuot gerrE!nd beLed turud ofLle strotgetooeatrady giee!. Tbe rhiruFr ro udo$ pffrr-ate rsrcr bu! .bouldbe uilir.l ."ry if Lbem{dJ n goirg ro be dt on a ftezing microtomc. Celai, ala& Stordge It will be seo from tLe fornuta tiat cventhc strongestsotutior ofgelatir corains ?5% q re,. Lfb'ocls rade r.om $.'r iot,.dob r";pord ,o rir fo; aDvtderh of rim hey e.rll srDr* oecme d-rodd. .1!e) .nodd LboerdcalM)', bc:drd .od b a u q u i dJ . h $ d e o ,h J r l ' o t n € J n , . m - L 4 d y s N s . o - i f d n i s ; o , r v . i t " b i r in lormaldehydcao96,ro bl. ; distilledwater m n| flre iDf,jh"doDof Jcry Lrgc "lcciretu. rd rlu ,ubseqrer.sccdonirei. derlt qi'h br Co"sh rJ wcsor L (reaoji, ueir $ ork on sjlcoi .. ,tE tb;. h lnir ( s e r e r n v e r )r J o e b t o * r w e r cm d e o d . u t o n r . p c - i a l djdoroae, wirlDur freains. The sccrios vcre llaced on -d *"d !.ps, or stifi'card; d.i"d, *i,h"d, $ m a ( l o r o l i cn o u 6 . Calatin, Se.tiot Crui flg S.ctim of gelaiin ;nflrrated materialae rnosteaily cut or a &cezingmictotone. Ttris is ldgcly.l(ase 6@iDg rhe l,lock to the blocL-hold- "ro.aitr" qoi"t"t m,rbod ot "dckins $e bro,k dom ed ,Lo be.ase ir T{.3"".4$L siw_ additioral fimss or }rdncs ro r1r block. vid rse blo.L. $r.onoh - blo.t r no, Co,ft ir r mercrrheldro r-heblo,k_ , hold., by ice- rd n d or coDdjdoturhe hddnN of tk blo.k Jd its ,rearbeir witl formu! n o.ihpq r.e. lJ'bc bto,tt i. bor hdd ooue\ tlere wil bc a sprme ro .r qLicl D au. uevd d.rins rd dhg. (r-I l/k o i.a,", -"y r, a " ,3 Dro{m-do\r.grtath. too wear d get"tin)otuaob,or roo.l,oa a.;Te h thc foEl,tii S@doi r@y be dt virhdr nuch dirtcllry bctwear ro u
Fftzen Seetian,6d Gektir Sutions

a7

J. The wa block containingdr gelatin blo& is lotared to tie nicrotone bloctholder by u5ins a IDt spahJeaod ftfted vd. 6. Cui betwed io dd 20 p. Placethe wu seaiom with the gebtin scctioniside upon water. TLe gelatin scctiooswill drop out ofthe w* to the lonon oftL. dnh. The w* shodd be d*dr.d otr&d 610\'1.vay. 7. Treat the gelrtin seaiols from tim on o atredy hdiatei.

Gelrtin ectiom nay be cut in the uual way dd riacd ufon a wm slidevhich hc or it a dir layer ofliquid r% gelariL Wher the sectios dc corectly .lgned the slidc shodd be placed on a cooi !l.tc or in . rcliig@tor ot the gelatin se!s. A11owto dry slighdy. If the leboratory is w*m dre slids shodd bc fLced in ro% fornalin ro hard* the gelatin, sd fron dh on t1t sectionsnay be treaed or dre slide. R,EFERXNCES B]!.G,J- R., 1944,Qrott.J.Mkt. sci..A5,L GoucE,J., dd q.Aar('oKtr?rr, J.8., r949,J. Rof. Mi. Sat.,69, 23r. C!aDN, E. C., r96o,t Rot. rii.r. sd.,78, 88.

CE.I@rT

URTA FORMAI,DEHYDE This nethod hc tle following foturcs : r. It is not. rifboDing ncthod. 2, It is . proces jtr vhich m dehydrationor cleuing is rcquired. 3. It ir suit$lc for most tisus, but snall piee ody should !e *ed. Not norc rhaJmo.hthiclne$. 4. b.Altration t ks lLcc at about 9-2o' C. j. Slocb {e mde by t*ing the iniltrated speinen fron the F6igeetor ed laviog it on the bcnch at a room temleratre ol r7-2iq C or highd, rv e still within thc tube ofresin. 6. The most vltery ad soft specircx suchasol,elia nedusae,mal l,relia nay be infrltratcd ud blocked witi litde visible dlstortion or shrlakage. 7. Once tle seciios a.e cut they shouldnever be dried, but should be neted like colloidiu sectioo, drough it is posible to stick then to slidesusing gelatin. 8. The stdd{d fornuLl is inflndy vdi.ble. 9. Sectionsmay bc cut on aDyt)?e ofmicrotome. ro. A ldge of sectionthiclneses lron r-:5 p may be obtainedwith ece U+Frdnah!,1,)d. Re'ir Wlrar t:re: ,Id fomildehyde uc brought togethet vith a sligLt tnce of alkali to act s 4 cdtalystthcy pioduc a gls-clcar rcsin. It is thernosetting od om the ieactior hs gone to con?lction the produt is iuoluble in alnosr all reagoc. Thc lection proceeilsh two rtcps; first there is the lornatior of noronedrylol-uea, folloved !y dinethylol-ue NH'

co

/

Co

+ CH:O*CO IIH,

NH,

NH CHgOT{

\NH.

/NH.cqa'l

+ rclllo ? co

\vn.cn

og

A nkture of dreserioducts vill react in acld conditioro to produce the 6nal had, glaslike product uro-fornndehy& resb, which nay be nodiied for useas an infiltratior nerliun for the cuttiDg of tisle se.tioff. watu+olub]e producs are obtailable who the rtution is stoppcd [:1imy either asa syrur i! wate! or o. d'y po-da to which w,ter is addedwhm required. Ia this conditioo the syrnp or the powder mry bc storedalnosr ildefilir.ly but vhen the .plroprbre catalystor hdmcr n ddcd the sohtion selsto a hard .lear mss

uea FonoLdz\de

6e

It i in the half-vay ward{oluble conditio'l thar theje producc oay bc puchoed. Tlrn n4in ue i, D *oodwor[ing indBries wbre t]'eir sred "dhGire prcoed,:s. @upled with qricl srr.ins d Gml solrbiliry o:le thm indis!tuibte ,o rte borr builder, tle plywood mdactuer, od rie fuhiruc meler. To the nidotonist dd tle hjstologisi rhe fo owing de tie uin teruns of .

r. 2. 3. 4. j. o. 7.

the resiis s luclaed ue water soluble. They may be potlEeriad, or ser,o. hddeqed, by the ue of hnd eidr. The hdddiig n done ar rcom iehper.rure. Thcre is no het ofpolluerizarion. The solid or po\Ddized lsin ij gls c1@!dd .o1out6s. t he pol}teri'ed rsiD is Bo uble sd inIutbLe. The liquid r6in nay have addedto it glycerin or glycol to .trer itr hrdness

r. The polymelizrd resir is subjectto a ilotable ozing or fraauring, so th* in riftr .r wil breakidrrro o,tre3.p.rrj.Jrty wbd i! btockfod s;d , blo
90

SettianCxtting in Midototy

The normal octhod ofinEltntion witl yd such$ r{.Frrr is to raisethe temocratu,e of dreovebudl |treh $ me r.. to tuflrae therpecino * rns elox"d rmp** tue, od tho to nale a block by allowing the wax to cool with ihe slecimeniflide ii. Tle uea fomrldehyde n*hod ha beo worked out to retese the aboveprocess. That ir to intrltmte the slecimm at a lowa tmpoatue (5-e" C) od tho to mke a block by elev:tig tle tenpenture to room rmpcBrlre Gj-,f C). The vLolc proces nay be caried out at a.o@d J ro" C if reqfted. ror snall spedom, whn the hilnatioa tine of fte sleci@ i, le$ n\d the geliis iine of lhe resin, the hii$ation my be cmied ort at a room tcmpehtee ofarood 2c2J" C or 1ower. The natffial utillzed for modifying du ue fcnalde\dc rein r poly*hylerc glycol. ofde mge eaanablethe nost suitableare thoscwiri nolecular veishts of 4oq 600 Dd r,Joo. Aa interdting fearue of rhei $e is rh.t rhey are incomparible vith a uc! fo@aldehyde syru! of a 60% strdgrh ($ sold). wto sucha synp is hated ud thc polyeth,vloe g1yco1is addedrhe !re. formaldehyderoin is throm To prevot thls the polyethylde glycol n nelted, somewatc is added,od tlen the cold uca fomaldehydc synrp is stired in. The resultis a dcu, cotourles solution which will remdr for four nondx at a roorn tenperature of abour 20" C without deterioration.The momt of kter fof rhis lurpose n.y bc innnftely ldge, but ir praaice tlut which is snallest is tst. TLerc cxists tluefore in the solutior uea foduldchyde r6in predrsor, polyethyloe glyol, od watei The wrc is ssstial to the conplcte solubiJiryof both soli.l. whcn the uea fumaldelyde is aaivated by thc addition of a hdddiis reagert so@ of the w.tcr is incorporatedinto the L a d m i r g r s i r . l h i sd e d J ! . - h - e s l c ' . . \ t i h b e f q r b e t o l u a o n o f d c p o l y erhllcoeglycolwhichL fo ceoo$ of.olutiob! ioudpolyeLhlleneg.yco..lJreesulis a whitc or trmlucent block of sfth a h{dnc$ that it b.y be read y fri into €at sectionsdoM to r // a! dy roon tenpcratue, for tfie mN n no bngd hearsendtive. It will dry od shr k however becaue ofth water wlnch is stil1 pree*, aad thdeforc tle blocl should be tui {hen lady, or stoied iD a.orked rube. Sinildly the sections6 cut shouldle rlaced imnediately into vlter or alcohoi or ay mixrus of sdchfluidr. They shouldthd be treat".ds loosesrecimeDs. For sc.ialsectionvork sectim should be vshed jn water od tho plaed or a slide covded vidr i% The uea fornaldehyde matrix h* the wcaknes th* it s!.itu with dost of rhe osd dyessucho lacnalm, cosia dc., dd x new staioingmethod!* beo produed ro cope wirtr ilis p(obl@ (!. ea). olrhose rer&ly aerileble Heidolainl haenarorylin ued in the sholt dme rario, toloi?d by acid ftctsin vil be fodd ro give llq dd p(mdd. eyJb. De ll]s fo, .rdir8 aregivrn oa prgr o,. of tle "l'o,r itube I abLeColDj-e whi..hD" / be prcpd.d od u cd witb \u..ess rhe foltowilg n suggBrcdasbehg oDewhich plovides a cotuist ncy suited ro most ti$us dd moshcq*ements. It is posible that very nary ofrlcse ni*ues bay be ilvetigatcd dd sed in the future, ud it is *ggesred thereforerhar rhe oDc iehich follows should bc refdred !o a LIF r,Joo, $ ir codists ssentially otlolyethyloc glycol r,5oo od ure fornaldehyde syNp D,27o.

IJfta Fowavehyde

9I

UF r,joo Polyethyloe glycoi r,5oo, nolten Ue

torn:ldehyde srfr! DzTo (60io)

r cn. r l n . i s , b eb r r f o m u t r . T o i r . b u ,b . , , l d e J g m o f d i h e # v l b ) d d r o h . o r _ t u d c h l d e . o ! { 9 n . o l I i n d h , l o t_ p r o p & - .t h e , eR o r e . e m bD t v s r d t m e o u o r r h er ( , h . b ! r " , _ r . L l $ E I ( g r . o f d i m e d v t, r d r _ " r",.;a.11+ iAai",. t r M r ( t r h ' . h r h cv r e u " y * t h eb - r r o n u l I u r , r w l b " n o . " T,i,rst ro Ldd driug $.1 ,her.rore r o , . r i r g . . n p . , c r j c c , ,u L b e f " u d L l _ r r h c b a L c to,rrrr \vrlnours) 1JJDo", witt b" ,ujLbh for Ino I worh, brr dre rwo Jdirives are given should dy Nr dcciden!.t!e btock should bc kept fo six nolths to a yeu afte. luking. Ifso !hq- should be Lept in a corked tube. Ilanleftet rr.J gn. AImoDi4 o 88 S.G.to pH 7.J l\e ue, fornaldelyde.6iD.{up D,r.o ad a nadojng roludo" qo ro $ir} rl t r o , n ! e b u l e a . R c . i n o u sc h o i c a L t r d . B , " y d o h c, o u r ] -" rn the ver) 8'dy b^,.!6 i. ppuedno vriro.oh h th* yroou,c ill,i.J:,* Theammonir@ c oiide in the hardde. whar nixed with the rein srup sraduailv ------, ;dizes bt;nsils ebout a choge in ?H which i suficior t. r".,r* th:;;:f. ror th€ pulos reqded here rhe hardenershodd fe addedro fotu . r, dd !o dyofift fomrLbon lsng D_ro, in rhe prcportid oton. d,op of bf,do1 ro rJm. o re.i ivrup. Thus in Lft r,jm Lhcre"rc do DJ. ofrc.ir "riur. Fotrvdtop. or !&dmtr lbourdlre_reirebc addedro cvcry ro, oJ. ofdc hin.; k i. ; -od,l c b o o r h d d e r r r o , y r p . h o r j d b e . o o w e dr e e * d t e ro f d r e o e r _ ' cqLrge ot otheriJdidrc . uDlcst.ror .ddil\es r6e., tie pH oi,nc ni,1u,e. r. Heat.the rcquired eourit

of poiyetlrytenegtyol r,5oo in a be*er until it is

:. Add de $.kEd,d! rtr mixrureulit ir n quie clcd rd i..orpleLetv ml\ed. l. ^dd rne fele s)r.,pr!tu ring the ni$lrc welt $hne doiig ro. .., llr pbcnoldd.5M rhc .oturjoDu, rhephtrol .ry;rJ $e d-rctv"d. L^dd rne 6Fsr wr) ro ndre up ttFr \olu.jon.i\ ro mrt r be-Lrr betorc[Md , wu M* a $e flrd. fhe dry bture, L 4eh 6red up wlh :I::-.al.p:."?,,*f3 rDecoBotuoB n the ordefdradv eivm.

SectiarCuttir! in Mictoscoly

92

nhni'ation of foduL Ls r,5oo shows dut it hx approximtely 66% solid!. vher de rsin gels md hrdm a fat ptoponion ofthe 34% water is bor:ld into the resir, but a certair momt evapoate. The biock thuefore sluink, fut in practice &is tals so noy dai's to slrink that setiots may be cut which show rc shrinkage Itjlnatian ,ithLrF 4ao r. srirg the 6xd tisnes dolra to wter, sd wsh vdl to reoove oy aci& suclt s acetic.Tli, n 6senti21. UF r,joo 6J ml. wals l5 ml. (or 3oo;Jcohol) Hrdoa z6 drops Stn thorog!1y ud allow the bubbls ot a; to float to the slrfae dd buJst befoa rddiry the spainer. 3. Pi.ce in d refrigeratot tt about g' C, and leave for a6ou! 16 hous deldditrg on the sizeof thc specino The tube :rith tlis mixnre nay be Ieft x 9' C for about 40 hou, but if longer infiltration is aeded the sp*imo shouli be trmfcned rc a esh! md+up dxture Movenelt of tle corked tube should be attmpled by tuoiDg it upsidedori so 6at the sPecinendoestrot simplv lie withour dy movcndt in dE !sin. to fomula ttF r,:oo, Plu h,rdener. KeP in the re&igator' for a 4. Trsf6 minimurn ofz+ hours,ard longd ifa ldge slecimenis being ini6laeted Chdge the cin *ery z4 hours. The tube contaioing the specioeo should be stnred or iovened and tho placed upriglr agab 6eq@d) r-houghortthe i!f,imlion p tvd Jt r trbpcr.tue of 5 ro" C lne 'ai! ddue Mlb .dded bxddcr srx qel rd hddq, rd with LhefomrJa s givenctu may et. pL.e u"-d rotu:ll\ e tiis thereloreb.ncr to chrge .le Eixtue s r reguJarroume eachdav bom. +s Blo& Mdking eith w \5u' r. Srine the tube fron.he refrigeratora teve or dr bendr -Arruge tle s!*imln it witl a nouted rcedle so that the tue to be ot will be .t the bottom ofthe tubc. A tube 2 x + in (Jo x 12 hm ) is .oovdielt for most smalltissus At a room tempdllre ofzo' C the rein vi11 gel ovemight. tf grater speedis .equned the tube mry !e 1ut in m ovo at 3o' C ud it will hardm withh four 2. Tire resh {i[ shriit away from the side of the tube dd it tuav be renoved bv,!icLi!e a Fdte inLoir dd pullbe ii our oftie rubewirh a cortsoew nove od oay be rlmed oil) wilb a omdged *itl g. "*l,cot 'J*,. rrI'*i" lfi! i5 rot hard enouebte.ve it or the bdch or pJaceit in the oven ezor b1&1e. for an hour q so.

BlockAdhzsiorto Bhck Holler r. Trin tle Uock so th.t . good arer ofbase is provid€d, asshom in the diagbh.

Fre.28. Trinncd {rei-foturldehydebloct z. Dry the b6c ofrhe hock by lssing rhe Uock over a very smaltfrne t*o or 3. Fst6 the blo& ro tle llock-holdd !y uing esterws od a hot spatulain rhe uual \'ay. Tle llock {ill harc tuces of;oisrure ia ii, hore ttle needfor drying ir slighrh d in step nso. Should nace of noisture rcmain the watcr tolcroce of esterwu sill pernit the ru to mke a goodjoint betweeDrhe hetal or wood@ mi.roromc block-holde! xnd rhe block. puaffr wu ued in dre sme w:y fails to hold t1u blo.! ro the holder lecauseotis hydrollobic

i

.rll ."ided on na1 nicotone.. +e llock ro 6r n Lhcdrping dcv..- pr M-ke rle Lloc! - .bo< r posrb.ero p
Cuttifg UP Blo.h\ r . f . . e o f r t e b l o c kr d J c o e c i re n b y . u n i r g i r t t r e: r . r w " 1 . : $e ,l.lao h , bes .-r to r F p.. r " "b. h c.rio ",e-equict. Y-'e: piaceboeath dr llG a sna[ dish ofNarer. 3. contifte to cur sedionsud a! rhe),a$e. ou dre lnife helf dled to drop into L b ef a r e , b j " f t d \ o , ,\ s g r L - 1 ,w i i , i s n J t b . u h . { ; . n ) . r j F , " i r D r ^ - b r s " r e , . r h c \u i l l r r u t & . e l y t t , - e n o - r r d b ( o m e r o s r n d ?,rmr. 4. To $ain dE sectios pou esay rie s..ter dd give the sectiod $re; o! fou vashs vith nore wattr to renove trres offrec pol,rertvlor glycol. Trcat $ Stdiflin! UF Satiotts.^,Lthodr r. Heide!.hain'sshorthethod, uing abourro ninutcs in iror ath jolurion foiiowed lv :o nii ure.b Hcidenhir\ Ldm",o{)ti.. Thc *ce,. "hin in !t)cm"ftix w b a l m o e u d r e l t . r ! ' o v e db 1 r l n i o n r l u n o F . - e n ur i o - . 2. Coute.srain wirh a solurioa of acid ftchsin. rhis shotld bc weak _ about j. 3d!g scdonsup 6rough Jo%,Zo%.eo% ud 3b$luteerhylltcohoL. a. Xrlfle, oi any orher duiDg agen!. J. Sals@ or lollsryrsc mouru!.

Sediot Csuingh Microtcopf

94

Mahod z. ALiar hh.ka oacolire ftl AS Makc !p tlE fo oving stocLsolurions: Aki.n BI". Alciuble I sEl ' . !{hahe well a ^eEcacrd.o.I%lq. ,9 rtl.J '*

Elter.

Wdrolift RcdAS V*oLbe redAS

a'";' *4,''v" 'q

r sb.l "nl ^, *at '"1 ntteo lsl"L'

Alcid ble solution W*olhe red ,,

97 nt. 3 ol.

This ABWR solution wil kerp for blny f,ooths, dd is a doublesraini4 $itrrion which nay be uscdon uy te of section.Wirh almo$ all sectionsit is at irs bcsr when sectiomre stainedovenighq but it wilt give a brighr picrfte in 30 drutes if dE fletive ; .rpropri*e. Attention is speia y dram to rie leeers AS afrer wuoline red. Th*e ae other wsoline redswhich will nor worl, and it is escntial to lave dr AS dyetufr To get r corect ihprssior ofthe cl.riry ed lrightnes of thii stainn ihould be usedoeem*ht ar 6st so riat it nay be sem at is bat. Comective ti*ues, blue; mucin, blu*greei nuclei der red; cnoplam pbl To Stdin r. Bring sectiols doM to 5o7o.lcohol, or to water. The UF scctionswi1l be in 2. 3. a. J. 6, 7. 8. 9.

PLce rhe sectionsin the ABWR solution ovenight. Ritue n1 jo% alcohol. ?oo;,qoooalcohols.bou{ lo.ccondsir each Alsohte alcohol No. r 2 ftinut€s. ,, ,, No. 2 2 ruut6. Xyltr€No. r 2 nhutcs. ,, No.2 2 mi!!tcs. Mout in xylde bals.m tr xyl@e lolystlrae.

Ure fooaldchyde sctior ue lst ot at about 3-4 4, ud the abo"e staining tim6 are bdcd on se.tiohsin UF of n\.t tlicknss. Shonld they be nuct thi&et a longq tine should be givo in the vdios reag&6. xx6s wuoline red w*he out of the !.ctiotr on the vay tuough ahealcohoL, but n is insolubiein water ed itr Method, ForFat Alobc r. Fbh water briog UF $ctions irto 30% .nd Jooloalcohol.

Uta FomdLreqde s5 2. Stain vidr @y of the uual far stain soturionssu.h a Sudd blacL, sc,rlei R, etc., rJ 30 oituter, 3. ndrg the secriotudown to mtcr and nout in Fa*.rrls nedim. L[.thad 4. For F.t dtul Nidei r. Stainlhe sedio$ in Heidoiain haem:tor1tin, a alreadyindioted. 2. sdns up ro jo% slcohol 3. Stah wnh ScarietR sohrion for rJ 30 hinures. 4. Briog donn to varer dd nomt ir Fmt. Cawal Notls ahautUF Sediots r, xvcn tlin secriotu,about : a. nay be tmdled with esc ir the sme way as 2. The malrix is i$olublc dd rhffefore sectidN my bc takd inro &y ddid lequned, apad from very srions eids or .tkalics. l. UF rftiob 'lould be uared i1 dF ,,. tt u " J ro ,aove b4tui. flsrive<. A long penodof !in? i' "r,ong ioure :ol rnoru.hower-. wil I"\e J trdo.y to nake the matix lrcak up. a. The lack ofshriil€e dd rhe pres@ceofrte malrix inp.lts ro a riick se.tioa the appearare typical of a gcladn se.rioa th,t; a slighr lack ofslnp contrct. To counter this sectioo should be cut s iiinly s p;ssib1e.Setiom ofz,4 p 5. Scctios shold nw* be dlowed to &y or drcy wjll curl up ud &sintegrate. 6. UF sectionsmat be cut on arv type o{ nicrotone. 7. If thm n oy chemical"dvotdg. to be gaincd ij1t arion with UF mi\1tues m.y be caried olr a! doud 4-ro" C od hetd at rhar temperatuc oril the minr gd. bd h"rdcb. 1r,, lu,dcnirg rr low renpcrrruo n y tale a_ro 0")s. b-ri I m) be 54rded b).ddilc ho,enrdtrer Lo$e br-ue. lbe min poo. lo! cod'delriob b-c j. !L. if nde b"rdser is _dded be hi, D"r'cel before. of rhe slecinea is conplete. The govedi"g Actor i', ;re iinftatl@ $a or ttre speonen. 8. Inilrration ar low tdpetatures mut always be longu rho bjltratior ofde sme speomd .aded out ,t room tempcratue or higher lecaue dmece io tcopa:ture goemlly nms inftose of dcosiry. r! doesvith LrT r6nu. e. L1@aed viscosity ofdy iniiilrarion dediln shortd be @@tered l,y apprc_ p r i a r cm G ) u e , . o g i e $ e . F i r e n . l n - " u i q * a r o . " " p r i r q _ * o r d t u n a n e q H . h r l m - , l - . " , , e J r u o - . o n m g L h es p . . i " ; o ; p j d doM d; 9clce b Lx.-gu c\ery tc, mi , o urlJ pron ore f^.r peoe:"doDof!l-e.pccrrd. r o . s n s u r p e t u ' . r . r u l . b , t u d s o p r o o z q a , t b c D n l . , . c o o n r h eb @ c h at a roon t@pmiure of:o" C oi belov. becaue nfllratio! ofrhe sreioen I .omplete befoie rhe mi:ture tccong so eiscols !h.t n can no loiser lss tuough fxed lisud. Ir. Fats,re retaiDcdin dE tf dedrod dom ro dr sh,Icst drcpter. 12. Mucin, dd hocin-lile Dareriak are hcld wifiin a UF wtionien* than they rc vith ws r.tions.

GTPTE

IE

DOUBTE EMBIDDING Double infdt$tion tcchliqu ue enployed oo tisue whae thc advotage of two sc?aiatei,Iltration media conbioed nay prove nore srcBful tf.r cirhc! sed s?{at+ dd alorc. Thu the flexibility ofthe hiltratioo me&un, aad the holding togethcr of tisue, which n foud in the ior viscqiry @llulosenihate te.lDiquc nal le partly gainedbt employing it iD a reak $lurion, precidt tirg ir in thc tissu6, od ths coDtinniig to infrltatc wilh pratrn wr. There js tLs withid thc ti$us a sdal but advdtegeousanout of ellulose ninate which h+s to hold rlc nriou pare together ad givo slpporr, but which srifl leirils rLe inf,ltratior of ine wd, dru mbliis Uo.Ls ro be 6t wLich show lle iibloning proldtis of wd. otdre methodswhi.r havelen producedthar ofPeldf Ge24 is thc nosr widely Cenoidibtdafib Pehtj s Methol r. Biiig fied vas!.d tis6 into absolureerhyl alcoholdrcugh the su,l giaded z.

Methyl bozoatc Low viscosiry.elhlose ritr.re

99 0L

It; an ailvutage !o r@ iii! solution ro rhe bouom ofrhe rule which hol& thc specimq in absohle alcohol. The cellulosesolution is havia than alsolute alcolrl, vhich vill therdore rise asthe sr:od fluid is pur in with a pilere. Thc specimenwill rise or the heawydrid ad rdain at thcjmction ofthe tvo duids. Wln it sinksto the lonomlroca{ to the n*t stcp. 3. Fcsh netlyl bazoate ccllulosesolution. 5. Renove the specimcnmd place it ir boae. Sel:enc is not e solvot of celtulosenitrate, ard thcreforethe celdole n lrevdred &on le.ving rhc sreciDel1beaBe it ca$ot disolve in the belzena 7. Saturatedsolutior of pra6n w* in bmc 8. Molial pa€6tr vd. 9. Slock * uual, ad cut asar odiauy gu6n

at 3o" C. wu blo&.

The doublebfiltr.tioo methodjr vhich the spe.inen tucompletely;!.dltrated vith .elhlose Ditrate,6t j! a lard bloch ed rhd raker via cl orofotu and ced@ood oil into rahffi! w* fo. s@tioninsI nohecommaded.

DofiIe Enbeldkg Ac-{r-EsrEBW,|x MxrEoD 3y this netlod (Wiggleswonl 1959)sectionsnay he ot at o.5-r 4 in thiclaess fron sna[ piecesoftissre, r-2 n'i'. iit s;s. The nerhod.oBists ofiniltEtiry rLe specino vith . Jol" sotution of ag,t h watcr. Aa agar block is mde with tlc s?ccineo in it. The block is then dehydnte4 ;nfiltrated with eter w, md fally @t in 6td wd. T[e adaotrge of tie nethod onpared with celloidin-parafin teclniquc of Pcted, is lhat it permeatesard surouds thc specioo with a harder but lesilient matiix, thereby pehitting the lloductior of thin seaiom of dificult ruterials such* snall icects or of insectihtegMst. Sectio6 are cut vith a ruor bladc holdr (p. ia4. It i5 not 6y to lrodlce a J% solution of agd in water. Tle rclubilitis of agm vary, and it will bc foud that the Japdde phdud n rle b6t for dis lurpose. To get the agd into solutioDbring the watd to fie bon dd stir in the requned afroet ofagar powder. Ktrp the fluid stired od toiliry rotil de,gd pass into solution. Ifit wil not pds into solulion plee dr Leakerinro an artocLve ar abour roj' C and it vill th6 djssolve Allow it to cool to 60' C dd placeinto n de specimenfor bf,ltration. Keep the agar at ib; ttupc€tue for the ap?ropriatelergi! of tine. Iftfu agar geli it vil haveto be put itrto the auto.lave agein,for it isj$t 6 dificult to liqui& ge cd agu, J %, d ir is to liquiry the dr} powder h water.

r. sring a sbal pie@ of nxed, v6hed mtdial doM to Nater. 2. Placeit ir 5% agu h vater .t JJ {o' C for one hour or longer. 3. MaLe a trougL for conaining liquid agarby faitding a pieceofadhoive ellophue tapeuoud a snall piccc of brss rod abou 8 m. squareby ro m. or so in lcngth, so that the tapc projects abovethe rod od by about 3 m. The bros wirh itr at*chcd tapeshould be narned so that vhen the €r solution is poued irto ir r1rcagar doe not set imDcdiately. 4. Pou lnto tie warnod blocLJroldcr sufrciot agar to cover the slecimd. 5. Renove the specinar 6on t}reovd dd ph@ n in fie agu in drc blockJroldcr. 6. Olidtate the stecimo Eder a binotuh microscorc so that drc facc to be dt lies donnwd agairutthe !ras. 7. Allow ihe age!to set. 8. Renove .Le celophane tape fron the block, ud trin the rgu bloclc wit! r scalpeiso that n b a litde larger ihm wili be tu.Uy rcquired. Iffkdtiofl of theAZd Blo.k o;th Elter Wd r. P1a.ethe trimed

agd block into lo% ethyl alcohol lqueous.

3. ?o% alcohol. 'Cellosolve' z parts to r. a. Zo% .lcohol ed ethylde glycol nono ethyl ethd, Iqual pu* ofTo'lo alcohol ad ellosolve. 5.

Sediat Csdin! ir Mnhtcopf

98 6. 7. 89.

r lIt 70%.1cohol.2 luts.ellosolvc. Pur_eeth,vloe glycol ooloeriyl ethd. Threc chr€e; Celosolve sd ster wd equalpans.I! m over. Purc ster wa\ ovemighr

Blo& Mokiry r. Make a block containerwitL bras rod md adhesivetapea for drc agarblock, z. Pour into the contaiDd suElciot moltcn ster vs,

heateda few degreesabove

3. Allow the wu to cool slighrly. 4. Add the bnltratcd agr block. 5. Otiotate tie rgar llock it tlc li
DoubhE|tkdd,g Ctttif!:

We! Method

In tli nethod the sectiou arecollecteddirectly on to a {ilm ofwater. L Take a rcveslip hestring rr x , rm. :. Sner the top of the covosl+ vith g1)'6;n-a1bunen, od the lottom with 3. Foten the covolip to tle oicotone lnife in mch a Iay drat it is horizontal udjut below the edgeofthe knife, d sho$n below. Usc 'rlorirne ro hold rhe coveslip losition-

Ec,20,

RrTor-bLde bol&' :fd.

'peorted.ovrdi! fo' a dtrg ,c:'*.re' qeL meLhod(W;bgl6sodb/

q.( bo.L b/ lbc

4. Cdt a dr/ sstioE or two ud love in posiiion on lie blade.Thi! is necsary to preventrhe sura@ of the block gefthg wet. Ifit do6 get wer ir rll lick sectios ofi the ldfe blade on the vay !p. 5. Placeon the covenlip aboff retr dropsofor % $dirn chron-glucosate(D. w. Haering & Co., hr., Su Antodo, T*a5) in vater. Tlln is d dti-corosive. Sw€! thc €uid u! the knife to the sectiorolying there vith " nre c"n 1 hrr 6. The dry srctioc will sprsd out on tle fllid, @d s otier ue cut 6ey will slide domwdds od o to the covdsli!, which n renoved when sdfnciot sectios havebcn gatltred 7. Doin ud dry the coverslipwith it! sectiotu. 8. Tleat $ odinr-y wd se.tionsfron then on. flis nethod wa! prodrced pattly as-r neu ofcuttirg din sectios soitabletor the osmim-etiyl gallate stainingmethod (Wigglesworth. r9J7). but it hs gener.l

pE:ji'M, 1921,zcitt.htfi

REFERENCES oitktkh|rtuhe MiktuskopleantiJn hiktutkapk.heTechflib., Jtt

38,142, WrccdsvoRs, V. 8,, r9J?.Pro..Rof. So..Land.8.,r4j,135. WrccEswoR?F, V. B., r9J9,Quilt.J. MnL sti.,roo,3r5.

C4lrs

19

ISOTHXRMAL TNCHNIQIJXS In it6ltotior tedriqu* where hot wax is ued with nmalio or with add tissucs tlterciqsooeduoruon oftemDain r. ir $c bcriooMhi, h eL1 e bctnen rle d*r-h of de uimal dd Ge 0tu1iftlu.iob or$rcof i6 ri$u6 in 4.oiiJ blo(k of wd. The he.t history ir roqhly s indiored beloq tholgL a detailedrinc scdeis onitted.

60 5o 'c ao 20 1o

FIG,30, fludrtios

TIME H . a t h i s t o r yo f m o h m o t i a nt i s s u e s 'n thcL6r hntoryof m6-n;r tisus asnom,ll]r pE!trcrt fo. $didnis

Wid invcrtebraremar€d.l the heathntory would be a ti*le diferenr W}ile it is ostomry to disregaldrhesefluduarionsoftehpennre it will be cle* rh:t r.hcyola, u inlonDt p:n ia nidoton ) ud Lltology. pmjcuhrt/ where *eptioually 6r detail: requ-edir tlc 5u ed ,edon. A matcriel vhich is fluid or sehi-fluid, suc,1r as the @jo.ity of living rises, n nuch more readily penetratedhy i{4riv6, wam txarivd, &d is the e;e @terial $lidi6cd ed old. Ir tlLB nrch lorger to colou z roo nrl. block ofsolid J % gelarin in rar.! w;th a o.r% soluti@ ofnedlyl@ blue thn it docsto colou roo bt. ot tle srnc gelath solurid in the iiqlid condition. wit} oamnalan ud avi:r ni. er rl,e cnp oyhn' of 6e!iva, Mhing Cuj&. d€bd drioDduids.lM solvft dd nor,o qd ai, t4.l ,.-r.ru;..-.;."di.s with that of the nimal in life nor only redu6 ile rihe of dl rhe prooes, but produc6, raoolh lookirg 'ecrioDLorpr"ble widr dd oq@ muJ beftr rba s*tios producedfrom tisslestrearedh the usral way. Such a_teclniqre rcquires nore precie and aa@re tihiog of a[ the srepsas compded with the oodal routile, b@!se the lerneabiliry ofal *sucs is so nrch g.@td that it is reLtivcly e*y to erry sone of tLe pro At onc time Lie u ofi.ooeroul 6h!:que( .^ aLrosrs impo-,biJ:q bc.aue tle urarqrrionmediaa,il.ble ud .,LLb[ fo! !hi, 4d $i,i 5e
i€stricted to parafil! wax ald irs modidcationsbut with low heldls poinr 6ter rttu$ avil"bh (r8" C melliig ooir,) rli, approaclro b.n.' ...ao^ .o-e. ro! the iDveltebrateor cold-bloodedaniEEl i5othdbal netho& could be {fied througl by fring in the lsual way anoom toperatue, virh a 6!al in6ttalion with .eloidin or wi$ u."-form JdcLydekG (p. rsj. bur *p";eoce would sugggr "di? that for gtueralpurpo"da low srdls poiit ;r \ woutdpr;dk. ,u-h " ,-,n de in rhe finishedsectionrhar linle would le iost by uiLingsuchm iq6lrrarion oedi@ eld thoud it vould ne.n a dep.rtue froh thc previou sleps,,]i of wLich were $ouef4ar, ud rt room temPeraiue. It is stil nddtaided hovevs, prticularly with maonals, that isorhermaltechoqus whicboroJu,e a rmiglrr-J.ine h.rr turory oChisrologjcal bJe"J showrlnd"rJ rd 16, lrril:.|s rhd Lheo-isoclcrcc me Lod.comonv used. lbefolowirs Bivc compndve dmr. uirg a hJf r.t kianey.to' tti-e, piece: nuch shorierties my be giv@. All re€ents ucd at 38" C: r. Souin ,. Jo% erhdol aqleous j. 7oolo etlDol eo nl.l utrosorve ro Dr.J a.9o%e.huol 9j nl.l 5 rru.J

Jlhols 30

,,

6. Pue polyeste!w,7o/3o

) A vdation of the isotLm.l appro&h is given on p. ro2 where detailsof sinulte@!, ivtion ed wd idfiltntion *e eivei.

Cli!"IER:o

SMULTANEOUS FXATION

AND WAX INFII,TRATION

The pradln wd heitrod ofinEltrarion sd sedion dning h* guided the thoughrs ortbe fl.,oroni{ rd lhe tu{olos:,r sjn." ,8oe,rd tr€,ioo. ai", d,"tion..1..;n rd infiJ|ation,re pro.e,,o $ hi, h to r L"se cx1sL h,v" b"- *i.d"d * ..?,,; md distimt. V/idl advff ol new infltHion nedi4 such $ polyethylene glycol, sd +e putioluly its 6tcs, theren no remoutoday why the fou. to lbde rrc@k; -G;,i n u 1 n o r b e . o n , 5 i n c dw. b n r * e _ p o) e r s - s t ) r c t .D , ; , . , r * J ! i " - , . . u . " , o , L , -pp,or: no,e rbr ny o-bri Dl]haEon m.di m. thcy hrve p-ored o rroublc.om. i! na$€niDg ad adhesior of se.rions that ir n osidered pruddr ro disegrd them ad to coumtate more or dror esta"swlicf ue dispenible but not i v soluble in water, but which are solubtein w.rcr-at.oho1 soirrions. Ot the w** : jr,!'e.ftr Ltr. Dprc"d- bol)tuk. " a F. {,) ru r gF"r dral in rh r,t olr. bdau.e ir r n.diJy sol.rbl.ir J,orol. *iX r. m!a- -ucl ^ ,ooowJre in LJ,e Jcohol, wJ di$olve dcnc &i4 ed vitl tolore both fornaldehyde ud dercuic lercHoride. such an aplrojch rculd nerei be vi$atikd !,ir! r{aftn w4 because it isio restticted lrr lrs luge or sorvcnts, Sonc cotuider.blc iesq.ch hs shown the following tormuh to Lavea excellot Pirc_Ialnat wd* Picnc acid oysrals,dahp Lopropsol 40% fomaldehyde Polyster Rd, 9o/ro bi\rue, nolto

J gm. J5 nl. 2J Dl. 5o ml.

I$olopsol ba breDued ., J deb)draingegrnriaLerd otrdlyl Jcotrot.bfta,6r tr doe(Dorprodu.e,o lBh a tuted rfltur 6e JcohoL. ddr efied oh Li,ru6.The mir.rlrc is comrlerely soluble at 38" C lut floauLrs .r:8. C. Medtrfamol_uB hoprolyl alcohol Dnt 6d vlter ao% fornuldehyde Mercuic percbioride,aq. sat.so1. Polyesrdwax,9o/ro,nolten The aboveis ! cler liquid at 38 40" C.

70 mt. ro 01. r5 bl. J ht. Jo nlt.

ii will be seeanom both thesefornulae rhat sone ofthe rcaqentsde emDtoveit in a dual role. rhe wtcr prescntin the forhatdeb.dc in th" p;"_f".-"t -; ;d

Sin barcorsFirdtianatlitWaxIijtttatiof

rol al,o p,er1L in dr n.e,. t-fo-J-wr pFvenr ne)ll rr.ue. b-ue subiecedro , u d d o . l e 5 ) d , r d or D d . " r d r es a r d m . e d L , " . e t a " . f l , " . i * . " . o p . o p r t alcohol; dre isop.opyl alcohol a.rs rdrry asa A\.rive, p."ty * " d.hyt"d"s;g;:i,: Padysawasotvenr In gdcJal n nay be std tL.r dE dosr videty usedixati,cs aree$eftftUy.okous. s d , a d ' er $ o . i \ " b J b o l .. o n t , , o b l , r o o o o " r a i ! t J e p i . " . i o : r o t wu, u d cc t r l r | \ . i b r ci n . . c r i L 5 . e d - d . , r e di r r I . , - r y . A r p 6 , n r . , . d r g n J t' o , r h . r p r r r r j c c , o . . d r * i t , . . . , . " + " , " . " . . . f f o m r D f a t r " r d ( F o tI p ! 1 i . t u i r t r o . d J p . e " - r o r t . . b c g J i \ e r o t e , fdJ r u e . o t u o 1 : ( J I g I o . d u , 6 : . o n c e i , - d .\ . . , , r e g " r d, o t h e p h ),.ic-J e n c : \ J r ,m r : F " , q o : f i ! - \ - " J v f . { v o u , d . , . ! g " . , , ; , , i b * . r ; i , i , f,, sectrodshowte; nrrDrrgc ddgrcrrer \ofhe,s. The nedrod could he ntdrned to sit vork on tg,m$ in which c6c a mtio of .o o r. oi:rot- ^.,r , S h - p - o . eq L i e . I u b t c . b ee d i u e d s r o L . t ? L h -"r m e r t . o o d rhi U1d\io$. uy r e l . $ [ d d r . c o r m p r o . e n , c bor\ " r L l e m o e c o r. e n o o J : p 1 r o * , . . tJ:dwtagcs 1re s toilows: r. c.eat.r specdin proccssingtissues. 2. crcatersoftncsofti$ues. 3. It Day be lrd isothmully lo! cider wrd-blooded or cotd-b1ood.d din.l Nlcthadof Us. Mahnaliat ot Aridn Tissaa r. R*rok rhe dgd reqLlnedfron the dnhal wine sdlt mrn artt ptaccnr eirher ue trro wd or $e herdlry wax !t l8 $" C. teAve fo. fivc hoB !t thar tempcrnr.e.Sft arhle ,als. r ws, rith$irriDg ifldsible,lbr z_3 hous depending ry.; nti-,"f ]1A!. oa trre sre oi rhe spccine!. Iour b.rhs. :. Mrke a blockb rhc uua1war,. 4. Clt $ with_ort wd material,dd floar sediotu oa to stideseither usingatbuhen \{ith 1096loloa1in asthe flanoing fllid or ushg aniopcain solntor With sull piees of tisrc the sinultmcous fixarion od ws hEltratior netirod cu lead to I sredihg up ofprccdue ro a roinr a! whicfi it qay laLc 2a tous or re$ to ?rod&e slainedmorntcd secrionsfron 66h Dareriat.If vd could set{rstu the hcthod could nru ttat naterirt llred at dne iu thi?;lysre. mnnre woutd be ' o r r.rbmd rd n_or o b r n " ! o n b / L J c o . . t o . k_ , , r , " . " . " i " e , i " , ; . ; U ; p o r ) 4 . e rw d b e .d so v e m i g l rh , d m , g L op r o d u . e goo.._,o- d";bbo, d"M i s $ d " r n . b e . .w h . r u p r e . e . o r u . . u: "u.c h , i . r , l u o d s u r , ,b' rl eh (eendr r l o d r " r L j d , , f l .e d ) o o , . , l F e m r . o \ . 1 . h q , o o ; i c e td . , - r l o J c r r o _ c|tmiiJ .ooh-eu$ re prcsc,led, , ,LrL. t. - ,+.', *-ir".i." " a;._J,

CFrPfs

\VT1]
?I

W]THOUT

FIXATION

It l* long beo sratedby listologistr dd cyologists dur .c@te, @refrrl6xtio! ol tisues is the only baJison which the study of hirology od cyology may be condlcted. Thb approachis soud emqh in sone ways but the biochemisth* *eqre y reverled ooti* viewloint. How ofto ho tLe Listologis!or hi,tochenist appro!.hed the biochenist viih a pro!1@ *out stainiog rcoction ofhistological s{tiomi ea?lainedthe situation dd stat€dthat he could not get cstarn expected €heni.al iadions, o,1y ro !e greetedwith rhe q@stioD:'AL yes. 3ut I r*e ir tlal thesewere fxd tissust' TIe suggestid my drere6re be qde that the nethods of fiation pnctised ova the last eighty yus lave taken tisme so far amy Gon tLeir BruFl conditio! tlat the biochenist od d1e opoctrenlt ue &illiog rway fron each othcr iasead of a!proaching. Thc lra.tice ofnrGltntior witlrcut fix*ioo is sometling which requira ia adran@ a defnition of f*ation, rd u uderstudirg of itr n@iog. Pe!h.?s it may be suficiedt to srggot tbat a fxed *stre shouldapps in sectio! fola unds thc microscopeo closex posible to that volrme and shlre wlich it posesed io du living conditioa dd dBt it shouldalsoyield &enic.l rerctios vcry clow to thoseobtained vith lresh mterial. Cleely toth 6rd ed coDtituti@ mul be arreisA In the pa:r the moryhology olthe se*ion appared to be the prbcipal bd on w!.ich to as6s ihe herits offL\ation, lut grstd efiolts slould be oade to eis6e rhat thc chcnical rcactiou oflreh ti$n6 ue .lso retained. Thae mry be sone doubt as to wtrttrc the fom ofa tissueor orgo my !e prewed ud cund into w* vi&oul ia sone vay congeding or preci?itatinAtle proteinsofwLich fte tisue nay be cooposed. (this tale !Lcc, evtu if only s a roult oftie he.t ofthe nolten ws, sucha co itioo nay be onprable with fintioo, aad the tistre3 tuy Bpond ro staibirg toehniqlB in the sae way * those ti$les flred id a hore conventiotul turmer. If coDgelatioDof protein were to be acepted ar a cnterion of fiation, however, t1'! condition ofrgor /drii highi be conidered asevidene ofnedon. Thir i' never acceltedd such,dd iftissls were to be caried into wu ud conpletely in-Eltrated wiii evm les corgcllarion ofrrolcin &u is food jn r&or dotu then a conditiod ofhiltration without i-gtiou nioht be rchicvedVlut is oeded h hntology, cyology, Listochenistry ard cytochennsy is r Eore gede epprcach !o fixation of tissue. The violeDt telliqus employed at pment shouldbe rexaninedTo acomplish h{ltatiou without trvtio! tvo diFercnr .pproachesnay be o& ud loth take advartge "f 'dch bodeh infiltntior media $ polysld wd.

Wat Injlhitior Wii@st Fi*atki

roJ

Tte EBt nedDd is baed upoDdE solubiliry oflolyerer wx ia :lcohol ard water, ud iB success hiigs or the !.eipitation rars of proteiD and tte penetrationntes ofvd6. Ifa con&tion were to be orablishedh vkch the solutior ofwc pmetrated lhe tisu€s befo{e ihe alcohol plecipitred de protein then thc tisue would be pardy indfated befole it w6 pre.ipirated. A bath ofpure oolter wtr would continu the iniltration viihout conthoins rhe t&rior. The sMtrd nethod ir bsedlpon fie se ofvs solvots wLi.h de Jio d€hy&atiig agds dd vhich do not preipiate pfoteins. Ofthese ethyleneglyo1 nono ethyt etheris most suitaLle,though ndy othersue av.ilade In lotl reo\nlqus it is esdtial to use polyetcr w* becaue rhis h4r a nehirg point (38' C) vhich n equivaldr ro n\at oflhe temp@ture ofnost rumnals ad bndr dd thdelorc no coagulationby hat raks plae in dy olthe risslB. Tiisus de rakd |Ioh the G6hly killed oinai wbile stili mrn, md ae put a!rcugh the vuiou fllids at 38" C. {/ith funher voik on thesemetho& it should be posible, once tte teclniquc ha bm chorouglrJy nnre.d ro prodrcecobdiliob ; whjch conEoled dcgis of fxation cotld he appliedto uy tisre. Ttuee fluids are srggeted x giving diferenr degre.soffxation: z. Macury-fornol wx. 3. Pidcfomol wd. xthylene glyol monethyl ethr, 'Cellosolve'
celosolve ?olyster vu

7ol3o 5o gn.

To 6e this @dmd the folodng stepsmy be tako: :, .. r. P enove r !T,I pire o' brue f-om LheGeJJ)-killedw"@ ILnftl or !i!d. z. Placei*o ellosolve wx for 2, a, or 6 horB de!{ding otr the sizeofrfe tisrle. a. Mlke a block. With a dos sliceofa rat kidney a hous axatior or dehydrationin elloplve w* n s$icient- The total titu th*efoE for sucha pieceof ti$ue ro bc tato into pue tobste! vs ud blocked,is e*ht hous. If thin slicesor lieces oftissue ae ed the time my be brouglrt dom to tue or {bur hous.

Sation Csttin! ii Midos@pf Mdery-fothol w6 kog.opyl al@hol Distiledwatd ao% f6na.ldehyde Mertuic pe.drloride, aq. sat.so1. Polygter \w, 9o/ro, nohen

zo n[ io bl. rJ hl. 5 ml. jo m].

wa.h all tie duidr to t8' C aod add the molrm w*. Thir nuia widr its contet of alcohol, fonnalddlyde .nd rErclric pmHoride (HsCl,) could !e cdidered a a f&tive ot6ir sndgdr used for two to four hom i! acrsnildlr and sedy. The rine rlEdule n thc sames thar for the cellqolve ws.

PnaFfowatw4, ?idic a.id crlat.h, ddp hopropyl ai@hol ao% fom.ldehyde Polyesrerru eo/rq nolten

J 8m. 75 mJ.

This solution is more vigorous tlu the prc.eding two. The tisues rre thcreGrc closeto the conditior efter nornal6xation. The oirture should be ued ia the sane wy s the tuo lrevious Suids. Sectionsshould le fattmcd on mylolectin olutioro ir the way rcomoded b. Iz4. To pddr &ration by alcohol whe LG scctioir re being tako dom to water for staidng tiey shouldbe takenrho$h the foloving steps: r. Xyldc and cellosolve 5o/ro. 3. Cellosolve70, wns 30. 4. Cellosolve5o, water 5o. j. Purcwatr. Ifi! is cosidded thar xylde my af,ecttlr sectioo nr uy way, it nay be omittcd, sd ihe se.tiom coutd be placedditecdy ilto pue cellosolve.lf the did6 de w.rned siightly the sd til &solve be!te!. lt will be fomd that a{ier the se
Ptatanntng!h\ \tl Rrt J\,, .1

IL

Flc. j2.

lr.

C.llosolvc*r\ shorviw\omc shirllsc oflldR.r sr,uuli

Mercrl,\-foxn.l

\'rr

shdulgr

-qlo ola{htiikrse

br

somc slight cotrft.iions

rtr turM ::ipt ^[ rfl ^f nr

Frc.ll.

li.ro-tirn.ol{lx

shosnrg rc gloDcn,hr sunrklge rnd rdy 1iftlc elseNhere

WM Injbtdtial W put Fixation tctT celololvrwax seclionstows sone shintage ofthe glone&li or of rhe tisu round then; the nerory-rw section shom no shriilrge of rhe gloneluli, bur sorle ttuddry ro sepJ€r.io of rrbulo: tle pidGfolmol wd KioD s[ow. Eegligible Ttu ;i'." i-ti""--* fl"id" nry bc 6ed witi odd tiniiss ed wirh tuiatior of cotrtet An ingtrite a@lei of @iarioru is posible, atrd a vidc rege of degred of tuatioDis ebvnrsed.Ti5.E seftios couldhe orodJed "howins s,.ded drgre6 of fisrio. frco !h$e $oqiig lidlr cvideDeofuch,o olhes sh"wiig lre co;ptelely Th! degreeoffxatio! would !e nesurcd both &hic.tly

ud morphologically.

Ctl-urm 22 UATRA THIN SECTIONS FOR THE ILECTRON

MICROSCOPE

Ifflndtion M.dia Jot Ubtathin S.i,io6 wto rctions *e requned s di! s jer,ooo A only rlose infiln.rio! nedi! which are very dase od morphors sil be suit ble. Crystalline ncdia, such o p,,"6n w,r, l.-I Jr D.sery codpecrne." rd (oD6jo! The media nost co@only cnployed are lhe dylate resiru,od the epora rcsiu. Anew one- urefornaldchydc rcsjn is suggcsbdherc,aso *perinentai medim

The * oftiese resinswasfrst put fotra.d by Neard, Borysho od Swodlow, r94q md * naterials for ulhath sectiomdby have a grer &alin rhei( 6vou. They re olouleq tnsldert ed of closetextrre. They nay be nodiGed by nixtuc ofonc vitl .nother, or by the ddilion ofsuitable plutian sch o dibutyl phtbllate, etc. Thcir lrincilalwealBB lie in fle hat they vil Dor tdetmre thc srccim lntil it h$ leo rcnpleteiy delyduted; they show sone slddrage during pollmeriatiou tJreirpolyneriatioo is generlly :eonpiished by the useof he.t (afproxnnat+ ao jo' c); ad they show a tardcncy to bubble Thc most widely ucd of dr tuth&ryhtd n ,-buryl medEcrylarc, blr nay workers considcrit too soft md it is comon pra.tice to add to it ro 20% of etiyl nerhacrytate. T]E laacr is much hard- @d givs to dE foncr a sritrues which embl$ better scctiod to be produad at the lowe! end ofthe sectioncuttins rmge A ni*urc ofgo ri. rlutyl de{lbcrylate and 10 n1. nerhyl nethacrytatewould le m a@Dtablebiltue to host microtomisLs. SuoLis the rctive condition of a1ithc hetiacrylic .6is rhat thcy wiil polymerize spontucouly if lcft for a suficidt iength of tine. ?obheriation is oore r4id in bdght light, rxrticululy sunltht, thm h dahres, ud it is rcel*ated io mro conditions. To prevert pollndtation the monohdic methaoylatcshave hydrc quinoueaddedto thcn, ud dri re.gdt is preent in tlrc resiu o bought. Bdorc the resinscan be ued the hydroquiDonehs to le renoacd. Thi is done ly shakiDgthe resin up vith a 27o solutiotr of sodium crbonete in distilled water. The two guids are thar sepamtedir a seprating flmel dd tle wasling is rcpcatedfou tino. The wohing proes leavo wate! h the liquid ndoner evell aGu carefinsepa-ri@ The picefte of water lads to polynerized nerhacrylatesof such vcying d$sity that they will not prcdue good secltiou.The *eta is renoved by addiag to the Mhed Dotuner a snull uout of calcim cbloride. Filtntio! rdov6 dy solid matdi.l i! lhe rsin, wLich is rhen stored io a glos v6sd in r rdrigsator. The 1ow tempelat@ ad dah*s retrds spoatueou po\nerization, od dE re,in is ready for ue who retluncd.

Ult. Thin S{tkflsfot theElectftnMi.rostapc tfkdtku

roe

ltith n-brryl Methaqlax

BDns i{ed, wslld natcrial ioto abrcIlte ethyl alohol by !6sing n through the ulal r.ics ofsradedalcohols, u:%, so.l",zo%, qo%, abiolure. A t o , . . e ' l , ) lJ r o l o ' r d n o n o m e r irc- b . q m e - L . 1 L r c e q u Jp , . n . 3 . p u F D o D o r . . ' t / - b . I I nc d u d . t " E w , r h( _ . o " " " . . h I n " d . J . t t r - . 4 5 L , n cd o f e r L J $ o f o ' . F n o n o n - r , n r h , c - r c n i n r - . I o r c g - ' r .o f _ n o o o e d . l o . zg r r . o f : . a - d . t r J o r o b e v Iop. . , o E J - . ] I n . i s d , e . " r J ' r r q 1 j . l r . o 0 \ e , r .t l p a o o l d . r d L o , , i d- e , i nr o L t ep o r y n c r i r o d . o [ d b r r . 1 rn o . e d . J 6 | r i - es d g i L i n g . l , oou ] , e , r r " r e ,D d r h , , , L c o r J . t . t u l db f . u F e di n ' oJ , e , e .n n o . r r l q o l g n l r . t JL h i .r - o r o o D -r h er w he po&ers of Dcarallzcd rsin io lhe hixn!; shch iitl lead !o a firuI blo.l of : h v*y irg ludro,e. Ln"t.erioningwiJJbc J$oJ iloorimros,b.e. 7. At.r d-. at lad r-\j! I- br.r .Hding o; r ,u6.;-t I,bS oC .n. for rI thc sdned-in bubl,ls ofa; ro flo.t to rle ro! dd bu4r, into-" g.l"ti" No. Oo capnneardr y !h@ db innltated spainen. Thn is bcsr d; with a tneiy 8 . I(rlo tl,e .or, iro n in Do.ior L ',p dre .f Lle wiir the , .zedr.. o. Pou D go,l/ so Ll-rrdre .pccimm < nor movedo r o.po rion. bur if., is gendy nmoeure it lack widr a drara-out glassnecdle,or m ordimry momted Plae dE 1id of the olmle oD rhe imer pa* which now conrainsrte sle.imen in rdin, and !la@ in d over .r 4J-Joo C for alout ?4 ho6. Tte rsin,ill gei od lnrden to some etteot wirhin 8 hom, but conplete pollnerization aid hddenibg ta}6 alout 2a hous. lfo renove the gelarin rcak rhe carsde in waln va!o, md gcntly rub the rr. Undei a lov povcr bjnoculu niooscole tib tlE dd of the btock sith the sle.ihen itr it to a squre r x r nn' or saller asbefits the specimo. cleuly rhe sruller the beltel r:. The blocl ory row le ideired jn rhe microtohe block-holder ud is ready for It may be e adaanragero trsecaralyzd mi{nlres Fon stcp 3 onwards.

TJre detailed iteps ueesrry vifl dcped on the rute of hicrotome. I! ir nor propoFd to_gointo the t)?s w!i.h rc low avail.ble, rd rheretorealt dDt may be saidiuc is th* tLe nulen iastnctios shouldbe fottowed with greatcue ud doM to the last derail. Whatever rJt nake of nicrctomc dr f.lt;wing .equircn nrs :. Fix dr tlock niniy in the niootorne chrk. 2. Takc a newiy_brckcn gls knife dd to it srick rtrc littte metal bxth generaly provided ly the miaotome maler. Srick ir ia position wittr p*fi]n-w*, to

SectiotCtfring in Mioavopl which ha heenad&d about zol. resin or dry lalsarn to give it sdhdion. Use . hot spatulaor other sinilu istrunent. Makc surethat tle edgesotdE bath do rot stick out beyond tLe edg* olthe glassknife Stick the melal bath in seh a position that when it is filled with flrid the fluid will neuiy be level with the .dge oftle lniG. capillary anractionwi take it light !P to the edgewhcn thc sectios arebeing ft!. ShoDldthe edgeofthe glss LG be touchedacidotally during this proccs throw it aw:y without furthcr considention and makc r 3. rix rhc glss lnife with its bath in position in the Lnife hol&r dd n.Le surerhat it is pbad ercdy a specfiedby the nakers ofthe icnunol a. check the clearuce ugle ofthe hife. 5. check tlle height ofthe kriG relativeto dre corect oler*ional lcvcl ofthe blockcuying un of thc nicrotorc, shouldthis be neexr;. 6. vith .low ?owc! binoculd fuhed jfto porition give a secondckck ro thc edceofdr- slx. r{Life.cL' Jlseclio$ u,'d, rbc binocul"r. r. ravoce ri. oiie to Lbeb oct by m.m oi Llx co-nc ,Jju'turr s by \sd ud ciop the knifc car.ic 6mrly whu thc k ife; "1nost touching the biock. 8. Try a cut or two by mdipulatiry the fine cutting nech&iso of the hi@to@ Ifthe srccinen lp?ers io be too f:r away, bring it fouard vith the coane ,dinsrdentuh1tr 6ne touchtr Dade e. riL,.p tre krue b--b \ith fuld. w.th meth*rl Lre re'm. w"ter oo ml. * erone ro nl. is recoinnoded. ro. Sart cuttiog, ud when tle block hs ben 6ced olfthe coloLu oft-he secriom shouLdbe notcd. The apparenicololr of vof thh se.tion is dE to rerr:ction phoowr: r.d n nr1 be ued s " g,de m rhc r-hi-ne ofdre .c ion Seaiodt whichue grq ir rgpuucc *e sutabte r,. t l-erc-p*"ion ud rcn uq"dooofde serdobt}oJd J o be notcd whor Lh' i. u rlreieiou ollo 609".t d y be -hr $e ecrior wi beMablcro egain ridr flamss, td tle k"ife 'hould bc moved to dotha Part with a sharperedge, or altcrnativeiya ncw laife shouldbc nade. rz. Shoulda voy thicL sectior be cut by acddot the llG shouldbe Eoved io n\et dother pl.t ofn i5 used.A much thicker s{tion thd is requircd *ill blunt ay knifc, whethcr ofsteel or glcs r:. The liquid in the brti shorld rm cledy ud eoil1 up the gls surfaceto the . & e o 7 r o . L o i . r h e p , s o c e o f r e o r b r h c . r - g i v e t o L l - er m r e . ' i d , c d s , ' 6 e r n . i o " " h i c b l e d : r o r s x r e rn m i r s A g s ' l a u l e o f s o n c hom strading in idurial regionsshouldbe diserded becauscdelosits uron it f'om * ar p cvor .ooo$ dowirg oi lhc *r- niru. Mauntinr Sectio$fot theElethaf Mietttule Thc scctioc rvhor ot td floating on watq G w€k detone atc nooted on snrll circular netal grids, or on sn.11metal rlats vhich are sLottedit the niddlc wirh a g'p "fot --. v";o* typ"s ofsection suPpoitmay be .eqdiredfor di1lereft electron

Uha Thin se.tknsfot *,. Ele.tto'1Midasope The sdtiod @y be floareddirecdy on ro dr detal sullort or tie supportnay bc coveredtur widr r rhiD film of ro.nvir lcsin. (shawiniscn prodrciicor!., ljo Fiflh Avdue, Ncry York.)

Make u! tlc foltowiis: Foinvar resin 4 gxl Dioxe 996 bl. :lo nake_a thir-fin take a &op of Forqvr solurion on a gtss rod dd getly touch it ro ile surA@ofa dinr ofclcm &tilled water The f6 slurion witl;!re; out nd forn a drio film. Another ray is to dip . micocole slideioto rhe lo.nvar solution ed srdd it oD od to draiu. This shouldbe donein wdm drr air ro p.evot nottlins ordre suface. Wle the resiais dry the ourline ofdre netal sullqt on whicL the 6l@ is to rsr nay b€ cut thiough the 61o to rhe glds vith a dissectitrgfted1e or 6nc sc+et. The resin61n sho d tho lc smled amy at tLe bonon ofthe s1ide.rhn frd shouldbe gmdy toirered into a .lih of.leo v.tcr. The tlb wil lift otfthe slideo the water rm lerween it ud the glas, and drc cut-out suppor* oflornmr will float iee ot the suface. It is e$enr!.1ro lse clcanslidesfo. rhis tshnique or the itn nay srick ro uy gre*y patchs of the slide. Ttre il,n doating on the wte( n ?icked !p on the netal plate by holding rhe plare h tne forceps,lovering n bdEath rhe surhce .nd tl$ rdsins it fron r toirioD l,clow tLe piece of6Ln ftquired. whm rhe iLn hs dricd the plate I readyfor reccivingrhe section. Tla cut sectios are collectedlron the suf.cc of rhe fllid - in !h knifetarh itr the s@e way d oudird lor thc collectionof the Fofr@ frLns$vd in the lceding !daga!h. When d;cd in cleo wrm air they ae readylor thc elector miooscopc. Absolutedeadhcs is esotial in all rhn work.

Thsc first cee into $e n! r9J6 vhe Moldc ud Bnch-Anderson"ud Gleuert, Roges ed clauen, lrolglt foNrd approtnab nerhodr. The epoxy r$i$ heaet\e advutagc over acrylatesofshowing lss coar!&rion or voluc slnirhge after polyrnaiznion. The lisue qootcd (clauerr, A. M.. dd GlaErt, R- M., reJ8) ir 2% lor Ar.ldire etorf lsin s coolded widr rj,2o% for nctheryhtc. They m trsparot ,vclowirh r6iu which n.y le vded i4 hrdnes !y suitablcplcticizo such6 diluq,l phrhallare.They a.e soluble in ed@ol, dd vil tolerate as duch d J-ro% wlrer in !,\e dtemedirn. Thc chief waknss of epo\.)'iBins for ultra tlin sectionworL lics in theit high 'Gcosiry, whch nay hed rathd long tuiltratioD lcriodr. rn indutry they ue ued for 3 vuicqi ofprrpos6, dd their ldhe3ivelropertics ee videly recognizeddd &vdsely ued. This rropdr)-, rdticuldiy vhd coBidercd in co{oction vith their 1ow slrbLage on seuing, givs great value to dem wheD dificuft objecb for sectiodingsuch s hair, cotton 6!!es, etc., are uder cotuideratior.

&ttiotl C"uin! in Mido$o?y The.oldd nethodr of settiDgqrorT reiu utilized srrorg mins, which would undoubtedly,b,wr r.lrene e6.d uponpror.in,but de nost recor ,etftg agoa ec or a mu.D 16 uo10t clr"i.cre.

Wltdtiat

with AtdlniteM (Aero Res*ch Ltd., DBford, ce!.idge,

Engt rd)

r. Bring thc 6xed specimd ilto alsolute ethool. 2. xq!,I!!.ts ofaholule erhdol sd Aialdite M ro nl. Hrdetrer 964B roq. Diburyl phthajlare r nl. 3. The puc resid hixtue d given in srep2 in d ovd ar 48. C. a. Make u! a fresh rupply of resinlcordinc ro the fomuta atove :rd add to it rJ nr| of A@elfrtor 964 C. Srir we[ end add the s!6imm. Keep at 48. C lor >j LNs if the stecino is nall md soft, or for z+ hom at .oo- t"-po"t,," ifit ir lurd. J. Ino r \o. OO geLd! c"D,uleohc rhesorcineou.og " 6re prpene.od Lhm ld rp rhe epsLre. Thiq is bsr done ki.h a pipenr wticl tD. be{ 6led u,irh lesin."lae rle ri! ofthe lipen at rhe bofto;;fihe carsuleod gottyili the epsulc fron rhe bonon tpwrdr soth,t ao air bublle areprcdu..Jq perhittcd ro "jck rc thr sid6. rftbe speci@ moveak) 60- $; -'ddtc .ir!e 6tted opsule gody move tleo br.L .gair, $ing lhe 6ae pitefle. 6. ser_l-be res 6y krcpiig tle ep*J., witf ie co"r oD.iD s o!@ a! 48" c for 7. To rmove the gelarin pl&e rlc €lsule jn wam water, rd brush awy the geratrns rt sotreiJ. 8. Trin the @d ot dE hJock$ dL rhe speciJnenis i! rhe njddje or a , bm. :_cuir" p.. :}i1 udd a bilocvrr nia;.cope usiDsa oie+dsed safeq r/or bladc.TIe biock n r!tu reedyror cuniis. CaitingtudA e MBlo.Is rbe tudng of !h6r blocLt L &e !@e 4 lhe curiile of Dethacrvtetes eiver on p. oe.Tbr sc.tio6 er tut ob io puc w,Fr (cluen;d ctaen, r;js). Epoxt tus ib Modif catians It my te fouad h pradise rLat wfile a blo& of .Aiddite' dts we , ihe Daterial in it my nor do so. Cledly rhis is duc ro incomltete peaerrationofthe catalyzed od acelented roiq od it uy be due to ditrermdjpaebation of the dtrc (D.rn, r9J9). He puB fotu*d the view that the isdod pfletrates the riss@b€ner rbd do6 the r6in, dd thar wirL a tornlt4 where rhe r;in aad thc hrdener m prsot in eqnal xnouts (Glaen @d claudq re5a) the rises becom poneated

Aba This S&tio$ fat theELchallMilotope

rrl

with a nixture containingincoEcct protonios. To courtcracr thls todency le lurs lorsaid the folosing ni\tue dd tining: r. Sring 6xed tisus into absolureerheol 2. Absolute ethdol dd Alaldire jo/5o at 48' C for 6 hou . I. PEe'Araldite 72 hon6. Chmge wery:4 hours 'Araldite M r5bLl-,. 4. ,. . -r t sork ir rbr []urc-orz.: hour.,rrh frohrsir ffirre 'b48$ rre'v ' 4 Djbunl DhLhJrtr r nJ I 5. Add o 3-",1. aaelentor to'the alovc mixture ad soaLfor 2a houa ar loon 6. Pla@the slecimo in a 6esh dd &elcratcd mirture ud bake thc bloct for r 7. rr tdlding of the bakedblock sti shom ir to be on the soft side bakeit lor a f!fther ts3 dq's at 48' C. EttiliMkl The 6e of ure!-forh,ldehyde reiu s infltmtio nedia for rhe cuning of seciou betwed r 20 I is giveo jn Clapter 17. Their e s bdltration hedia for ihe dtting of cctions bctweo rcr,ooo A is possible,rhough rhey shodd be regarded s experimdtal techniqus ody. The resimde soluble waro od havea visosity which is su6ciatly low, wher Eiied vith otler reg€$ts, to tueiit qeful invesr;gario$. Uscd alooc, without $ftding agents,urea-formaldchlde resnb .ie brnde, but tiey rnay be softoed sufi.idtly to clt wcl. Thc folloiring lomula is suggestedc leing suitable. Ur 600 6oopolyethylm glyol, nolto water Urea foEuldehyde sFup DzzTo Phdol dtsrals Trinerhylol lropde

35 d. ,o nl. x5 mt ) gn. ) sn,.

Tle lolyethylde glycol should be hcatedin a bsker uril it is noltm. Then the water should be added,and the other reg&ts in rhe ord* givo. rhe seqme of rnixins shouldnor be alereA Ha/dder , Aj@onim .lJoride rr.J sm. Ura 3j gm. ware! to roo ol_ Aroonir o 88 S.c. ro !H 7.j. idda6 The sho!1dbe addedro the lsin in rle lropoitior oforc &op oftudorr to ocb nillilite ofDzzTo. fl1!s roo nl. ofLrl 600shouldlave 35 drops oflardmer. sp€cirnedrtuy be brolshr inro LT 600fron wat r. Ttre liadcne. shouldbc added bclore lhc srecifro is introduced.The resin ed hddeDersLoutdbe welt hired by

rr4

Srcti Cuuin! b Mictos@pf

stiring, and wher rhe 6ubb16 introdwd by stidlg Lavc foeted to thc top tle spccim@shouu bc .dded, and lhe rubeplaccdin a rcFigerator ar 9-roo C. The tubc shouldle corked,hd it should!e $ned at;nterals to naLe swe tlat tle oecitrm is beirg b6irrrted. A Jow ,rift* s suggekd on p. ,J b veD $iLrble. Shouldt\e speino be kept for longer tla 2a hos lhe flrnd shouldbe .hanqed. To nake a blocl pou into " gL6, rrbe $rc D.wly dixed and @Ljlyrd rsnr-and ,ddrhe specino. Anrg- rh 'pecimeb* Lhebo,ron ofdr glas rubeod leve ou thc bencha! a rooh tmpe.atue ofa" C to hsder. Tbis tehperature is dot diridl. Any tem?datue will do, bufthe lowd lhe slow6 vil te the re'h ro h..d@. Shodd there be dy .dhtage ia iDfrltratioo dd hrdeiing at J-ro' C the tube may te ld h thc reftig@lor, dd the lesin will hardo in 3-5 &1s. At 20' C dr iesin wil hrden ovemight, but should more repid h*dming !e requned the tube nay be pur inro an oven ai eronnd 30. c. Thil wil hrde the rein in abont fou hom. The tube ued for hddding crnnot le the !s$l gelatio tule baause the water still premt in tne resin solutior will sofrei rhe gelatin. The glas iube oscdsholld havc ar intemal dianets alont r nm. lreu thd the chu& of the niaorotu. whar the raia slrinks, o it wi in d@ cou;e, the blo& sLould dt dely bto tte The block vilt get hdder day afte! day. The point at whictt it i jut right for dltiog wil dependon tu plcvailng coD&rios ofloom rdperatu's etc. sectioro should be doned o! ro w3ter conraining2% glyceiol ad 2% acetoft. I1r glycerolis ttree to prerfl 6r comple.rdryingsd ortrg oftie :ecnonLttr. Tle r.etonen to redr,e fie 'uf"cc rdioo of lLr slv.erolv?Ler. Re'in Wlnatkr r. The vjscosityof dy rdn in the liquid condition is invqsety propoltion l to its tedpdlrue. Therefo.ci{it i ihpondt ro iDnlbatea s!6ioen at a low remte..tee e largeindede i! the infitratior tine shouldbe qivo. The a.rual extdioD of libe i somefiingq hj.h shouldb- s dked our by rll ud edor, bibg sr
Uba Thii SedioBJottheEledtobMicrcyaj'.

rrJ

thfl the ienDioder could be .dded in dror fotu. Litde i5 lost in accuracy,and a gr*t deol ; savedio !ine. The tubc my le clened our verv e6ily wilh cottoa wool ed acetoneor othcr appropnaterein solvmt. 5 . The nsuring ofancos reshsmay be madeeoier by hearingthen, lut exc6siveheatiig shouldbe woided, c this my alter the characterofthe polynetized prodrct. It would be hadvisable to hear dy resin higler rhu abort 60" C. Bubbllng ofa rcsnrduring hfrltration or se$ingis duc to volatile elenertr which. at the giyen tchlerature, becomegscous, dd arrcmpt ro leavetlle rcsiq. The incldshg viscosiryofdt gelliug rain may prerent thc tubbles evq leavine. T|f !o ,rile clemdB nr. br prr)m! ir dr" ,.r whe r bougb,or rhe) nJy b; prcsot s a rcsult ofbconplete renoval ofsuch a nuid d absolurcen\Dol. Tlc prernce ofsuch reagos may alsobeconc nore obvids $ . rcsul! oltoo nucil mtu]l5t or accctdato.. Thi hay hatetr thc speedofpolynciizarion but it wili alb *uxlly itrcr6e the tcnlper:ture of the rcaction,so rhat fluids which vould becone in orloratcd ioto dre gelling r6in ar say4j' C, tuy becomegaseorsat Jj-6o' C. Thc remedis suggestedfo. pcnntdt bubl,liry of speihem is to k) make surc that no mtemediun is presmt, (r) tse les .ardlst or acceleoror,(r) condut the po\mcizarion tader sJighrprcsure. A presue of 4o {o m. of lrelcury vill bc found sumddt ro lreven nost fornx o{ bubbling. (,/) conducr thc scttnrgofthe resin at a lowe! te'npc.arue. Tlis will mcm a loager setting Win\ someresnr ra.es o{ noiture iD the gclatin capsulemay mo&fy or softcr thc oursideoflhe cured block. rhe c.lsules sho d rhe.eforebc usedquite dry. 8 . v hie tr i. r, F 6"r ,-J,. 'e!b ,tru 1o ym.z, ior b.comp tuotbte od ;nfusible, lew re €n*dy llec frcn rhe sseljng o. softening ellects of such ligdds s acerone,edl)l cetat€. etc. l{ suchfllids xre rsed to dsisr in curtirs or in llattoing setiom they shouldbe sed in reLrivcly snali percentages in water. In ey h€fttution hedih, vhether of resj4 or of w*, wh€n slch a mcdium is a minure ofditrerent corNtiluenrsir is los$ble rhf due my be a diFelerial iDtakeofthe rr.mte elendts. This leadsro r miarqc vithin the rissus whi& n dif,ermt fron ahatwhich is ousidc, ud f the difercrce is at all ukcd it nay lctd to vdy loor sectioDsof ilc sir.cin.a no nntter how good seciioruof thc oltsidc r6in mr} be. This is a ponrt I'lnch ntst alwars bc bome in mind, od it will be foud.in pnctice tlut initial dGltnrion of a specimo is bettcr accompljshcdby uing a ftlly otalyscd od accelerated rcsin,dm by infrh!.tins fi$t with resil on1y,thd with otalyscd resinnirtuq ud lrdy witi a catalysed dd [email protected]{ture. Thc tinc dd tenleraftre ar which *ch frlly oelysed and acelemted nixtures srould bc ued nay bejudsed only by expdioce, sd a sihrle pre liniur trii vitl tle materiel dd rhe resils ia hud ir advtable. As a reult of enpnicJ proccdnre should it be stablnh."d tlar rhe caralysed r o r . e l e r c d r 6 i n B d 5b c f o r ei r 0 " . p r e r r . r e dd r e" p e m e o .r h " - e r . J y r t o useks olallrt or ecelcrator. ud to naintair tic smc sermg renperaurc, or ,o k{D lhF Dacfl,ss or.}oe "sds ,he .r-e bl|o tow;. tha outrtrrioD

116

SediotrCt iititt Midoeo With tissu which u rough dd dificuL to loet ate a p$liminary bath of r6in wirhout catalystdd eceleratot ruy led to the productioE of very poor se.tioff, b@use with viscog Biis oo@ lhe 6in hs drered the tisre witho"t beiig ac.onpanied by se$ing.gors diFuion of crail'sed and aod@led lesii! into the tisEs .k@dy occupiedby p!re lsitr is so slov llat gelliry my preede

ro. lnadequatehixiry of r6in, catalystand acceleratorwill pioduce a block which vil have eas of eryiis ]aldn6s. T[defoie ell binlres should be stired mchdioly if !os.i6te,od if Dd #6 ecry gt* de by bdd. rr. Sriring of rho r6itr vith the s?e.ibo in ir shorts rhe time of iqGtr€rion. c@d. agit3tion orey khd shodd be cnployed whoed pssiblc (p. 2r). I\EFERENCIS DA6,J. M., r9J9,Ndr/r, r83, 3oo. Gu@r. A. M' dd Glauelt,k H, r9s8,J- Bio?h8. tud Biathefl. Cftal., 1, 2. MN6E, O., ud BncE-ArbsoN, A., St 'r. Sd..g.redl nidobiologlbd.t6idttuaksr, Cdl,rage, Udv€rsity P!s!, r9J6, 26r. NEwMrx, S. 8., 3oRKo, E., dd S*rfurcw. M., 1949,J. R?sd.t. NBS., {3, r82.

CEDrrx 2j SECTION ADI]ISION

AND ADIIESIVES

CLdt', C/eos4@ Mi.royape slids v/ Wlbtffir adh$ive i5 ued to rnLc *sue sectionsstick to glss sktes it is esential dnt the slide nus be grecc ftee to start with. OtuNije lhe secrionsare tude,ly restingon a drin fin of gi;-sq ;d when rhe slideis placedin xyloe t. renor th! s d o f - b - . r ' i o b r h e6 l r o f g r e ^ ei . r e n o \ e d" , ' h c s r m ' m e , r d r h e. e c n o n vill w6h of tfie sldc h dre cd,.r Muy lacton contibute to dre acidental sn*ring of oil or grese ovd r slidc, oJ oa, p:-..on 'iom .be rog*, i. one oCJre mon foDmob Dd Lhemos r.edJy o r e r o o r e d I. l r " q b . l . o ( c u r .! h - r . o . n ! o b "q h o b ! v f l . r e f u l t ) p u r a t o r o f slidesthrorglr vdou cleuing baths in order to mure their g.*.ei; condilion tlcn wips thcn dry wiii a hmdkddnefwlnch he hs hdd€d r;equerrh ud whch rs a fr rrout o per"prJliob ; .. te-ed rr- ovcr ir. The prodEtion of gr6+free slido is dificu1r, od eachworLer hs his oM steial -r-c rpprorcb. ue oirc"p sd Je.-s"nr. i oo Lbcwholero Le 3voidcduija; very s p e t u l . / e . ' : r m n n , " h " w r v a t r ' - J c * o r . r ( L - e r s s . . r t c r , . r t l D rJ r t r r l e i , rltr wix rb p r rl-c.Ldeererly ud wi.hou' bbbbuls do$ nor 6d rh,r l,T&e rlide is thd.fde greoc-free; it gmcrally meu rhat tr"c*;f thosere.gdts stiil r!mri!. dd bv vifre o rh ,..1,r"tr r6,ob o$criis rup,cj,yp*mt wa;r ro u sn@tlly ovd a tufi.e wllich is still sneued widr sreae. To olhnr dr bestlo$ible gr6+fiee slidespr;ceed d5foUows: r. Trkc a slidefron the lox asieceivedtron the supptie$ea n'l a drop or ts,o o w d d . r o m i . r r d . - w r c ! r . $ . - o o t H y s d ; " t u \ r r e k . , L h " t; d o ! " ! o rhi 5uPPtythe bener. :. 1"le,uch "1d", -bd ol. e b indu.ral m.!i) L.ed .pirn.. sro,ein dri5duid.I or '.s Lrre a sLid-ou'. drr. dr wilh r rie, b,oer,dd ir $oJd te qui,eJer od stiLigreae-frce. Use tlc sade fitrs pap; i..,l,out six slids udthd slait with a dev trlter paper.k is edvisableto 1ift rhe slid€ tron tu corr.ide. vith

9:.*r.lrLy4 &'g"r

lf, prdimir"n,r* -hoe. rJe .Jd. ro be g.e-/ bv dF wrer going sro b,ob, upoDI fe rbee o owrg c \od, are re.ommded. r. Placethe slides(a gro5sor t1'o) iDro Polrsium diclionare, ro% aqueous 90 m1. coocdtrated subhuric &id ro bt. Add lle n.lphuic Add \en slow,1ro thedmon remxue. Kecr tlE dj6 i! rhi .o Lrion fo' " ridmr of,a hou.. U N 'hrn se b w.",, t,",,f* t"

IIE

Sedioi Cuuiry in Mictosopf

d;tilled wat$, md Gon therc to indstriai berhylared spirits. Storein rhis fluid aad dm o hdicated above. z. Ifhot water od soapor detergots ofany kind arc usedwashthe s1id6in runnirg wter ovem[ht. Tha t.osfer to r % hy&ocHor;c acidin water or to r % nirric acid jn vatd. Mrke surerhat the slids re scprated during this nethod or rhe detergot will linger betwc@ the sLd6, ed will everoaly 1ad !o se.tiod wchng olithe s1ide.Storedre slidesh e5ft alcohol,ud dry a above.The acid bat! n necessry to neutralizeoy races o{ soapor detergemwhich nay s!i11 3. Sodiurn hexmetaphospiratemd so&um metasilicatcstock solution: Sodim lcxanctaphosphate 40 gm. Sodiuh nct4ili€re (Na,Sior.jH!O) 360 gn. r galon Leaveovemight. Filt€.. To usetlle one lIt ofsrock solution ed ninery-nineof water. Boii didesin fiis solulion for 2o minutes.Wsh s'e[ io w!er, andtresGr to r% hydroclioric acid.Ri$e in qater ud tm$fer to eJol"alcohol fo. stomge. Sert;ofAdhesiotto Sl les eitho"t Adhesires No @!tu hov 6o.ough the inihrarion of a tissuevirh noltm w* rhe tissueof a sectionwill alwys nake sonc d;ed conrrct vith $e nncroscopeslide rho dre slideis dticd. Sone wokea coosiderth:t this conractrlone n suficiot to nake thc sectionstick to !11eslide,and drat ro adhcsiveis necosary,providing the nifioscope s&lc is gra*tree. Thcre na1. be occaions too whm a prolein adhesivesuch as albumcn m.y be detrnncutd !o the staining!rca$6. Should i! be cotuiderednecesry to avoid adheivcs r\c following stepsnay bc r. Take cleu, greoe-&eenlcloscope slidesod float on ro tlen sectionsor ribbons, uhg distilled water. 2. Fiatten tle rdiod. 3. PartJydrain dre dide. 4. Atuge drc secrions. 5. Dein the slidc coopleteiy. 6. Dry ovcrnight a! a t@perature soue ro' C below the nel g point of the wax. The method ornot bc gururecd alwa)'sto yield succ6s.It n.y be ihrrord by substitlting roTo ldnalin in w.rer for distiled warer when natroing lhe s€.tiotu. Drain the slide, rrd€e tlE seciios, ad dry ovenight a bcfore The fomalin harden thc protehs of rhe s*tiom duiog d,-ying od dis nnks drrn le$ iiablc ro lhe sfrFub i!C"oe of w"r(- during'ub"carhr ,Lining oreiidotu. c .. or againfloat i1rewax sectiorxor ribbom on to a clsn, greoc-frec slidc wirhout ,lbmm. Flatt6, drain d1e slide. ud anoge iie secrio$. Dry overnighr in e $hosphde offornDlin. A bell-jrr ovcr a rack ofslide besidewHch is a small perri dnh of comefcial fomaldehyde will ?!ove quilc $ital1e. Arother nethod n to t.le a gat sectioD,placc i! upon a cleo slide and pres it

SatiaaAdhaionaadA.dh\i"e! ftbly

try

dom widl the thmb. A tbin liece of paler nay be pleed ovs tlc section

rhe mosr comonly

lEedfotuu1a is thd olyq._,.

Fr$h hen egg a$md A d dc l y . e r o l

Jo nr]. Belt .nd srir jo rl.

lilrtr though 3 Brchm creen 9oafltu paper,in r danp atnosphere,asshom in the dilgmn. Add a fev crl6tals ofthynol.

olbunen flil.olion Fro 34. Fntlrtion of alblmenin a moat atdo$hdc ro 6e Mayis aftunn a very smil uout sholld !e placed on ! ctce ilidc, crefujjy 'lbbed ov$ |treslidcwiLi - tuec-, ud rr e^c6, wiD-d06. t dc solrt of at6utu pur oD!b" side shoutdbe Lhc-,rcofipi, b%d.ro; much,JbMm wnl Ieadrc.eft"1. c;nbg on ,fistid-. ., ; ,; i,e; b,ctg."..d. c;;d nou! of distiled lvater is placedon thc slide ed a ribton or sectionn placedon the vaier. A 6oc seblebrush b sefti for hddling the ribboa or fiE loreps may bc used. wilL o,atra wr dreseciob re 8dfttredbv wlfug re J de on r 1or rlare or on $e,:i{,ce oflor w-rer ;n J distr.rhe.Lidei d,.in.d ,he.e.doo. rc noged * leqGad;dd rhe didc ir dried ovemisht at 4G4j. c n o ove, or a! roon roperaAIbun.n Adh* irn Vdi dtiou It vill lc fodd tiut if rcdioas uc coming off the slideo inprovehenr in adksion will be eficcredby snwiog the slide wfth nbuho d aheaayindiared, d1d ihm floating the scctiou d rilbons on ro rle slidc usins ro% formalin in"ead of wter.

Satiot Csaifg il M;d@n tl"tm. dr.i!._ad d, Lhe)ectiou r gvo abore. Ihn tectaiq( mks borh ltrr alburo ud $e s I on proeh n'orc r"nmr ro thc "ofrding efect"ofile ,{ious r@gentsued otr the scctiotuafremard.. A1ot}tr nerboJ i. !o mou' rlc .*ior '.irg JbJmetrdd dlriled s"k( bu, to dt n?n ; s "'To.pl.e,eof iorn"ln ,,Dou. fhis D"y bc dobeby ptdcingrie slid6 in a rack uder a beljd vi& . smll olcn dish of4o% fcmal&hyde. Dry Arctlcr nctlod n to he* tle slide virh dlhLuo sheded or it ovc a buer buner urtil the albunu smokes.Allov the slideto cool oq flood with distiuedwater dd !rcced s alreadyindicated.Tle hish hearconvedslhe elb@ into d aloost hsolulle n*cria1 which uitd *e1l vfth dr protein of the setrion ard is most A wiation of tle slidc snoring reduique is io &lure Mapr's albmo (z mls. to e8 nn. of w.ter). The fuid nray be usedh. dnh ed the secriod flo.t€d on to it, or n nay be uscdin pla.e ofdistilied w.ter or a slide.Drain, dd dry lhe slidesovenight. Bokeis Alheli,e AlLak'.n 4 p o , ' e d o l b r B r t d n d J o r d e ( r o - lM ) , 1 t r e J o r o - n. r o t u t d t o d a yx ; r \ 3 i h e n j r w r 6 ^ D I l o R J r d a r s . r i o ! . d l e e ( 8 8 1. T o d . y f i c g \ e r o l playslittle or uo prt od thc albunen rclution may be nade up $ follows : o.e%rodiun clloride,aq@ous roo ml. Sodim?-hydroxybeuoate o 2 gh. Dissolvcdd add lresh egg elbrfren rm ml. stir fii! mir.rue vcll, dd rlm certrifuge it rutil tie supe@rdt fluid ; quite clee. Decdt dr sulematht, sdich is dre adhenve,ud ue * Mayer's albuner

Caot;o,tal'i,n Pc.i.rs the nost iidely usednetiod is tlat ofHaupt. celatiD, b6r qualiry r gm. Distillcd water roo hl. Dt ohe ", Jo' C J1d .Jd '. hrl. gl)c5ol rd z gm. ot ph@orcD5uls. 'I he adheiveI oulJ b' soored orr a niooscopeJidr a indiarcd .or all,Mo. Rlln 296 forridiD, .queou, on to tic slidq flatto od dry rhe s@rios in rhe uu.l way. Shodd rhe sectiod fall ro stick drying !l1d in d atnosphere of fornalio, wi inprcve lie adhesion. Cdtbalry&atu Athdiw ccbohydr*c adltsives shouldbc sed oily whol erbob drarec glyol sraining methodsde lot being ued. They have *cettiodal adhsive pover, ud whd rhorouglly dried ue strcngly

SectiobAdturio" dd Adtesi,es Fotuula of McDoweI ed V6sos

s^':+ l.otd eter

3sd" !o dJ,

Mia to a pare ud add to

Thm add Hydroclloric rcid, conc. o.5 nl. Soil lor 6ve minur$. (@trd "dd o. gm. tlynor. orbolydr"e merhodui ias;yto_peo (Steedne. ,oj8). ._A!o&tr r. Boil otre lihe of lvat r and .dd to ir while sdl boilins r.j gm. of Nipa e$d

,,,("'r. r,bor",orjs rtd, rrerolen rtudin8 L'"*. p."q,p;lA Xl:: urDorge,nnet.

.,. Chr.:oo nl. of rtn $ urjonro,oom kDp- ruread add I sD,.u!,o_Dsi!. om r "u,otuio^ ! "i|lbddn.ot"e__dL[a;ou i rc\ry bio flk odr 8ooDt. $hia. "t_outob- bo b g duiig lbc addjo.o;. J . L o o t .d d D e e g ( n r i " r e , d yi o r u s eD . o .of 6}er. +. -I,n.-;ocDi ,o.jbb bero,ejbg. ad M a few.[oe: oo ro a ctan -,:r.le ** Ho"r rbe .etuon or jbbotu oD ,o dJf tiqrJ. I Lnen, drair --jj:::?f" .wa oorou.hry. andge_t-te.e"iou. od dn tle .rid".Do oor ltu\e ev6. of rhee\ie pecrDbrutr@ o -nr.Ljdeor., orJJ,pprr ir Lte ,bn ofvery 6ff doB ude;de nd6copc., I bc 5rrdcri.outd be dned oveigbr. thL nelod b- be@ roud Lo Irue dtuc n,ten.t adnereqhm :ll o$er blloo( h"ve fried.

,

sran,trry ,4th4kt1 of sktio| It tu) hJpptr rhd a p-ebnitm r1J of,tde.. rpoa shi h de ho@ad dricd wq r-.Eotu,sioL. ur"r ure,(dou r e.wasnrgofl txed with Lhetoj. ofa higbpcrcor_

::;"H:frn* r. :. 3. a. 1

*diniDs srid$n isaqadvanbg" to pd!,a*

tri",igr, lhio

Disolrc o*'the wu in xylene. Absoluteethuot. Abrclute ethool ud ethyl et!* equ.1pu*. r % .elLoidiain.abshre ettool ani eGer e.tll p_*. RoovctieslidefondrccetoidiDsotudon;dlaveinthea;foraGwseondr. E e , o r t c s l d e o n . . o ( e j - J o oJ c o l o t .f r . * ;; s l d ch . ) b cr T R d i ! r b el o r r J u " 1 f o m r J r , ," o . E J ' ; . . r " i d _

lte.ellojdin p,ov. obvioL in the 5rL yuro. mouteo,Jdc. bdne de ,.Shoud son rtuougrrlbiorLre t-obot rd e&_ _rre"r,\pvtuve b(o b.ouSh. uD o rb;tuR .r-obo. tbe u rc qijt o;,ol,e aMy @.elloil]!. rhm pt,ce J; [de iq.bsotrle .@eor, heD rytoe, td mou! b bJ q or ool) ry_se.

Sediafl Cs$rlir

sttio Alleskt -

Mi olralf

&,nnary

r. Us clru srs+fee slid6. 2. select d adhcsivewhich suitsthe tissue,rie inflnation ms ud netho4 od the stainiogoethods to bc ucd htr. 3. Never ue alkalie io ay staining method ifir cd be avoidcd. If thcir ue ; esotial coat the sectionsl c witL nitroccllulo* befora\ud. 4. levroe of uing evo nild .lk lia such s lithiun @bomtc for rooving picdc acid,borL on ti$6 belore hfiltration md on scaioro. J. A low melting poht vs w ned for i* seaionsnorc erefd atterltlor to adleioo thu will .ectioro fron a high meLirg point wu. 6. Do not se caibohydrateadheivo if cubohydrate stainiogteciniqu€s de to be 7. Al*ays usethe nininm

ofoy adhesive. REIERENCES

Bre& J. R., andjooAN, B. M., 1953.Qtdrt.J. hiq. &i.,91, 23j. Have\ L. W., r93o,Staiflteehr., 5,97. McDo\',d, A- M' md VAsos, L. A., r94o,J. Arh. Path.,29. 432. M^\ER,P.,788t, Mitt. zaol.nat. Neayl, 1, 521. S]I@MAN,H. F., 19j7, Nd,re, rZ9, r34i.

Csa?rER2,1 SOFTENING MATXRIAL IN fEE BLOCK; HOT WORKi CHOICE OF MEDruM; MICHANICAI, DI\4C!S: VACUUM INFILTRATIONI PRISSURX SLOCKING sofenkg Makial in theBh.k The hirotomist freqrcndy Inds nalerial hud to @r. The hardnes hal be felr rhrolghout &e microrome s 6e blo.l pss6 the hifc edge.Tte sp*iben may n*: . gmtins mie, dd it nay show further eviddce ofh.;di.ss in iovde.ins o; 5llitting s ii ; bcins crt. ro ru-ltr ho " *oro gliy DrJ i,rcd. dr proreia or dy V^ imcDin ba ,ril Rr tlu c,pnny ro iTbib" $rre-. sd .hougjrdas i\ cobjoe,rb lcduftd (om/ paled wi& rheir uturn condirion ll{Eiem reo.is to lernit a sodring ofthe btock 6 a meatu ofsoflening rlc slecino. For pauFn blocks: r. Iae-olltlc

sfecin@ on lhe hicrotome. That ij cur scctios nltil lhe sf{ind r d $ , t p m o i r n t e , e l e do F ^ h i c hi 5 r e q r n e d . / l.re" \ed:.i! z:. Plae rbebtockb ffiB Dd .mw for.omf hou,. - dcmi;br. 3. Dry the $rf.@ ofrle blocl od proceedto cut secti.ns.od; a crlain nunbs my be cut bceuse the water or otho sohrtioowil qot have poetr.red norc drm a certair dcpth. 4. Repat steps2 dd 3 agai!. v:rior..olu onj . !-!e !trn "dvr.ed a bu8 supe,iorto w*er for $r prpo,e. A juo \o .t n o, p '60 e w_r* L good for pramn \d btocL. Bal_ ( 9ar) -e.omasG |ne u c or w.tr lo, o)% J.olol 14.glyce,otro. Lebd@ tr94atbe. s')(rol 90. &-]nr ro. l!. ,bo\c . ofreninCnud. rry be rocd.rfeJymti par-trn \6, bJr *rey 'hoJd Dor be 6ed qrb e .,tr wd or wli ootyster w*. q,6 ,he lattet two pure water orJy shodd bc !sed, ud it rhould be,pp["a o" " p,a ot"onoo wool. As wiri a specino ir a Llock ofpuatu wu the blicL slnuld ie ot on the minoronc ubl Ge .pcciha is viribledd *po.ed. A \brl pJd o. colrob woo' \oJed in w"rc. ..olld bc pl,.ed on dr f,( oCrL;b,ock-J te,tioreboL r*o bor". lbe drrle 6fw*e, " t} ero qq ud polloro wd L mr h f$tr, $d ir L wi,h pmffi,. Thr sur6.e of $c blork .hould be d.icd belore.unia -urrilg h_t6 rtae. clycerol will !rcver secti@ssricLiogto a slidcifsuflicio! jr pr6d; dd rh;ugh it is excelert Ib! soitctringiissuescareshould!e tako to seea\ar it doesnot acmu_ RIIIRINCES Br@,J. R., r94r,J. R. rnr. Sd.,6r,7J. LNDnuM,A, C.r 1944,Srdi//e.l'-,19,t43.

S..ti6 Cc$ing in Mietusopf IIot Woth Thc hicrotohist who volks in a hoi roon, wtorhd in thc tropie c clscwhae 6!& hinself in a vcry di&cult losition. Id this respectit sLoutdbe ciw tlat he h* neverbee giver f.n t!€tmsr Ifhe hs to work in Hong Kong Mrlaya or Arizona he is intuiably fo.ced into niGotomy which ii uo6d ir prirciple od difta:lt la phctice, be.a$e to coute. the @biflt temlcratse ]le Ia to ue high nehilg point w*$, pani.nldly palaFa wde3, which hrdo his mteria| nake it dinidJ o cur, dd produ@ excessivedistofiion in it who it n vieved e&r rle nicroscope. It is xs€ssentidto tie microtoni.st to havea cool roon with a connollabletenperal8e hriarion foh alout J-2o' C, 6 ;t ; to the tissueculture worka or to the oioal breederto have steadvad suit,ble tenD@tu$ in rhen vork. vhd thir requnenem is net dr stadrd of histologicalp-re!{atim will shov d imediatc Whoe the conditio* *e sch that secion oniry ho to !e doneat room tempentures doing the &y ofz5-38" C the folowing hetLods nay be adopted; r. Cut the secriw in the esly nolnilg when ihe labomory is n its .oolst. 'Pespex' 'Lucite' 2. rnclose thc miaotooe h a oe of or with dm holes i! i! od blocLsof sokl co" imi&. 3. Try tlopical esterw* M.P. Jf C which wa designedto enablescctios sd ribbou ro te producedbdwed rz ed 3J" C (63 dd eJ' I). 4. Try lhe wa-fonnJdehyde resh nelho4 vLich tub16 sectioasto be cut at any tenperatue which the operatoro stod, The hoter the roon the hrder beconestle blo&. It is not a ribboning nethod. J. Ior temtohry s@6s placethe block, the tiootone od the niqotome tdfe h a refigebtor |or 30 ninuts or so. Al$ the fatreiing fluid ud the cla nicroscope slides.xver in a roon at z8-32' C this should give rJ mioutcs or so ofgood ieaioritg. CEorcEoFMiDluM Poly€sterwd 9o/Io widl a clting poiirt of 38" C; tiii @ry ribboning, 6y dattc!itg, agd good adhdon of sectionsshoold!e ued f.t all soft specin6. It is Mtial, howewr, that section shouldbe cut in a cool room with a tenpeBturc betwed r72f C. The block should be lermitted to hdden ovemighl May dilficult objecs becone qrntee*v to cut in tlis oedi@. Esterw* rq6o n reco@@dod for all gmaal worL The 1960fomu|tion giv6 a product wlich showsquicker Ettiog, qui.ker hddding dd letter dattding of pctiou thu tlu rg+z formulatioa. ?raf,n wu las advoaga lor .outine vork in bulk. Its strongqt $seB-leliability be serioudy challoged by etc wx of .ontitution and easeotmilularion-nay celloidir is wqulled lor vay rough objcct s@h s ar&opo& ed woody o.t!!ia!, hut it sLodd be ued ody wlm was havc provcd to bc uuitrble. Th$ is b@sc n reqnires!o much lorger to penetrnc tissucs.

Metlaaiul Deuies

nt

Freding nethods sLoda bc ued wLen sped js osc$ary; for fas, nerve staining, somehomones ,nd sotr eu}tes. Getatinhold tisua together left{ rid a pure frding nethod, ad it nay lc urd fd 'at., roae hoh,or.. go gi -bDa,ru, sd ,ome roug\ n"rr; -ls. Urs-fom.ldchyde .esir ii Ljte celloidin but ir is rater mluble. Hocc frts atc presered weil ud in sone cxe wld $luue nucins ate prcs*ed which arc disolved in tie ws techiques. Ir do6 nor leictrare tisslessell, dd should!c ucd naiily for shal pices of tisues, about 5 m. cuhed. water soluble ves of the r,5oo polyethylenc g1ycoltype are atrractivebut so e.enain lvnh .egad to dr f,nJ prodrcrion ot srainedsedionsrhar iiel should !e tsed only on hnnportaDt mleiial. Foi secriotulvinn vithid ile rcei,oo A roge n-butyl metLadylate Nirh ro% addednedryl nedEc4'htc is in common nselor most ti$@s. Epoaf resi$ are good but f@etration is slover bcausc of their grsrer viscositl'. They shorv cxcellcnt .dhenve proreni* dd hay vcll p.ove sryerior fo! difflcult objccts seh 6 h.ir, M.cI NrcAt D!.!rc!j Where large nunles of tissuesre to be sectionedo a regular routhe, such as in large hospnals,it is a grcat advotage to ue onc of the inshunents designedto ele ri$ues tbJough dE alprolriate rage* and into wu wi!ho!! mlch attenrion once the hsbument hasbeenst*cd. For snal req reneDtsmcclunicalaidsof this khd (:mot be comidered,but wlere rhe vohne ofwork vauurs it the instillarion of suche itu$uneir inaediately a.celerates prodEtio!. The timiry ofthe vuiou chmges6on fluid to fluid rnay be varied. ed jn nost nodeL stining or shalnrg dcvice fc enployed to hastenthe intdchdge ofduids. All this is donewitlout ay tine being lost, loue tlc irotruhent wil work through lhe dght, rd tisues * dehydratedod iufiltratcd by the follo{ing hofting. The sme tutrrmots ue alsousedfor rcurine st.ining of ris'uc Mictaton'eKbiIe Shattehiry Mothibes A nuber of thse is availalle, dd a11aresood in dtn i,av, blt somerequte as much time s?emon then during laife shugoing s would be slel in tie usualway weic the operator to honc tle lnivs ly hdd. Sone ofthe highd p;ced nacliD6 produceexcdem restlts with a ninhor ofrttotion, od areweli vorth puch6i'g should the oorey be evailabie. VAcuuM INTnrniroN !y lar tle larger anoot ofn!filtratior ofri$us is donc .t the prevailing atnosphc.ic !rc$u!c. This is becarseit is cheaperud more convoienr. The ini]lr.tioa ofvery luge nu,ben ofpiece oltissue h v&uun ove$ r'ould nece$iratem expaditure ofmoney out or!.oponion to drc resuhsa.hiev€d. Nevenhelessva@un inilnatior hrs the admtage th* it rcducesthe tine of pdctration ofthe inEltratiod ne&ud, dd s ! resuftjt keepstissue softertho those

126

Se.tioi Ctttin!;h Midoyolf

treted in tLe uual way. SucLtisses hay look belter in sectionlotn tlrn thc soc piecesottisse put through at atncpheric pressure,tlough the di(erocc, exceptia @rtain special6d, is gderally so slight that it nay 6@pe obseryation.A fa-ther advetage ofredoad pre3sureG rhat disolvcd air in the tisss md aisoin thc molto wu; renoved, od that ah bubb16vlich my iavc becomelodsed ddDg alis poc*es may be inducedto lave. AI this leadsto better poetration of the matcial. vaoun work of this kind shorndbe done sendy. xxGsively high vrcum will L{ dd distort tis@s, dd if the vdm i suddeiy rcicsd it mry leavedr bubbla. For gmer.l histology it day be slid that v,.drd innltetion is not ftcss.ry, lut that for ertain qp* oftissue where air bubblesue dilficult to prevflt and diilodgc such* lung, it is uei:l od nay evd be gsential. For iruect in6ltration it ii nost

Prnssur! BrocdNG Sone heteriah suchs ha;, conon f,bres,od fabncsgarmlly de di6c!1t to penetrate witl bf,ltratio medie. This iJ due panly to tie detuity ofsuch @tqials and pardy to the high visosity ofthe tesim which my be mployed d i!f,ltratio! Eedia. Witl other nareriah slch a powden, looe edds, dd nrjlemls the resinnay show Jackofa&oion x well * poor poeration. Where tle cor&tior of de specnno will stad it eoun iidtration helps 5 . start ud it nay be srpplemted by oaLing the block ud* presue. This o be done a stitable contaihq ofnetrl $ the pr6ue chhlei rhe specihenir r6iD in a g16stule is pla@dwithh the pressurcchobe, and a presue up to roo lb. to tlc squre inch shodd !e naintaired dunry dre [quid, gel, aad hudened phce of the resin. Thls will prcduce a block of closeco$titution" dd a$ilts ia lenctatiog [b!o!6 or ]minat d hat nd. It is of particuld .dvutlge to ue presure wherc tlin utioro atc requircd of roo r,ooo A ard vhcre comllcte pdetratioD dd good adhsior are ncesary. A simp)e screw-prcsuc devicc nay also be ucd which will give gretly inceced p.e$uesbf sone rhoMds of pou& pcr squrc inch. This mbles vncou roins to be forced into loor matenalsucho &ys, od soft mincralssch o gnphie.

Clalrlr

2j

MICROTOMXS AND M]CROTOMX KNTITS Microtobs are isifth&ts lor cutting thin slic* of naterial whi.h are to be lats exanined mdu a miooscope.Their coDstructionis bascdlpon six lrinciprl Glturs: r. A hetal rod with a 6!c thr6d. 2. A tbJeadedhole tluoqh which the rcd nay be trfred thu advmcing thc i. A lnife holder in vhich is 6Dly clercd dD bidorone lniG. a. A dcvicefo! miis the rod a medured amout. 5. A devicefor ooving the rod or u *tcnsion ofthe rod p*t the oning edgeof the nicrotome kaife.

Thesc rcquircmmts are incorlorated in r vadcty of diifdenr &sign so that rhe following type of micotone ue available.

@

u TbeF re simple tubdr microtohs which rc hcld in one hod wiile sectios de cut tsiig r nicrotone knife in the other l"od. They are ueful for core work whdc great acdra.y or ddue$ of sccriotr is rmcosry. They re frequmdy emllold by l,oteisrs for cutting rcots ud stem. The specino, such* a sten of sulnowe!, is plaed in the weil of tle nicrotone. Tln rve11 is rlen flled vidt bolren paufin w*. \v1h the vr is hard it n trimed aw:y 6on tle front of tie sten od sectioroare ot by s1idiry iie hile acro$ dre specind. Admcenent of the s!6imd afitr eacl cut ir oltained by tming tle milcd wheel at the nicrotooe bse. Plugsofc ot ru)' bc usedc a sqpon nr p)aceof paratln w*.

Sation Cxtting in [6.tos.itI

Trj5 rj u:d_D . + rye 1vly d, rhe Ldd miclotone, bur it hd the admrase rtat i! tuy be bofted ro the bdcL. Borh hdds uc ttrerefore fre to nanipul":te tle nirrctorc kdfe, r@ove rhe s6tios, etc. The inslrmenr may atsobe ;ed to cut .cloidin block, in de safu way s pldr Eareriat_TLe ldafin \!d should be rimd away 6on de blo"L trr su.! a d@cr !h,r |Ie micotone krife neer, Lhe elloidin blocl r-houthving to ndr ova or dr bough a M. of pan"6! wq tsr(p.78).

The rockiig hicotone ddvs irs nmc fron the rockiog boriotr ofthe uppcr a4 s scctid 2r€ being cur. Ir ir a f,fir-clss qualiry i$troner, d my be obrained in a wiety of foms; sooe deiged for sectio betweo r-ro 7r; mrho to cut medi@ tlin sectios berweero.r J r; dd oothei ro cur ultra thid s@tios bewd rcr,ooo A. Orh6 re noditred for ldge scctim, and sone have aiapratios for

Midotan8

and Mbotoh.

Ktir$

Thi! in$lumdr is meu{:dEed jn hdy coutri6. rt b lsuaily a msive, heaq imtmt dsigrd for acrrate work, od for dcalhe torh with snal dd laree pie6 of dsue. lr is aLo oodrricd fo, ,J a-uj! \, ft;o,L be wc.u io .-o {. I r,-. fae advancmdr ofthe bloct to the kdfe is achicvcdh sone desi6 bv necianimt o e s b a e do Da d - e d e d , o . , b J ri r o $ t r , , i s o r o d u r ebd, . h ; . J ; * p * , * .r

s. Sledseat Slidils Mkhtos.

r3o

sttiofl cttting if Ml1otuty

Thn is dorler istrbdt of hcara consauction designedfor cuning naterial inf,ltrated vi& almost oy ne&rn. Perhapsits nain advdtage lies in its capacity to dol witl larger objectsthm thosecut on a rotary nicrotomc. It { alsoflequendy enployed lor cuttnrg scctionsofcclloidinized nrternl. Serialscctionsir wx may !e r u r w i l h i i , b , I n o , . . . i l yr $ . \ r h . , o c t n g o , i " o r . d r i . r , d r e .

Though nost hi.rotobs hay be nodificd to takefreezlnganachnents,e Seezing nicrotone b constucted d a rule loi {oding lut tlrt q"e ofwork. rhe block n stationary;rheljiG moles pastit dd cutsthc scction.A!!rclriate tuter ard Sezing device areitcorponted to keepboth the block ard the kdfe !t the core.t tempera.'

A most riorolgh dd detailedaccout ofnicroioines is givoa by Richrds, O. W. in'The ell'ectiveue ud propcr orc ofthe mic.otome. Americu Ortical Co., lnsrrumebr DrvNorLlJdllalors, NcN York. I4rcRoroMnKMvEs Thc sharpnesofany microtone hlfe is depodot princilaly ulon its contositiot, ud rcordarily upon the nethods ued to produccdre cutthg edge.Tlus a grdnlar mrte.id wh * .te.l c-,ld ne"er producethe fic smooth itpc of edgeloed in a brcken-gls-kniG, ad egulI so a 6ne qoality steclknifc fnished on a coarsehone wotld lack the snoothes ofa poorer stcclkaifc tuished on a firc hooe.

No mner how Ingh the qurliq of stceldrc cdgeof a nncrctont kdfe is dcpendot for its tual condition oo tlc ski1lof tle sharpoer. And th*efde, $ 90% of qe . succese, ud po% of r.hcfanures*perioced in s.rior cutting .re duc to the .ondii- tion ofthe nncrotornekrri1i edge,it shonldbe cleuly undentood from rhe begiming

Midota,i$ at'dtrtidar,he Ktir6 that tinr ard cde slent oD tlre con&rioninq of tlc nicrotomc knite n wel dd i{variably well remrded. S ' . d k n i v e d e o r r o u rk r d , c . r d i g . o c , o * r . d o n : Microlome

k ni ves

llla

wedge

pr.no .oncove

hollow rozo. blode grolnd ho de. Frc. 4r, Miclolone Lnivcajn ao$ section

For slupoing, the wedge, ud lhe plucconclve rype neerl a neral back fitted to givc d:e corect bevel ogle !o rhc front ofthe knifc.

Eo 42.

'wedse tyle ofnicroton tnjle vfi brct

.1'In a sharp knifc de bevelsconthu fom*ds edt they neer ar a clean point: rvher tlle tdfc n blut tley ate comectedby a orve.

l

Rc. 43- !nl,*ed

r

I t

cros sectionvjew ofu shr! and a blet tnife edge

Sucha blut kdfe my be comideredto haveno edgeat all, and in practie quite a l o r o f h r r . ' b . . r o b . r m o v c o b ) r o n i n gb l f o r er h e" d g ci . r c . . h c d .

r32

Sectifl Crfrlng in Midosdpf

edge

hone Frc,44. lnlargcdaos sctio' aid of. blmt tnife edsewhd on a Lonc Slurueir a sLeeJ LaiGm.y besemby e1cin sooee*. ud b) riryr low-pow* binooltr minoscopr in ollcs. Ir is resDtcd s J shiniry G.[r Lixeor b "hH'g

dorsor pakrs l&

fic *y,"&e 6fFt"ir;-h-

tt; !df. ; b;t.d*iJ , Lishr

shiniDgon it, &d palticuhly whd the lnife is heid abovea d{k b&kgroed.

ll

t

^/,

do.k b@kgrbund FtG4t. Mcltod ofvicving t!if..dge tor bbnt dd! -

The [liG shodd be tilted ttrough o uc of about .ts' mtil tle right position is

Slotnes le& to dificr:lt sectioningdd to dificult lilboning. Iteticts borL rhc scction @ge ad tlE ribb@ @ge, dd it prodkes danased sectiom. Thc diagr@ below shom . blut lnife relativeto the llock whicl is shom above it. The gaugcis setat 9 p, but the blutne$ ofthe lnG ls *cL tlut the ro part of the sectionconposl of ftee mimd - Nos. 7, 8 dd e - w be sevgdlqg!!p!$!€d. Dd wil 'e,eJ dd.sed d$u6 udcr lhe dide(orc,j tlle or de srvo drgree ot blrtrd hJd pemi, sectioblo bc cut n a gaugerirckndc of rodd 8-'o a or abovq lut with the gaugesetat r a, the ovcrloging block would be ody 3 4 clear

Mimilbes

dsd Minotale

Knire'

of a\e blunt lnife edgc- the oth$ 3 4 of r\e sectior would be conpr*ed sectioningvodd be inpossibL.

IJ3 - and

i : 1.,2:

,.-.

q taiac cdge.elativ. to . sdion ofgr

Sectiorxcut with sucha kdG wodd duve .osiddlbty dd dbbooiis rould be cN4tsl49 gr.+esdo

L do r rrk"d rr r-hb.e.rioru.olhir wi r brsr knife

rle rhir ebd ot lhc lotal *. doo rag^ ir .dLc.d. fl L {idr e "bip krife de ,.(llon rog- of " givm u* o"y be 4-.o p, * r coomrerpcrurue o. rr" C. Lut uth a blur kuf" thc rd.Cc nigbtrelJ be s :oa. A ldfe nry hm blot 9atch6 ud slur! patciresalong its edgc ud this tea& to ribbon lro!|ld 6 shoM on p. 60. -qve rcrr e tui,b to a koileru) i' id /dlrp bn wil rLo le-,eir Fthr nube. .A d ra-eeu doj(ood upou it. lbse wijl tea,e,;r"rch n ",ts alorg :.ecrior or dbbob which will le ken d vhite ndkr h dte wa. r -od,rc".roi irthi.jig8-d .dge is Lhe -oop or I ock*. t, i\ duc to ?r6sue \uc! ". dutlon e redleor ti- orccps.,grir- rLeedgeofdre hJr. Thispr.he,1,d,U p:n oftho.edge out ofline wiri the rest ofthe edqe,$ shoM below_

!!o

a7,

Scoop or pockd 61db!

il tnile e.lee.

If the pocLet edgcis out of the by 3 p who a s*tion is @r ar ro /r rlEe is liirlc Doticeal,led@age to fie sectiontpart fion a line l1$irg alorg the ribboa lut if tbe cuttiig ihicknss i5 red@edto 3 p the pocket rvi11ot wery secrioninto iwo pa$, prcducinga:pLitr6oD oi riLoon. A big !1ock oeds a thicl, snong ldfc od a hary mieotone. WLo a block is pdsiritrFt;iise ;fa midotone hG there ue stros fores at vork leediis to a

r34

Sectia Ctttingin M;iotupl

tendocy fo! $e krife to be forcd out ol lhe blocl. ,o prod:.irs o bcoopLere sF.tidirlli;;liob s rhicl<doush rd lhe knle isrtdnlhthir. b-d, b*L -;-d. the block. Th; back ud forwerd movemdr hay be repeted nmy tins before the conllete sectionis cut, ud the sectionthereforeh*-a scri* of tiick rd thin Knifa or evd isnhflt vibrarion ofthi kind occu$ when roo gra! a 3thin is beiDgihpoFd ulon the kDift edge.cencralty this excesivesffain n due to too larse a blocl rehlive b dF lt.rng+ o be klf.. t4 Lhe prorohic"oarupb{6s. a'cr tlc xaior is dur of- pie.c ot Lvo in potyerr w,r. lb. bto.L f"; ;;ded 2J x 20 mm. The se.rion w6 c$ ar 6 I. The miclotome knG ws very t|in. wlen ine thicknds ofrhc sectior w* rcducedro 3 p a whole secrionwo oo longer produedj only r showd ofbds. This vilnrion ofth kiife cdgeis ofto rcfenJto blade.daptor or ho1&r hay be for soall secrios it /eood rhough tle sfetyle! ldhowsprotuu:rced vibetid on big oncs,dd ftercfore its uc is linited to rheg4ing -?ntitrid @y .lso !e folnd on . brdd-new lnG of strong deign which i$ . beo sharpenedto a featheredge.This is a condition where the levek de .ortinwd beyord the poinr at which tiey should neet, c a thin srrip ofsteel,:s sho*n jD eddged detaii below.

'::l:" *s'

I ) I

beYel

F,c.49.

Cros sion

of}nife

edgc $oving

bevcL contnrued i!!o a ferthd edse

Srch a conditior my be reboved by sof! od gdde honins (p. r38). A bl(ed edde; me which od$ whd ""e sid; of rhe kni:rch$ !@ ,i"ir*"j -*" ,rthe other.Thij lads to u edgewhich is slighdy lmed over rowrds ihc tedr shdpded I

bu-.ed ed9e

l * - -

l'.-"

Frc Jo cro$ sion oftnifc cdgeslowingile effedofucvE q9!,ue qfq'C !9.if1g_ It my be felt by psing the nnge6 6om rhe btck to the edgeofthe Lni6. A3 the 6ng6 leavedte edgea slight dr.g uil be experiencedduc to lhe €gged, hooked €dge.'Itis feeliis wi not be foud on lesringrhe orherdde.

Sectior oflivlt

slo\rnrg i[c ef{r

ofbifc

cdge vibniion

MidotanesdndMimtone Ksir3

r3J

The bevelsof a lliG weal iL, straightne$.f they de puliel ud evo on both sidesof the hG, the lrlG is str.ight. If they are mevd, broed in sone plec€sod $rrow io othe!., tlo tlD knile ld be.ir un*enly gromd or is vdted. h sone cares the warping @y be so grearthat the hi{! carmot!e honed to a mre edge Wto a lniG is ?ut on the horc for the ftst tinc the bwcls wlicL oe prodred by de sluqoet, o distioa froo thoseprodned !y the naLer, shodd be esnined with great cm becaue drey will give a dlret guide to tle srrzighoes oftle blade.The diag@ bdow shovs a cured 13i|e ridl broad bwel5 at ach od" There wiil bc conapondinglt snul ]evcls on the otta 'ide at the ends, md plobably some

&c

jr.

Cwed

midobne

laife snowils bev€15otr the @erc

lurge

wtd adjGring rhe position ofa tiife on a hicrorohe rhe clqrsce ilsle,4,ould bc ng+q!e.-q.j dge{&oqr the L@ bevel of the knife - dut L t}e !a_"1*l,j$-i,

si r L

, / : ' l

1 2 3

beve!9!9re 5

Rclrtidxhips

b@cn

mituionc

tlife.nd

blo.! i*cd

from tne,i
Satiafl C"fting it Mitloscop!

136

Eext to tie llock. Tiis poim ls panicuhly inportot who loivs ae beiry ucd which reouire a bacl lor hofie. The cleuue msle my v.F/ acording to conditiors preveili4 at the tinc, |!t tetwd 7-ro" would be &ceDtablefo! Dost work. It is esdtial th.it microtone [nives sfodd be kept clea dd free fron rust or tmish. Most laboratorie arc fairh rvam ud &y od i! i nor ofto th"t rtrst maks its a?pemce or lncrotome kdves, bnt qone fiequody the funs ofacids suchd acetic,formic, ud hydrochlotic nay be See in the an, md this will leadvery readily ro imisliis, wllich in t@ is lollowed rapidly ly rot. In suchcondirio$ it i, inponut ro k*p thc knilc lighdy snered with r thio oil During seltion cutting thir oil mut be conplctcly *-oved. xylde is a suit.ble cleding ag@t. SteelMietutan'ekires - Sharyebibr Bf hdnn Tlis nethod I stil tLc nost videly used,largch bc
with r badly drmagedlni€e drc edgeofrvhich hs gone to lieces the Pro€due r. MaLe the kniG ready fot honing by 6xnrg to it the approprirte Lodle ud back if requLcd. If a lacl is fitted nake surc that it is alwaysthe sane back od that it i. alrars pur o. the knG ln the smc way ud to the see rositiod litlErc ir uo -eo oitellng ot od &on the other ofthe back, makea ndk on ir with a [1e. lulricdt uPonihehone. :. Plae u appropoate I. Phe rlr iie on tl'c'ooe oJp-o.eed Lourw backod forurd ^ rdicrLd The honeshoddhedure at lest 12 x 3 x rin.(lo x 7, x,5 @). TIt stedald sir ofhoDe, apPrcxibately 8 x 2 x r in. js too snal for good vork, thoush ir may be ued jfnothiog irrger is avallable Ir vi11 be sed 60- ii. dirgrm tha! lhe ktric is not only noved up od bo-e bfuLo_1eru"7 ^ "elJ I his L ro m*'. ri t]ur.!h:.knf.: is dmoe .r;p,-"d ,n . .ns i' logtl, rd nor ju t in LhenddJe. lf tle kdle i. jut p r * - k d , p s d d o a * i r b ; u l 1 , r . , " o o v e m e n:r L e r o m a , o D c v ea. . i o w b

L"t*. 'r*.1-"4

. *"r.*

hr tlur r]'. l-gLtroiiiebrfc.

Ib- .dqe of the Lnlfe yre eo6 tlt b,l d boMg dd rt lhe eDd ol ean '-om the hooe. L rumed.n .+ b"-I wirou, Lftirg r s-te t'.itofc

Misotones8l Midotow Krjtes

F c. Jl.

FIe J4

Conev.

Honing a njmboe

edge oftlife

dB b !oni4

Fto rJ, Tlmjlg th. nicrcron.lniG

,17

kbfe

on r lEow

loe

ar tLe md ot. lEotc

r38

SedionC'1ifg i'1Micto'topf

Thn me&od of tmins preedts aciddtal ddnege to the kdfe cdgc, ud alsoleadsto a snoorh r$rlmic notioo. A very badly d:lrused lnife suchd is bcirg tr@ted luq should be honed 6nt on a tue cl|orudm honc. The nDkds should be *Led for the fuest they slpply. To tle hi.rotobnt tlis ir the codscsthe vill ever ncet and n is oily neededwirh a very jagsed lorG edge. With the ldfe in suh a bad condirion a fanly heqy presswenay be plaed upoo it, but this pressue should gradu:tly becone lighter and lighter * thc kdfe imlrovs. The risht hdd is usedto nove tle lnife, tie 1efthmd apphs dE !ie$w, md shouldbe lsed either on the od ofthe kdfe or on the hiddle acordhg to the wishesofthe sharrener.The objectis to easue ihat eve4 gmdc Eesue n appliedto the whole oldre cdge.ad alsoto nake sue ihat it is thc r/edgewhidr is being groud ud not the back. Exasslvely heavy prcsure on the edgeofa nicrorome kdfc duing lrcning orod: e., io,'rr ede". oujnrlrr. wirb hh lniv6. Tb. edgebod5,ray ieD ,Lig'1, ro. ,hit.o. - ,1", *" .tqp-;g tction n oor reJly taljis place at tie edge blt at a point sone slight distuce i!. Tfc r*ult of this if continuedfor ary lorgrh oftime ir a thin prolongarionofthc edgewith.otrgHy !d.11cl sidcs.rbn fcather edgenay roain, droqh gradn:11yrcdrced, tlfough a[ &e other olerations, and it will fmlly lerd to vilration duiDg the dtting of sections.Or it may break of, leading to a most uplrudt jagged edgc. Thccfore hanry presqe should neverte appliedto a nicrotone knife dwiag a. when a[ major nicks have beo rmoved from the edgeof the kdG mduliy wipe it cleu and plae it on a f,ner lbne. Ir is qsdtial to se dnt it is perGcdy clw, othemise it may ary over oo to the fne lone a ftce of codr glit flom dr cods hore. Tlis may enled ibclfh dE fnc Lore .nd wil gsh pi€6 out -of the edge of the lnife c long s it renai$ lhcie. Coftioe hoDinguatil the come nrkings of lhe fnt hooe have dnappemd. A suitable6e hoae for the 6rul honing would !e u Arkms whire. a Belgia yeilov, . Wat6 oi Ayr, a rJadc c",boMdM. etc.ror tle rd&e. s of nalea * p. to7. / Tlc holirs " thi, 5tage .lould be sof( ud s"Ddr wiLh the nininuD of "presue. Inspeaioo under the binocular should revel tlt serations produced !y the come honc brconing lss od les, util e snooth line is produced.There will be ao higi'lights to be seo in t}is oe leeuc the hrife lrs receiveda lew edgeo a rcult ofhoDirg on tlt fint LoE. 5. if tle frc hone listed rbove h* been ued coEetly it is quite probable o\at ljn kr le wijl rqfic no 6 rEs rrturid. A slighLly,moot}* edg. Dy br producedLouerer bv yrofojre rl'e knile. rlLL.ho:Jd be done very Lshtly *d ro mor tu 6ve .nolireJr u"y.6ord be oee*:ry. lbertro! ts mlde ofleth*, fnet caBkjq od it shotld be secu.elyglued to a flat fe ofwood, Duiig slroppirs tLe hidotone laife shouldbe pulled bacl ud fomds alorg the shop with the brk ofthe hife tust

Mioatanz

dtt,l Mi.ntoh?

Ktrir1

139

Strops nay be obnined nr duee fornx:

concdve woon suppo.i

soiid woo d s!ppo.l.

Frc. J6. Types of shop

Looseo srriisy strcrs asfislred ni (r) lbove shouldncv* be uedor oicrotorne |'!.e. Ldc"d.on"!o'1.,.\) tu,,qop,slo dne\d bc u,o -! Jl. ior ud o . . L . - d lr)' . ) p ,j g o . c t . ! l F . . e . s * h o o r . o d b l o c ko f $ o o d ,i . , u f i c i e n r r o r o E d o F r l e e d g " o f 6 c { n i tpi r D c , ,rri y i t e d " r m i , c l y . I b e , " f o .i e r, h o J d -. , b. J*r'Lt {rcppbs .orldb" "'oidel if po..ib or.ho Jd be,-d'r-d ro'fe lrfunum or l1ves[oker cr!h wa]. wlen a nicotone hifc is shghdy blut it should never be placed on r co!$c hone od trcaredin dre noner just oldined. k shoutdbe honed on11,on the finest hone availal,lc,$ indi.ared nl prhgraph four, ofrhn accounr. GenerdlAdrne ar Mi.aotane k..i,!! L Always rouch up rle micbmme ldfe ar rhc bcgiming of rie day. A light rum or two cn tie hone will help to kap the edgekear much bencr &; a sotid hou spot at tne end of dre week. 2. Nerer lay the ldlc donn flar on rhe be'ch. Thse my be gd! dusr,erc.,rtHe lnich vi[ !m rhe edgc. 3. N*er stod ir oD ! btuch or its back, edgeup. The edgets difiicult to seeir this posiiion dd nDy rsult in a crr hud. The kdte nar alsotitl oo ro i$ side vith ddage to irs edge. 4. Ifthe Ldfc is not ir ue pur n ia its box dd closerhe lid. 5. Knives dronld ncvet be sharcdletwan workq. In a ct*s or anongst tcclnicios cacn\.o \er sl,oid begi, en " LrrF fq $h + .c is,otct), 1po .ibtJ. o . . L e o ! , r e L i - b l1F6 , t o r L e . h a : p n e .o f a u i t e i s i ! , b e h r e ; u d , i r g , r . , F n

RAzoR BI.^DEHoLD{j Mdy exceldt rro. bladeholdersfor usea nicrotone lni'er de availablefor se with cdt.in nic.orones. Th*e rmi bladehives poses a very f,ne edgc ud will .emlin slrp fq lonser dh rhe ave.€e niclorome kdfe. rhe holde,s.r; dcsisned

r,to

S.iiot Ctting if Midoscoly

that tle @or bbde is held betwctu two ih€ersor rlars ofmcr.L which cone aldost up to rheedgeof Lberoor bbde.Trr merJ !o[s i of lbe sse ,rrpe @d sia s the conretioDal midototu lnG for tic sivd icEu@L The razor Uadeprojcrs 6on its holdrby a caction ofa nillinetre, ud thu thc vilr.tiol of suc! e tlin slet of nesl is red@edro a hininm. .,I! practiseit wiU be foEd that hf,ltration nedia ofsn:il6ysl3l size .ur len r ,'wirl rer blale hold6 dd rhosene& vlrich have hse cvstali or vhicl arc uorpbou. rlu golyererw. oo/ro,wir-ha reLrivelysmaxf;rJ, *il -, -*h betto od will producc nuch flatts secrioruths vil pdalia w* with is lager crystal while esta w*, which cuts s if it were quire @oahoo, with ordinary niootone kdves, cu hrdly be or ar all wit! rzor bJadeholdm. 1t!@ purclasiig hicrotones it is o admuge to buy one of the m-zorllalc r well * u odin,jy nicotone lnife, beamc it is so much 6icr dd /{oldcs " quicler to slip in a new rdor Lladed@ ir is to hone r blut liifc. The blad4 holder dsigned by w;gglsworth (re5e) !o hold 'pal dd 'Schick', razor blads, Iu nuch b ia favour, ard excellot section my be ot with thir istrumot. It mry be adapte,lto dtfrr rocking or rotdy microtooes, od is simple etough to bc mde in dy wo*shop with reuoruble equipnent. A Dinor nodi6ation @bls the bladesto be witidnm oce e*iln o to be norcd aloag the holds vidtout noviis the shole hiG. For electon midoscopy it tuy well be dbi d morphoui i.tuial such* glxs, whm fieslly broko, N prcdue a soootld edgethd js posible rirh e greuh n.terial such a stee1.Nselrields, stecl exlnbie such a neltiry od flowing ofia crystalsduing slEpening (cettne! and O6tein, 1956) tlat they ble i*o orc mothe. dd e edgetuy be produd nuch smootherrhd rlat comideredlikely frcrn its greular structure.For detron microscopyrmr blade holdersmaintain a pototial vale r yet i*onpletely ssgrcd. ' Safetyroor blad$ de of the finot steel,od netho& to if"rpen then rrmrely hde bed put foMrd by Sjstrsd (:eJ3, rej6) dd GtnFr and ohltein Gej6). a nodifi€tion ofthele redDiqud (wissleswo$h, reje) is descnbedhere.

'Il,e

RmrBlade Holda

Tlis is a mild steelsuppon sh:pedliLe a nicrotone kdfe, with a zo'angle. It hs a wedg+shapedthic&oing itr dle biddle vhicL ha a m' angle,ud two soall pegs vhich 6t into slot in qs:azor blade The dn&tud Diddle lortion is drilled od tappedwilL tlso ho16 so that lhe faciqg liece may be emately md firnly scewed to it with a Bzor Uele bctwed. Thesediagm fron the Qaa eq l.wnal af Mntoscafuol s,ier.a alpeu here by tte courtesy of the pul];h6, The Coh?dy of Biologib rn{ and the au&or, Professo!V. 3. Wiggldwodh of Cdlddge. It @y he cobidqed an ad@tage to Iovd rhe pegsslighdy so riat the rsor blale rBb m thd, iEtead of th& psing tltough the slo*. This mbls the blalt to ic tcnovcd raore quiclly od eesily.

Mtoatona ard Minotane Knirs

Fre. J?. Holder for'sdick

Pdsom' or 'Pat' sfett ftzo! bl.des (Wigglesworh, r9J9)

The Rtur BladeShdteriry Jig (wigglewdrh, reje) This consisrsofrvo wedgeslaped pi@s of brds, vhich are leld rogetherso r.hat _ rhe ruor bladeruy be 6ff y astarcd rt the rip. Together rhey measrc 3 x 7 cm. Thejig is provided witL a hddle, ud virh pri6 otflat-sided steelpessofdjferdt h b s d x . L , h , q h m f r - k d . w J s v ef , c + u g t e . o i : 5 , o a o d d 5 ; . f t e " d d i d m q,itubi q ^he6 ro dr DFg( ."*r,,rgle from :5 ro lemrs tle ordu,rion of oy .of '5J".

Frc. r3. Rro! bhde +rFfringl]s

orisgtswonh.

,9J9)

Sedior CuttitrSif Milos.

U'eofshatrairylis

'schicL', 'pal', 'Pc6oui tyPe. If a or rit into thc sharpeuingjig blade of the f** argle of 3f ; requiftd ft the 3o"pegsinto the hole. Grind the bladedom or a sheetof plat€ g16s accordingto lhe mcthods of Sjctrdd G9j6) md Gettftr dd Odtein G9j6). oo to a shcetofplate gls pour a lappingfluid of the f" oving cotulosition: Lindc A slniletic sapphirepolishingpowda

t volune

Distilhdwte r rcl Gl)crol

8

|

(Linde A; ?roduc* Co., 30 Edt 42nd Sfteet,Nev York r7, NY, USA.) fte fhde; worked, edgefiat, ovd ihe sheetofplate gl$s witL tlc hpp;g fluid, in a seriesof zig-zagmovenenis d shom below.

-\-1 FIe, J9. Method ofhodry

a Bz.r bLde

'I

he bl'de,lould neva bc norcd wi-h rheeogeraili"g brau.c fij' ib"diaely hs beo '".cled L\e t'.ife should rorrghm rhecdgc.wJen rJc edgco rheg1,..oLace be tmcd ovcr $d b osht brk agjr b -Le.me w"r. whm r clcu'"rL ho bem proJuce"rie p .r. L "rl e" ad d'e uile i carefulli w*hed md ttu )appingn contirued with Li e B polishhg powder in the sanefluid. The bhde ud is holdernre do v6hed again.w$hss * addcdto the sharpening jig to give the requireC;gle of 35', by i*a:ting then below the Pegs,dd Pobhing is conti:rued on modier sheerof plate glds wii! ! lipping nuid of di'tiled @!d gllcool

6 nl r . tm l :ml

No ?owdd n ued. This final poll:hiq shodd le done gody ud softly. It give a very smatt3y 6cet at the n*gin ofthe 3o' edge.The whole procedurerequir* ody

Midotafl$ 6fldMidatoke Kri!$

r43

Thc.lappiog drid nay be warer to rvhich a Gw dropsof,Tecpof (shell chcni.at co Ltd) pls Linde 3 slph;e lolishing rowdcr (sjostrdd, iej6). rd uftrathin s€ctios rhe rTi6 edgergle siDuld be.bout Jr". THs is r iarge dgle . bu! n gre , "biliw. Dd m,b cs " goodedgero b- produceL T l r c n o \ r D m s C u , i n g . l d p r D i n g" o d l d b " . o . \ . ! - o o ! n . & d g t r r l e .r n t h . p-odu.riooof LJe fiJ cdg- t Fre .r no o e,.ne .hor d b- pJlicdro rhJr detrotder. c . \ e g l " ' m e , e s r l . J r d ,i q r I ' l l D u l db em d e d a -u . r h e. u r T . c o r !rcsue !o xach stroke should take abont tvo secondsiD a sidewa5 ard forwd &rection, dd tou sch strckd so;ng fomard ud eothe. rour comins Llck shouldtresutrcic!! to producethe rquicd fin.l facets,rvJ,ichwil1le about z a in widtJr. The blade shodd !e renoved frcm irs holdd ud washedin Disriled wlrcr 99.9nl. Sodim chome glLlcosat o.r gm. Sodirh chrone glLlcoete(D. W. Haering& Co., Se Arolio, Tes) is r.orrosion inliiiting rcagmr which pr4enrs elrly desffuction ol rhe btade edge. The bladc shouldbe dried orelirly without touchirg drc edgein uy i'av, ed i; is rho re.dy l o r e u r m a r i o nu d e r r l e m i c r o " c o r e .1' r, , ) b . , r . e dr , t , o r e r . s r v e e , r i n n i o n . b o s e \ e ! .b e . " u ' ei n r l - s c a c p r o o fo i q r J i q l e . . o , e , i r p c r . om u r e . 'Rolls' 'Roli

Raar BladeHoltur

The safcryru oi lhde is a dn*-bacLed r@or bladerathertikc a segnent of a nicrotone lnifc. A ho1&r to talc theseblade n descibed Ly Slolt GeJt. Popt HtuL-saaBlalL" Th€ roid divd hack-Fw tuhine conmonly udlizs vcy trnd and tough tugster stee,lblad6. A ncthod of $ing such bladess nicrotone laivs, afL arrropnate grindiDg, did slEpeing, is givo by Dllworr.h, S., dd Aplleyard, H. M., reJ4, iD A shalp ing machinefor a new r$e of dcrotone !nife', earrr. CI6s Knires The folovins rale of 9/d6erErLs should be bo&e i! nind: one nillinure equalsoic thosdd bidos G r@. - r,ooop). One miaor eryals tm thoudd Ansstron eits G a : ro,ooo A). sectioB for electron hicroscope study may bc cut lxnseo roo A nd r,ooo A dependingor the fnel n€nidcation rcqured. Srctiom of thl dltr6 -.od,e ,.urug edgeo no gru rd rt,e rdEr"ge trhi,h rle broko-sLs. tdf- hd o'ff !h. .,+l kaje j. .,!,' " "adrhou. eo;e edgethereforeshorvsno grddariq. Tlr .liadv*age ofdre gLs lnife n tha! rhe edgeproducedcmrot bc alteredor sciendfrcall,v controlled s cu a sreelrazor blade whd shr?d€d in the omer indicated by s.joslrdd GeJ6) and by wigglsvorth (I9J9J.

111 'W!ib

Se.tionAntiflg in Midoscafi

n is mtomry to regud glss a a natsial whi& showslitde choge t[rough the yeas dE edgewldch i5 requircd to ot setiom o tlin s roo A; so 6!€ tlat n soonshowschdg6 wh@ exrosedro a;, dd it is betr6 !o .!r the !Div$ d they are rcquned arher than to cut 5o ar a ribe sd ue then at sohe furue ilatc. Thc tclniqe ofprcducing broko-glss hivs for elecror nicoscope mir:otony ks introducedby Ietta dd H.rteM iD rgJq ed is widely 6ed. Wlo a sheetofplate gJcs is cut irto the oEea shapei! is rhe thictnds ofthc sLs' $hichic s.d s lir tudlgedge. ThLr r pier ofouner plregL,s will prodre r loiJi ed$ obe qund otr ilcl ii leistl: r pietr of lhrce sxteqrh ( F i!.) gL!6r wil [email protected] i5 r* in. in lostL The glcs knife is at accordingto rhe slupe ofttre rnicrotone kniG holdq, dd itr sohe 66 the nuke6 plovide ajk so rlat r]Ie .oE6t shale nay be eaily Foduced. It is 6s@ti,l th.t ttre mak6 imtrutions rc coefrily followed. To @t gl* koivs proceeda follows: r. Take a piee of quarto irch (G7 lm.) pl.te glss. Se tiat rle su.faeis deud dd dt &om it . strip of glas ooe inch .dos. To do this tue eirh! a sLlp dd nM gl&is's di@oDd or e srel wh6l gla$ onei Borh tools lBvc their adv; eter, but on &e whole the steelwh*l n dre esier to ue dd ir ir alsochearei. To [tle rhe(ur 6e a ftoden ruie sbicl is snrsbr id ,bout obe qu:Fr; of d in,h (o rm.t thick. Bedetul to ajlow ro, lhe;d po"irionor'Ie'dieo"d relatire to the sidc of i* holder, or to thc position ofde steelwheel lelaiive to its holder. Plae the !u1em de gls s dut the vhed d di@ond will be in the right pla6, dd briDgthe cfltter dom tlre glas rirh r fm ud st*dy novemert. Ifthe right oout ofweight ho beo plaed on the mtter dcle will le e sornd a if miaute c4,losim vere t lilg lLce d the dtrd is bovcd .long the glN. A fiz od d.ckle which rel5 tlrc litde. imediarely thlt dle cut i! a ftu6 @t and mt . sdatch. SuchI @r however good, wil pactiate the glas ody to a ]imiled extert $ shom i! the donpdyins phorosBph (69. 60). 2. The strip tuy be perted&oh tle sLer by placingrhe sheetofglcs on the f,oor. At one edd ofthe cut pl.ce ! natch stick so that it is approximrely ore quaner ofd inch bder ihe gls sheetbelow rhe @r Placeone loot on rhe floor wid the solegmtly placedoo the glas bside the ot, ud rvith iie orhs foot pl&ed jn tle sme riay buf on the srip side of tle ot, gody prs on the strip.Ifrhe tur ir I goodore &e $rip s J pmiiom Lbeslet que radny. \oer aneopL to bFJ srip, otglas !y hdJ prxue, udos r.he\ad. ue edequreJyproened. A sudda lreak oftte glos @y reult in a sevoe cut. 3. The one ircb wide strip of qwtet-p)ate glas may !e cut i'to piees ro 6t rte hife holdd ofrle nicrctone !y dlawing rhe gks dner aaos it et rie righr pL6. At dEt pa.t ofthe g1,$ srip where the lnife edgevill be produ@dir ; better not !o take the &dond @r light up ro rie edge,but to stdt thc or approxilutely 3 4 Im. i$ide. This gaealy giv6 d bene! @ting edgevho the glas is broLer It is ssential to lie glzieis plies vho brering smal ?ie6 ofgl*s becaue

Ftc, 6c.

I)eprh of dimond

.ut in qurtd

in ! sh$ ( x 3)

Midot4n6 dtulMiqotofle Krivs thejaws re loided

r4s wnh a throar wLic[ lerniB the g]as to hc gripped at tlc

j:ff"*."r*yJ:1"":.s" -Ii#::rn*6:,^HilF.': *:-y#T":?*T;Ieouerh";berowirprcd,Jb;;;*;il; To q n r-heeadof r-hiore;.h $rip o[gL* L [Ar btD<

otro.]om in ^,1t.T,*f-";.1*";:* *' *s No rud3arc ffJ "'ll*,x'i ?'i ,

;:* ;rn1*,:tr; ;t*{uri,_.._: xi:.H_Ttrd

iJ D-ut rc look clGely at rhe slcs - n ir beingb,okeb.6ur LhnL not -rI' nr1gbre bese oc6jonJly r o-I ctip or ruo o, i,"*, gr," *.rfty u.

Frc,or. Cftjne Bla$Lrv6 udn€ajig Itu fi-l b'-ts"g sboutdute pl@ jst befoe the hdte corus rbera tr*h cdgeb anitabte.

146

Se.tionCattingi" Mid6copl

The aboveimtructions ne relativdy simple to folloq but thir doesuot ner that ,[ the $d kniveslroduc.d re a[ tle sanE q dat thcy ue all suitable. It will be foond when the kdves ce mined uds a binocular miaoscooc that ruyof theedgaarcaor rtraigh(,udrhara bigbpn@trge hae s lirdeo saiglt piecepresenttlat they should be tluowa ilto ilE bEket withour delay. Only the very b*t is good erough, od rigorcu selectionshould be maintainedif good ultra thin se.tiotu de to te obtained. The completesids ofthe ldfe may he regudcd u the fra*, ud iftLey wac continmd right to the edge good mtdng vould fouow, but thc min soue of rouble lis in the ful breaki4 of the glass,vhicL @y give . slight cwe towddj the other faet, thu producing vLat is in efect a se.on&ry {!cet or a cwe. Thr n comistmt only with conplete A ue to produ.e good $.tions, and h the @jonty of coe a knife shouldftst be tied on a liece of resinridroft matelial in it to se that good sectioDs @ io fact be ot. If snali lin$ or cw6 d roticcd sone slight distace rway fon the edgeofthe lolft they nay have then origin in the efue iael{ ad srch glas hiv* should !e viwed with cue mder the binocular mioccope, dd ued viti canion. The atoosphcre of indutrial cities i5 conosive. It tuy also depositglotinous o g!i$y nat€rial upon th9 ldfe edge. clss !niv6 should thqefore be tuoken at the tioe thcy de to be ued. Once ued, dd lat for an hou or so, dry should b€

Thesecocnt of dimonds cct :o the requiredrgle od siape for ue with the rlta nidotobe Th& fahrication; a Eatts beyond the rcope oftle oiqotomist, and the only thing he co do is to puch*e then. REIIRXNCES GmN, M. n., and oNsrN, L.,1956, P\'kaI t chriEaqin biologkalresdtch.(Osre\ G., dd Polister. A. W., nd.) 3, 627.New YorL. Acadmic Prc$. LAEA, H., and Her]\lr,N, J . F., :-9sa,hoe-E ?1. Biol. Me'1.,A, 46. SJosrRAp,r. S., r9j6, PiryJkaItechniqraia bialagiralteedr.fi.(oster c., and Po[6tcr, A. W., Ed.) 3, a2r. New York. A.adehic Prcs. sro.|\u,J. F., !9J3,J.RoI. Mla So..,Vol. ixx[, Pad J, r7o. MwiceDrlswoau, V.8., r9r9, Qtfft. J. Mid. &i., roo.3rt.

CHAprlR26 TIMX SCFIEDULESAND DIFFICULT OBJECT5 It is usdul to hve sone guide to the idltrrtion tiocs requncdbv diFercntsize od dlfcrent Linds of spedhen. S!.h tinings should bc regardcd as approxinatioas beca$ethe condition, d€nsitydd lenetration ofa slecimcn by molten wn or rcsin is &podat on what hastaLo plate beforc n mches tle inilt.atioq nEditrn. rhe factor which induoce hEltation tines ue r follows :

3. 4. The deh)dr.tion egdt. J . The ia€th oftine in the dehrdnthg flrndr. The lengtll of tine in anl p.derative vhich nar havebcen ufd. The length of tine in the clering agent. medium. The temperatureof the infLltrationEediu,n.

9. The viscositl ofthe hflnatior

Sor* but not all of thesedetailsare lnom to the nicrotonist. Perhals on1) one or tro detailsolthe ten cited*e quotcdin papersoo f,istology. This absmceof conplete knowledgemakcsmy seriesofinfihration tines a seriesofapproxinatio* only. with hot s.u i!fltr.tion the rule is to h€ltrate for the sho.tst rossille de at the lorest possibletempera:ure.with aicors reshs dE iongestpossibletine shodd be Tle tine factor, fron fixrtion to iLe end of intltration, ir the no$ nrportmt si41e factor ofall. With oy tissue,vhaher d sofi a a clnck cnbryo ofa: hours, or x tough * a pieceofox skin, dE quickc. i! ir in being bfltrated and blocLedthe easia it will be to scction ud t1r bett$ will it appcu uder the micros.ope. The e*dt to Nhich .11thc hfitration proccses nay bc accderrtednay bejudged by referenceto the chapten on simultoeous fixation od rv* iiiltratroh (1,.,,,), and nlqllation i'i$out fixaiion. Thc ligus suggestcdas being sirable tims iD this ctupter arebgsedor expcriencewid:ont stlrring ncdrods being uscd.The tims ruy le rcduced,rco*iduallr by lsing the stifter ndicated in drc chaple. on agitation With any tisues o. mleiial nervto rhe midotohist - d thn nay occurevm aftcr tiicenty year' exlerioce - it is d advdtage to obtdn tle clenins tinc for thar pdticular piece of tissre or orgd. To obt3in the clearingtlnr the specine slolld be brought into absoluteethyl alcohol, rd givm t*o or $ree chmss ovtr $me n;utes or hous. Ttc objcct is ro hakc su.e rha! the srccinen is conpletely dehldrated.It js thei tlus{iEed to :jene md thc tinc taLen.Thc sp.cimd is e*nincd

r,18

&.lion C tistin Mictoe

ar &equdt intervals dep@diDgon iB iizc. Pdhaps every mo luut€3 o! ewry ro binutes. h sLould bc dmined in xyltue wilh the nakedeyc, ed it shoutdbe hdd dd a dllr. backgrcud, Nch as dE l.loarot floo!, with light shiniug oa to rhe spsin@ *on lhe window o! orie! ligh soece. T[e s!.cimer, who utLted with rbsolueaicobol,ir opaque.dd s\o ir is pL.ed in xyloe it ,en,r opaque util 'r cle xylme h* cooptrJy ta!6 lrr phcc ;. dr J..ho'. xylfte. kirD rerBcrivc ind* ofr.4e7 is clos* to thc refactive iodex ofthe tissus rhm is rbsohe erhuol, rd x a reur de specimmbeoo6 ftelucmr wh@ rrreyyltue ha bilrrted it Thc specioo lis LiL to appd s a wbire.oDJqleobjrc. h lhe rTJtue.Ir aLnost dnapped, wheil viwed ag:imt de d.rk bacLgromd. wh6 lhir chdrge troE opaciry to nuluceacy Lastako placethe tine shouldbe mted- A cobpdion wiil de tus tine. takm sh@ rhe ipecihn u frc.bJyput irro xyttue G;b rb"otLre alcohol,givcsthe cleding tine. It oy be asrued dut ny liquid ofsinilar virosity would pe@6tc that partiarla! !;ce of tir e in rhe sanc dmq ud $at thc ws which is going io be 6ed fo! in6ltation would take somethioqlike twnc dbr rime becaueofin gearr n'onry. Tbe clearugdne tu.lherfo,e. r.ed a s irdiotor ofpoetation mtes,aod it uy !e nuLiplied !y r 3 rins to give the fdetntion The 6e ofrylme:, an.id to obtaining tie cleuhg tine doesnot imply that it jr dre bestdeaig agat or utoc&m. wter the cleding tine ha bd obtai4edir nay fe lsed vith dy cleariig agent or virh dy infltration nedim, virh apllopriate ealdion dcpoding on the viscosiryof the rnaterialsued. The rcctrtte riniDg ofiiiltnrion to! uy piece of ti$de bay bd obtaiDed4 indieted, but ir is u advdrage ro rry two fiees ofrissre, one at twice rhe cl*hg time od the other at four tires, whtu they ft in hoften 'A. Tlat whi.h fin Iy @r' the befie! md shom rhe besrs@tionwlh stainedadd cwined uder the nioorcope is the most suitabletining. Ic dealing wfth new Datd.l the time lost in folovirg rh6e suggesrim ed it dlpli@ting or triplioting thc in6ltatior. tincs will be esily rcg.iled by urilidng rLe lnowlefue &quned. Ii sohe o!g{s ad tissu6, sucha cartnage,anlhpod qskdeto$, hair, chaele, cottob 6bre., et ., the pdetlation dd percenbgeof$d prdent a{ld iDf,tftation nay be so soall o to be rlnost ftgligible. Tlus is due ro rle natu.al ddity ofsch tisuer, dd tLeir cliting nay be nost di6cult. Ary exraordinary ineoe in thc infltiation tim€sin a! endcavou to rule thee tisus ot better wil do nothine bu| , nake the s?e.imo lrd6 ud olre refractory. In sucLcso it is advnabb to natch 1/ theeturalhdd!6 of th speim&by usinsahudqasuchas esterwg, oratqin' sucha uea fomalddrydc A hrd ;peibo shouldDeverte bfltrated in a soft wax, / The biggd the s!eci@, o! the gratc th. vi'codry of rhe bf,ltration ncdiu the gr*ter the neccsity for agitatior ofthe spedna during irilftatio!. J Moltd wd r. sDa[ soft objecb minut€s Pndnodian 3 fi, Hldta J Polltzlk rGrJ chick enbryo ofaz hous 8

1:ine Sched"les ad Difrdlt obid!

r4s

Tbesea'e specimero v\ic} are o,y to poer,e. The chid probhm sociared qjr! thnn.rbcir rodeoclto.oU"Fe ifLepi 'oo toDsi! bortu, ad \omcd;roftjoq or ttrel-)oit bodFr,evrDwb.b (rr i! sed. k I inponer lhereIorcto k lhe low6! nelting point w* available,od to keepthe specimu in it for rhc shonesrpoliiblc tihe. Melting at 38' C roly*r.. * t 't "'sly "d"*"!"d. Peted 2. snall hlrd objeds ?d. I$ecr eggs 2b. Aat (Fon'na) 2.. Gonli"s Pmetration of the naterial, ud o*ing good seaiom ue tle lroblM herc. rt is 6s@tial in el1tLe cdes giver, ard in siniLr cxo, to pie.ce or slne thc apecineoso r-!urfluidsm. set b'o tr. rhis sloutd be oooebelorarle ribe tor w* bf,iarrioo, or exe,dol trefc,eblybe de o, d-rirg fBJuor bur ,L rb hrve rc br rcpeared b d o r e t h - . p e i n m n p L . . d i r D o l @ q * . Y u l m e r r " g g sf t e e g g o , y b c rrickcd with a fne needlei! two or alree plaes, or a very fine slicemy he cur oF the srtue merelyby rmovhg a \-p or rhergg (se. A neu saferyruor bt de ir ruiuble 'o- il 5. id !,r.eopcruooo.iould be dob. ide! a low power biqo.uid midoscote. Ttre cgg bay bc firmly futmed to a bar of neirl d givd in de!,i1tor frog eggsor p. r5o durog slidngor pn(king. The vre d\ire n ", !f gild for turing rdeq.Lrepddr.'ion of de Ant (r//i 4). T\i! mdal : orc o[ Jr doj difi.ulr obir- ro ot in tr wboteoi LiE dinJ Lhgdon. lr r ss-jJ Lh* ir.lolld l,e(omp;k 1 dividedinto dre pm a cut rhroL€h thc rhor* and dothd though the ;bdonen, ;f r]1eprobteh; hMd pemi:. If rct thir piecesslrculd te sticed;tr rhe *oskeleton in apprcprine p1**. b Lle rheHonch"t qorm cu,/i* r*oblo r pieceof r-hhde. r! " \H bLd i lu tl"hJdre: od Gelof a nc.dl.. Byo ringir Drosm-tipie4, ech aoourj rm. u lmglb rdeqr"rcpo-r,rtioq of ietu-D,esduhim"relr o Lhehttlmdon mdru Irr the tLre oes cited beticulos ansrtior to the rqunmflB jur dvo ir eifrriJ ror.r,c6ful .*doninq. to droD\Lch sp6iE.*;b"t. i"r;6*;u" *d , tu"uy ro kke lhem.ax --pi.";. * * -u"idir urJJproduc norling but / Thescobje.r atl show cotuiderablehrdnes when in wc. dd dsr r6utt rlev tend to eitler lieak o! cruble duihg cutlidg. tor thi5 reson a hard wd suchs dter $u "to,Jd bc 6"d. Doube.mo'ddinC uing Perer6.me od w alsohetp.ud if bo$ lhesefail cellodh shoul,{ he m.d Estefwd 3. sna , sofr iopermeablc objects 3a Blos{y laffa 3r. Ffog €g

3

Polyste! wd

3

rjo

Sutioi Cutting it Mids.opf

h spite of th. sftnes of ituect larvae their crrticle is so inlcneble rhat they require sinilar treatmen to that oftie smal h.!d objet. They should be slightly cut oten or pncked ddins fr-{atioD,atrd this @y be enldged in the mtemeilium to facililate entry of tlat 11uidud alsodr bf,ltratim medim. T[is also rppli* to the egg of Rda tentddid nd otheramphibio cggs,though thc 6nal prickiry or slight slicing o.y take plae :fter 6&rion. The esotial difi'*ere betwear specnno in ttis group md tl'"se ir g.oup wo is that evay ellon shouldbe @de to Lce! the mtuelly soft spcinau soft ero when ir wu. Ifoosible th*efore sDc.imdu suchrs nud lanre shouldbe bfiaated with ! low n.ldng point wd sei s polyatet na-x, so tlat tllen natlr:l sofft3s should not be repla@dby the indued lddms of hkh tempaatue infrltmtioL Yoll, u cortahcd in the 6o9 egg rd most oder eggs,is &6cfl1t to cut bcaure rhe yo]l groules m rdarively inpernablq ard &erefore the mout of mxir them is so slight tlat thse b poor adLoion ber,reo the groules ud tle wq sur rooding th@. Thn le.di to a mbling which my well produ u *csive los dtring dtting md alsodeiis lxer stagesof stainiry. To prcvdi t!; cithe! estervd or polyeter wc should be used,rd single eggsshould be aeatcd with very gret c*e, nthe tho mmy eggstrsted orcle$1y in th€ hole dur onc or more nay prove to be succesful.Ribbor stahing is :dvoeted s the most satisictory way of dealingwiddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddd eggs.TLe full treatnent for eggssuch* thaseot Rora t!npddia is r. 2. 3. 4. 5. 6.

Fix itr SnitLt 6etive. 3ri!g up to 70% .lcohol. 7o"/oethtol od cthyloe glycol monoedryl ethcr, equi puts. Pue ethylenegly@l mon@rhyl erher. Ethylee g1yrcl nonoethyi etherud eter w*, or polyests Rd equd pans. Molten wd lor 6ve ro td miNtes. Tho rakethe cgg out ,nd dege it on a quirtd inch sq@e (6 ld.) netal bar * shom below. Thc bune( ii so ddged that ihe w* at the dd ofthe bu is nolten who the

slicing

f.og

egg

Fc. 62. Method ofholdrns . flos esg for slcins a window otrthe bp b pdnit betd infldrtion af w:

rirc s.hekles andDtfrdtu abkcts

Tjr

egg is plac€d on dE bd. The bmef is noved awey whd the egg lB beo coFecily olietrtated, so tUr tie egg is lEld by solidided vs. A sbal slice shodd du be cur of iie egs, udcr a low power binoc ar hicloscope. The bar is wumed, ud tle egg put back into noltor iw. Before cutting thc sliceof thc eggis so ddged on the bu. dBr the sLcewiLl$ be in d:e dencd cutting !l4e of tlrc sediod, dd vl@ dlc block ir tude the cut suface should be lmlel vidr the botroo of$e UocL. 7. Yoky esssshouldbe ftt slow\'. Thu foi . frog egs it vlll !e fouad that a cut wHch lsss through the egg dd vhich t kes ro rconds in irs pasage,wnl "row cs yo k goule mo.emr, \J, one cut "r $e boaiJ .peed. 8. If.ut in cstcrwu float the sectionson to a slidewhich h* teeo alidy snered viin Maycis all,uno dd which is floodedwidr r/. safroin vith one drop of ailin oil to eachIoo n . Ilattd on rnii solutioL dr.in, flood with distilledwater, drain agaiqdd thcn dry ovemighr at 3J-4o' C. Ths vill producea rcd stained section.Il a\e strengthofdrc sa6uin i wried by cxperinor ir vil bc toud alat no diFerertiation is necdcd. Sih!1y di$olvc thc vs aw.y wirh r-ylde, dd mout in b.lso. DPX, XL,paral,or orher noutot. witi polyetcr rvx scctioc float rhe sectionson to a clcu slide,uing anylolectin $ dte doating luid (p. r2r). Dnin, md drv ovcniglt. Ttr amylo-pcctnr holds the scctioumd the yolk grrnle sowcll to the slidedDt ordi@v slrining methodsmy be ucd. PolyesterNax or lm1fn xsrcr w$ houn houn a. Medimsizobjeds 4d. T .5.LtflbtiE tet5nit 3- 6 rer6 ai. T.S. Newt heador body 4a L.S. Rlr ljdley J ro Thesenlrcc objcctsh.ve concealeddificulties: tle ordlwonn haschaetaead nay have grit in t1rcintestine,od ihe ns!-t held o! body ls sonc bone or ot agc; tlle rat Li&ey ho thc upperpart olthe ftid which in the inlm of tLc Lidney foru a very tough 6brou rms. Before iiift rior thse lrohlos should be reducedin severityby clearing out the gut of the eaniwom Ly Geding it oq blotthg pepe! lor somedlys beforefration. Whcn n\ed dd taLd iato 70% alcohol,tie gtrt nay be ftrther clued by cutting thc lom inro snnler pieces:Lout r cn. in length. With a pipette blow out d), funh* piecesof grit rvhich nay renair there. This is donemder tie surfrceof drc alcohol.TLe newr nay be deolcfied, md the ueter of the nt shouidbe rooved wirh Ine scissor A clo$ to rle ki&ey a posible. 5. ldge obj€cts jd. T.S. Cat braja 6 rM.

slice

Jr. L.S.ShepLidney Ja L.S. Hlll]d ieg gelatin ,d T.S. Hme brain r-2 drys rubber laraFn

IG3o 4

Secti$ Cuuingin [ficlot.opf with obj.cts d lugc s th.se frstio! shouldbc donc by pdfui@ ifposiblc. rhir is the bestway of gening the filative right into ihe dcepp{ts of the orgar If thlr (:dot be done d efi.olt should!c n.& to force f,-stive inro the specimenby usiDg a hlpodemic syringe at wbatcycr dt ries or veis hay be awilable. The specim should tho be phced iD lrgc ootairc of fxtive rd nred lor a tioe uril thcy are sdF ooush ro be sliced"slice sLould be ot iehod sing a razd. Two sball shsts ofnetal seerely bolted to a woodd blocl<,dd fstded sorhar they ae venical, m,y be useds guidesto the slicing.The Liduey o. braiDis placedbervd lle upright sheetsof metal ud is slicedevetty by uriug the metd edg* s a guide. Hdd slicilg nay alsole donewithout mirg guidesofth! *hd. The slic* shouldbe about ro m. in lhl,kros. od tneyriorJd be pl"cedn Gxrive ag"ir. Duiug inliltration with molta ll,d dple f,lid should be ued ud stirr;ng or agitation nethods should be enployed. At leastttree separatebaths of noltm wu shodd be giva to makc sue thn lhe slicd de ?@uted. The luga the ue of thc blocl face the thi&o nut be the sectior. V'lile it n possibleto cut sectioGof 4ooA vith relative ede +oh . block neouing r x r m. it wodd be nnposible to do tlis with a block neaurirg ro x ro Ilm. ado$ the fte. wi6 very brgc picce ofti$uc suchd a sheepbmin or hrm bein it ; impoe sille to get a a'holc scctioo.t abouts p, therdore sectiomoflrye objecs de gderaly about 10-60p dnck. Thc tlickrs rhieved depo& oo the ilf,ltation nedio nsed, the s|:rpnes olthe kdl! ud the tmpoture of the rooc It vnt be foud that for lrge objeca lhe note dystalift the ne&un the nore usuitable it is for thick scctios. Thu lolyeter wu ir Muitable for very big secion rd so is odin ry parumtrq'u. For seciios ofbriio ofcat or kidney ofno, ester llax win give dcelcni scctios, lut lor lergd objectssuchs brain ofnn rubbopm$tr rw Is tlt admtagc of greatc d*ibility. For obswation with the naked e1etug ruy be nrfrltratedwith gclnid, hardmcd io fornalin md cut or a specially largeaiclotoDe. The scctios ee ?laceduton stiEshets ofpaper, dried ad vmished. This is a ncthod ucd ia the $udy ofsilicosis ofthe lldg by codgh dd Wentwofth

{re4e). REFNRENCE GoucH,J., and Wl:l|woRr,J.

E., rs49,J- Ror- Mio. Sot.,69,23r.

DTFI]CUTTOBFCTS r, TheVertebtute Ete ahdLehs Any object i'hlch conraitu loth very soli fd very hard tisns in it presehtsa seriesoflrobloN to the mi.rotonlst. In rhe caseofthe eye some ofdre problem shodd be setdedhefore sectioningbft thotrgh the ain of tlir book is to dea.llvitL secrior crtdrg it hight be srntableto give here a compieteaccout of dre techiques lecesay for dealingwitL this speclno.

Use fonnol-nrrcuy, {brnol saline,or Bolin. The e}r should be placedin oy of rheseduids,lut ln additionit sholld havethe n{ative injectedinto tle vnrcors dd into the aqleoushtnolr nsinga hypodonic needle.The oLjea ofths is not only to fix t|e tiisnd within the eye brt also ro h+ the eie to keepits shape.It generJly colapsesifihn iljectior ls troi nn.le. The injediou alsohelpsto Leepthe retinafron Fi{ for 24-,18ho!B- TLd b'*h ir water and placein a re{iigdator mtll stif wnh a very slBr! ralpel or new saGtl razor llade cnt rway a sesnent of tle eye on e&h side so that the mtqior chanld ji jnst rev€1ed, ind dr posterior chdler considerallv. Thaw tLe err od give it the treatnent apprcprhte to the fl{arive $ed. I! ney Jso be a:r advutage to give it further treanneqi eittr the trtative. IdeJly the eye should be nxed by nrjectior ofthe ceutral retirul artery, or ofthe carolid artery vhen the ele ls sti in its orLit. If ih. nrjection Dethod n lsed tle eye o o r J n t r J l b ep l , c e di r b e 6 u . i ' e " f . , h " , j e f t i - r . permit ahays useo eye lion a yomg minnl, leca$e the los if cncumstances dll not lc hard, od perfec! sectios dxy be oltalned. The vitlcols dd aqueorshmous may be renoved verv sendy either $hcn wasliog out dre fr:ative or when the eF is in the early ddrydnring dulds. If the dreous f,uid is renoved carele$1ythe Etina vi be dalnged. rheo ro ei% De\&ate tle e1e io gradedalcohol solutior [p to qs9l" 31coho1. alcohol ud polyester rvx, cqud ?aru. Fitul1y ro pure polyeter wd for a tnne approprirte to tle sizeof the eye. A frog eye should take about 6 hous in rhe pulc molten wax; a rabbit clc ud .lumm ele about u hous. A1l dresetines are quoted to! the !!re r'd widrout stirring. Wilh a rnechdicxl sdrrcr lhc line nay bc rcduced f r o n o n er l u J t o o r . l u l l . - r e , \ , \ n " y J o b e u c - . Ifit h6 beento$iblc to selcctcyesfrom a young miml tlt conplete cye should c$ virholt grot di6frorltl-, but if it is m eye froo a vcry old ainal rhe leus will udolbtedly give r.ouble. This mav be reduad by folov]lg lhe advicefm deaLhg

I54

SectiobCttting ifl Midatbpf

Th. Ele L6 The ele lens cosisrs esdritly ofr oorer epirhetiun c 1ffi @?srlei$id! which rc lm 6!res which areoited to fom lanina. h tlc youg uinal the Ie6 frbresde soft ad easilycut tlroqhout thc mtire b k of the ls. bur in older dinDls rte f6res becone very nrch huder, od in rhe cntre of$c lcDslhcy de so herd rl:r xhd cn on a nndotome afta bnltration with praflh 'u thcy sioply drop aw:y itr . shos'd ofdry chiplings or powder, leaviis perlals on]1.tirc outer pan ofthe los in!a.t. Thrs vhm se.liord in fin rvay sone 35,50% offic 1emde. my be lost, s viewed oder the microscope. It sholld be deady r{ogrized dr.t vhatever nedbd is ued pemnation of the cmt.c of e old l@ cirher ly the fxtivc or ly the biltration mcdiu is nevcf completc,ud if the rinc woc rako to nake it so it worJd coincidervith tie onsct ofprsbyolia in rhe bidotonnt. Tltrefore in cutdng socha 1eb the mic.otomnt is dealiig vidl so'nefiing which shows little rncc of tire iniltation nediun, dd which hay erd conlain sooe tracesof noisnrre. This problen shodd !e t.@ted ir r. 2. 3. 4. 5. 0.

Iix $e ls a given abovefor the qr. \I&o A{ed dd washcd,bring np ro 95"/. alcohol. &d polyester w* equlpam. 9J91,.1coho1 Pure polyesterwd ar 38' C for 24 horrs, wiri stiriing. Or esrervaa. llock in tle way givo on p. 5:, for poiyesterwax. Givc the llock two daysor more ar . rood temlcrarue of rJ 22. C to senle dom dd le.ome close-lnft. 7. Ploceedto cut sectiod 6tll the lem shorvssigru ofpowdering. e. Renove &e block-holdc with thc block on it lrom rhe nidorohe dd ltace a dror ofwater oD the su.faceof&c block who tlte block is or tie bcnch, or a lvct pi€e of cotton vool nay bc ued. L€avctor 20 ninures. 9. Slot the *ce$ wrter avay with llter paper,placc in tle microrome dd srarr ro. The lirst eight seriom or so will rct be very good bur atier tt2t a poinr wit bc lsch€d whtre rhescctiou at 8 a wjl c!! readitywithout pow&dng fd prcbably 2e3o secnoff. Powdering eil ttren stan aganr,ud a further soakingwill !e necssary. rr. Flati@ the se.tios on mylo-pectin sohrion o givd on p. r,r, dd &y at D. Li binging the rtrrios dona ion x,vloe into water for staidag it nay be fo,,ir 'h,r LneIb D"y b,ee . D in,o .tuI Dr(6 ud w- -*,y. ro p^'*t ! b b r i , g L f c w u e t i o o i r L o r l l e r e ." n d l l e u r b . o u e " b r t x l . o h o l . T t e ! idto r % low vncosity celuloseniftare in equd larB of absoluteethyi alcohotod ethyt e!Ld. Leaverhe sectio! for 13 niuuto. Take out aid hold fte stidein air. Wat h ir @.d!tly dd vln the fuid hc neuly evaporatedp1a.en\e stideinro Tlli, wil sel thc e1fuloq whtcl w;,! reuin- or the slidc, ud 7o9loa1coho1. a'hicL will hold dr ltu intrcr, Etil the celutoF is rmoved on rle way up by lrinsnrg !!e k tio6 rhrough edryl Jcohol od ettryl edr*.n* abrclureic.h"i_

Difrdb Objsh

r j,

13. lt is essentialto havc a very slrp ltife for cutting section ofthe votebnte eyc enfte or of the lm a1one.Ia this rspect rhe nzor bladeholda o doigned by wigslewollh (p. ra, hay le ucd witl suces. DFncuLr OBFcrs - Ox SN Mano.lid skin lEs lou hain co*imo* all of which mv erF dificu[ies i! cunirg: whire6oror. osu, hi. com lled ri..uc,ud el .dc tb,e. fte prcpomou \"ry rrob .rndl LoJaj,.Jr i! d'j-d-r regiob: i )d,J, nd €e. A( vi,ti aloo{ 3il dirul li$res skin Lom yoeg dirul js sier !o chr r,tn skin non old dinals, md ifrypical secriorsarerequircd for clas purposesskin fron young oinals should Skir is r*ually torgh, od evcn wilh rhe nos! c.rdu1 tretmtut ir c]mot be cur into section bclow 8 p wi& dry 6e. rron 8-r2 p good sectiou may be obtabed It i ssertial to se . vely sla!! hnre, &d s rhc krife lecomesblut €y quickiy wLo skin is beiog cut, a tar bladeholder L reconndded (p. ra4. polysdwd n alo rccoonendcd because its lov nehiDg point of 38. C hardensttre tisue les thu higher meiting |oint edes, and doesdot case rh€n to contractto the sameextot. Bouink fintive is to be prefeued. Very long inilhrion is trot advnable,,nd x'itir ox skin thc optimun time lies bcre-d J 8 ro.,.. )rirrilg i\ srrobgl) recomend,o drrinq Djilhbon, b'c-u.e rl.isd"y to,lcn +e rire b, $ bucb asone nird. The roond skin i5 iniltrated dr edier it vill be !o cur, ud it should be into wu wfth; de wee! ofi&dm or eadir, ifpo$ible. Two or tlree da)*safte. txrion The placins olrLe sLiDwidin lhe block rcl.tive to rhe ni.rorohe l
r56

S(tiotr C, inz in Mnos@pl

vrons b@se wlo lhe stjn is clr .ndwys b tLe knic it vrinHej b.dly dd mt becone dehnin.ted.

I

Frc, 63. Ox slir in wr bloct-wrg

trient tion

2

Frc. 64. or sknl i! wx bloc!-ftong

d ftc. 65, Or skin in w! ll@k-wong

Wronc beaue de full leosth of th. d to lh. k;fe .dgc, ilir s-!r*nt ploducirg sce$ive iein tleby

orierbtion

-r

d rny giv.! lrdy nsLt 6-rs. point dring cutilg the knife Eaiva only . p.t of the 6mL!gr! Bisbe oa the ,bi. wrotrc bmure the b:ir n i}e Ld ptrft ul the slu to be cui TLe hdl, $if h:n5 ftr tLe nlpd I'rt of thc wax ro nss r! fie tniG prrc! lhough rd rb! dDler ribbodng difiolt oi

denhtion

Righr. bec,G lhc ,Lr! plernB le$ io tbe krife ri ry ptrd ot si5tre tLe mt, aDd be6{s thc lans ar. suppote.l by tn€ sLh xnd tbeleforc do rc! te. tle uppd prn ofihe wd

ftc. 66,

Ox J.i! in ftr

Uoct-risht

ondbti.n

Dffiaih oliLts

1s7

rough, and aeratcd.This is becrlse yolk grmules, rlrricular\ tlose in drc ripe egg, re hard, denseud impurcablq od who heatedin conpletely waterles conditions, a in drc parafir wx techdquc. they tr$trne a hardnes individually tu greaterthu dut oftire biltration mediun. As r result of ths they novc rcadily asthe biltrated llock n bcing cn, od to suchu cxtcnt thrt they lecome complctely detachedfrom rhe s'd. The longer $ch naterial is lieatedthe larder it bccom6, d dy attem?t to obtain Letter sectionsb). frolorging tle h61[ation tinc will be follo*ed only by Ior most *.ork on yolkv ovarlcsud eggsSmirh\ fixative x fourd to be good Snith" fa nol-dnhtohdk oJ sm. Potsiun diclton'te (K,CrP, to m]. Forn.ldehyde40% 2 5 m1. Aceric rid, glxcial roo ml Dnrilled srter Maheup inmediatcly before!se. ILx for r: :+ hou". wash for n houn in tuing \ratcr. Storein fornaldehyde 4o%, ro i ., w.ter 90 n . r. Bring thc ovary $iough thc gradcd rlcohols to qs1/"alcohol 2. eJ9.; alcohol ud pohester v,* cqual parts. 3. Pure tolyster wd, at l8' c for 3 J hous Sdr or gotly shaket]| beakt coni.i ng the slecnNn. Chmgc the wax once. a. Makea block(!. lo). 5. Allow drc block to hldd for 24 48 hous !i a hom temlcruture of r7-2o' C. The loogcr it L left the nore solid ud lud tie llock will bccorne,lut at nvo d"r. ir h .rJdbe {tlire llrd eno:"l ot o p .er^' 6. It is d adBntlge to lse a ruor bLdc holdcr for.tttins thcscsectlons 7 . c t r J t r r . o . ' . - . p * d o fa o L u .1l 0 J o a : , h ( a r t r . . 8. Ilalten on .nylopdin solotion at room tcmleratrre, ud dry ovennght at roon temperatueor aboutlo' C but Dohigher. Dnnculr OBFcrs - lNsncrs The prescnccof m exceptiomlly tough, inponerblq ad insolublc out* coveriag nakes dre penetranonof isects by any nuid o ver,vdififtft nattct. Tne lroblem is funher complicatedly the presenceofair iruide the lody, which nakes the n{ority of inscctslloat on fLrids ruch d .qucou fiutives- The resistDcc to pnaetrationof f-xativcsis incr*sed ly the piesenccof a wat depositon the sufacc of nany insects. The hlstologist dd the nicrotomi5t nust ilerefore employ methodsshich will enabL n-rrtivesod otner fluids to pmetrate to thc intcdor. Various iluids have ben nggestcd assoftoing agene fo cl*it or tmed polyphenolsht tot onc is ro11y conpletely succesful.All haveto be ucd rnh care,in cascthey get into dre j!$e.ior of dt imcct and dmge the tlsoes there. On drc wboie it is prderalle to attcnpt to solvct1r pe"etration Foblcn nechanicrlly nrher ahu chenically. To aid thc variousfluids the intcgu'neDtof dre i$ect

rJ8

Sedian&ttisg in Mid^@fl

shouldte eirherpdckcd witl a sLarpncedleio severatpta6 o piecc sLouldbeshavcd avay with a lew saferyroor blade.Ifdb headis goitrs ro be serioned rhe brn&bt6 slould be removedby aetulJy cunbg r-bo o;,;d di*.ti,s o"dto -d E " rci$o6.Itrhe prn or prts .equireds senlou cu b' dn\effd o;of r}e dinJ this sbouldbc doe. teo e di$.xl d;seclionofo bou o. ro i5 wonb $r dne s!dt. The ue of hor 6xativs strd: d 3ori4 K.It or Srsa i, rccomoded in iomc oo vhse mechanicalteatnenr d oudiftd cmot be uriliz"-d.The hearshodd nor be tako above60' C. Aft.( a shon prelininary isriotr it is 6efd to divide tLe aim.l so lhat ir^tion nay be continuedwith opo s!rf.c6 for the iltarddse oforhc dti&, 4 vell s of the [r-tive. To pr,e " uhore inea suct 4 . hoey btr ( 4prrtr.trya) iflo trldve ud fiom tt6-oD ro Lep ir earne xo,,g\ou t\e re( offic prcelK hould tfud ro Do!tug Slodd rhe ?eru requnedbe dissectedout ofthe insect,no difrcalry wi !e expdimtrd in iDfimnon rd rufting.ud for suchrcfi orgs s dr g:r pot,erer wu n ,uitJbje.wili a.iod ia6jrhdon dne ofJ 20 binl,; depodrnqoo rhespecino.rf the isccr hs to be tepr s uhole s lo$rbi a hdd i!flnrtion necm ,uchn ercr ws shoufi re wd, ed if iiat 6ils rhe celloidi! nerhod shorddbe used. Who d insecth$ been6ied od brought into 7o"l alcohol i nay be fomd that b-in$g Inc speciho dom to witer o\ ml.. lhool cry.als 5 gr., may irJue o apprdrbte roftmbg -! 6e ilre8utur. lr shouidbe l.fr o,em ghr in rhr j o; rl,rlol solution beforeir; rakd !p tlrough the alcoholsto esterw* Iqjection of fFrive i qost scftl. A tDe hylodeeic na{le should be ued od suffi:ientnntive shouldbe gody foied into the isdt, virhour sing gre.t plssue. Tte whole isect sholrld .lso be placedin the fative foi aboft an hou, and it may t-hm!c parLlyopaed up to pernit rle ercluge of 6xati'e and oJer 4uidsLter. A fter the ?dtial fxation by injeclior rhere n lss iikelihood of the lody cootenrsbeiog fored out who the icct or larva b cur o?@ Gloss^Rr o! Tevs usD rN MrcRorow Ths is a terh prolosed byJ. R. Saker ofoford ia !l&c ofthe older ton 'cl*iry ag@i. Thc old tem cameioto gderal nseb@dsc the fluidr which wde usd prior to pl.ciDg whole specinm in balsd had I high rc&acrivc iDdc& e.g. clove oit R.L r.J33, dd s : .esult the specind hdne l6s oFquc. Tte clmge to tus ld@cf gaveit cldity, od aftcr it had bed wel pcm.tot with sucha fluid ir vs saidto be claed.'rhc tm cl*iog agot *as agplicdto fluids suchs *l.lde bdoE spedmmsvcre takm hto Ddarlmvu, If aI flui& sed belore rL specinm is dried into vE or celloidin or u@-torn,ldehyde were 6uids ofhigh lefractive ind* thde vould !e litde objecrionro thc term cledlg agoq bft to&y drh wd solvdr suchs efr)'l@e glycol m@oethyl elber, e6% erheol, erhyl phdplatq ed buryi phdblate; with elloidin solvots such d ethd ed .lcohol, ed ahyl acetat ; vith .esin solvdts such as alsolure e&dol, dd @rer, tie rcfrective idices :re so low that the speimenis !o longd

Gta$dryaf Tons

\5e

clearcdin the otd sose ofthe term. Ar all suc! fluids precedethe htltmtion mcdim, 'dtehe&un' hd nuch to .ohndd it lt a lsed on freqtert ocmiom in the term

Bri6-rp somc w*es show greateradhdive Prcrenies tlian othen. To somcertot this may bc hfluenced !y the roon tcmpcranrre,for tlc horter dre roon thc softer becones the w* being urd, md thcrcforc the greatertodcncr for it to srick to the knife As r scctionis being cut thc Lnife day Lod arvayslighdy fton thc block, od in additiontle !1ockieelfnay shov sone conprc$ion Belorvthe cdgeofthe nicre tome kli6 the llock then expods slighdy dd a r tesult it tolcha fie ktrife edgcor the way up. thus 1e#ng a slight depost of{ax on tle back or tle lnife edge.Each ucecdi.g o,t I't1d. up norc md more wax utjl thqe i sunicienrthereto interlere with the cut block face; to icore it, dd to roughet jt, thus lroducing rhpled Fctiotu, dd loor ribboning. Build-!! shouldbe tornc ln nind particulady vith 1ow-ndting loint, fine
Flc.63,

Cro$ section ofmidolone

tniG slEwing war tdherjng to the rer. bevd

Secauseit is hiddo t&iod tlt hife n is Gequent\ msuspectcdmd nay lead to the abodoment of a block or to the postPoneoot of cntting oldatiotu becaueoi the nistako inleroce thrt the room is too hot o. dre lnlfe is too blsi to lermit

Thi5 reG6 to lhe ditrcrflce whi.h oi'ts io dre ncsuc-ent ofthc sideofa seaion o coopred with &e side of the block from s'hich dre seaion is cut A cut scction resting oo thc nidoto're Inilc is snaller wheu neured in the direaion ofthe cut thm is the corespondingsideoltle llocl This ,lill'*oce is dre to two factors (r) corugation of tlE sction (2) conPresiod of tlrc nxtenal of wHch the sectionn conlosed. The true comprc*ion of ay hfritatidr nDterial drerefore nay be statcdonly after the eF*ts of corrugatior havebeetrrenbved by d.ftening

Sttion Csuin! it Mito!tupl

c o m p r e s so n = A 5 % 'rc 07

'onP'loi

or'ector(

Nevdthclcs in practicethe diminution of dea of rhc cur secrionesn L6 on rlc knili is frcqucndy refarcd to a codprc$ion, dd ii cctaidy forns a rough guide o to dE suce$ oftlie kdion fttting in the givd conditiom. In a goeral way it nay be said that the more cryrtalline tle infftalion narerial tle greaterthe initial conpre$ion, rholgh rhis n nfiudced !y thc tcdrenrure ofthc roon, rhe shrlne$ ofthe lffq the hui&ry of the a;, aad so on. Thoogh recoveryofthe inirtul ra dd nesurcndts ofthc block nay be induced in $c sectior by approprateflattdhg neanres thc iniltiarid nediun which shos le$ initial cohpresior wil te Letid thd onc whicl showsa grelr deal,evd tLough thc latter nay equalthe former after llattening. The les crurnpling and conpcnon a rction odugo* the les likely it is to sho\r bloko, dislocated,oi dislodgedrissues ud ells whd tna11y\newedunde. tu microscope. for thesereaohs the niqotoDist drd hnblogisr djslike iigh conpresion figues. ed every efort shodd be madc to reducet]En. The folloving figuresndicatc expcctedcompresionsfron foui irlihFrion tudi.. should thcy be cxceededtle nncrotonnt slould eunine carohlly rhe nicorohe knifc edgeud tshraor it ifneesar; thc room temperaitue;dd rhc purity olthe war ofthe block. Cohpresior nay be rcdued by sretchng dre ribbon 6 it is leiis cut. The maxinm tdsion shodd bc cvrted upon the ribbon, consistor with its still holding together.

comrresion hefo€ ltdt;rirs.

I*d w2x 1960M,?, 46o C Polycstr $aa ?oho M.P- 33'C r,5oo polyethylde glycol M.P. J2" C

of saxes me.$cd Room tempdrtue

20' C

e.7% e.ty.

The midotomist shouldn*er fall into the error ofbelieviog that a lnife freshfron the rmke! is , sharylnG, dd thar ifhe firs a lrdd new knile to the dicrotome md still gee excesiveconpresion rh.t su.l .ompresion my te due to olthing bot the knife. TlI is faulty reaoning becauenary a kdfe cornesfrom tle naka sh*pened or setat sucho oltuse augiea! the edgednt greatercompresion is obt:hed with it tha with a lnife well honedby the niootomisl Whd r s{tioa showins conpre$ion is placed rpn water ard warned it will ga1cml1yincrece in ue m some6to1l This incese rowads the blocl rea fron which n was cut is conecdy ca11ed deconptesion. wher the section .rea exceeds thc block areait shovr eposion.

I! iiis in$rmdt tic novenent of the blocL pxr the ldfe edgefollow the sane path loth domvr& wher the seaion is being mt, ud llwdds whfl the block is being retuned to a position above dre knife rcady for dre ncxt scctioD.h such a rysten the block psses the [niG edgetwice to every sedioD ot, ud I hidotome usingthis bethod is refened to asr doublc pss micoiomc. The dis:dvmtage ofdris netllod ofblock reton is that rhe block nay pick the sectionoF the hrife edgeon

flJs is the fat ff6ce at tle exttene edgeof a nicrotome knifc. lt rus froo one cnd ofthe hrife or hladeto the other, dd whde it mets the level ofdrc othcr side it foms tle cuning edge.The rgle fdned by the two lacex or bevelsis calledthe b*el dgle The wider.he dgle the sronger the edgeofthe lnife md the tongt it will 1*t, bnt the nore likely i is to producedrved secrio$. For ultra thin sectio$ for the electron midoscope ruor blades nay be ground to approximtely 5o' (Sjostrud, re56) o! broken glas hrives nay Le rcd with a bevel ogle olabout +s". For generalsection cutting fo. the l+ht hidoscole a level mgle of:r-:t' is cormonly enpioyed. The smaler dii rgle dre nore lilelr is the knife edgeia vibnt€, tlls prcducing sctiors wifi a nnmbo of tlncl md dm sdpes r-os them

(fic.a8l Holow groud nicrotone lnives are nade to be honed without the addition of a netal back. N*enheles i! wil le formd in pracli.e tln noy such lnive ate reeived from the ruken with der eddenceofgfl ing od settingd ifa back had beenused.THs is beca*e tle ldife sharyoiig instnmenti ued by the naker do not permit the back od the front ofthe lnife to be grced, c ls tie caseduring honiDg. sometimesthe bevel mgle of suchknivs; so great dDr ex.6sive comprssion or cullry ofthe sectionmay te producd when drey are ued for the fi.st fine. A lisht honjng will sooi !cdu6 the bevcl ugle, md be*o scctionswill follow. rus is of sonc inportance where a microtone is lcing uscd dnch doa rot imorporate a knifltiltiry nc&misn. The breadti of the bevelha a !ruing or the eoc of sectioningdrd ofriLboning. A Mror bevel sucLa o i-r.o rrD. eDaues&e sectionasit is beitrs tut qdckly to ecape the friaional draginpoed by a large bevelsuch* one of:-+ m. This lcads

162

Seetifl Ctldb! ii Mctosotl

to an esid sliding movcnmt ofthc secrioDwhich lcduc€st\e ruLu ofri6bonine dificulties. A Mtroq bevd E rlerefor . ddeble featue of uv nicrotome hife. witl ghs Ldva, wbere rhe berelsue vea L{ee eld fom rh. wbole side oflhe knife, bicdob bewm rir sedioD dd tte gtrs;urfee n ,educed by tufting rhc Ttc fe!, or 3 .ttd kdle sloLld br legrdcd defuly a \onilg pro.eed! beod. wid thitr tud ci€p !dv6 riev will ir&rte wheGer rhe koG hd bftds or ureeen glindings in it. Sh;u1d su& in;qralitis prove to be lrominflt rhc lnifc shodd be d*cuded o retumed to the suoolier. n/tuh pd,6! ws, tholgh ;oi with esterwx or potyesterwaa rhe frictioa vhich oon dullg rhe pcuge ofde blo& ?sr the hife edgeud bevelis suficior to podue a o\dge of staticeleariciry T[tu may calse sectios od ribboro to adhere rith grear temcir)' to the knife o! to lhe Djdotome. A nmow b*el redre thc fiction aad thereloreredre the inrensity ofrte ch.rgc. tuach theElge Thc edgc ofa microtome ldfe my b@one so Uur tlDt it is nothiog brt part of 6e cncmferoce of a cilcle vXe! sud.Iiife ii Dled rr"r " h."; i" ""h. bruer tl* ir ir r6Bn8 oa rle b,cl Dd thc ftoDr fets, 6 is tiicse v bo ir is bebg honed, the ftue edgeof tle lnife is not torching the surfae of the hor at all, ir is raisedfron it, asshom in rhe followiry diAlu:

re bevel <-future

edge

hone !4. 7t, Trueedg.ofrTife not ,tt sched duins horing leforc the true edgeofthe knife is reacted e strip of hetal rmiry alorg both 6ce* h* to be rmoved. Untiltle edgeis rea.ted no inprovenent in shatpneswill wlcn a klife is ftry llurt it mst be $o&d etil the rmo frot fre* agair net at r cled dgle. Tbs i caled ,resettilg' ile klife, ed in sooe ases is better lell to the kdfe nanufictua, but in host .d6 n hay fc donejsi $ vell itnot better by dy competedttec}licid. Nbbon Thn is a string or clai! ofs.ctioro whicL is produed s rhe setios alc cut be@usc @h sectim adhem to lhe previos oc. The conditionswhich aref"vouable to dt

ClNsay ofTms

6!

production ofribbom vlry sodirg ro th inilnarioD nedim, whi.L in tum nay bc nodifed bv tte tuilftated soecinar. In gedal wd o! wd-like trdia ribbon better rl!tr amorphols tnedia bur tlc optimu ribboniDg onditiotu for one inilt|,tior Eeditlln miy le quitc diffeledt tu ecb s*tion js ot rd sLrihs $e previou qe.tion lying on the hriIe, de uq ofedheion is daly tlar produed by lfe ltusn of Lb. .o-ioo ri-., *e *,.tno'. Thc if a blocL v;th . Ace ro x ro Im. is bei.c @t ar ro p the rdhesire ars is .edu6 this 'bpJc( r€ corspondingly redu(esLhe@prdry for dhesion, Badly trimmd block ue a freqqot aue oftrouble. * shom below:

I, fl.[J"D.

F&.69. N.- r .o4cdt hilmd

blo.t: ros. :, 3 rd 4led to poornbboning

Block one shovs the ideel trio. The othes show redued adheior rca beese of ' A blut loiie js ooth* nain ouse ofpoor ribboniry, even when tre bbck is of the section s it is being dt psfa-dy trimned, $ shosil below. The ffiiig prevens nainuioed adhesion,nd tends to separatethejuncio.

Ec, 70.

S€.tiotr b rcdion adherde

Educd

by sdion

dfrtuE

h this 6e rhe sectiotu?art\ sepmte alog the fu[ logth, so rh* de adh€sivc ra inst*d of being ro nn. x to p my be rcdued to ro mh. x 2 p! whi.tr in the givo conditioro mry be irurficidr ro prod@ea nbbon of more than ro scctioD3.

164

Sdtiai Cuuihgin Midoiopl

It nay be infded from the foregoing that the greater rhe de. of adhesGr the betttr the ribbonlng, dd t!.t in lairicdar serions cur at 20 a lron a block vould a&de bettd thd sectionscut ar ro p. Tirii vodd nor n€e$r y te trre lec.ue it neglectsthe fact tlat seaion adhsion is regulatedby rhe roon temperauc, or by the soinss olthe wd relative to dr speedof cut. Thtrs ir a rooD rt ro" C sNtios cut 1}oh a Uock ofpamtFD w*, M.P. 52' C, .r ro I do not adhqe ,s vcl to eaci otier asscctioqscut a! J /,. This is l€.ace ar rhar tenperotue tle paralln w* is radrerlnrd and dre grerter idpad dea ofthe rhicker sectior redststhe nnir&t defornation wlich uito cad sectio! ro the nea1.For sood n b b o n i r g . e a i o no.r t b ' c r t a td \ p e " d ' p f r o ? r Jc r o t h el , r h n e $o f , h eo r J r - r r i o h rde,IJn d(] a r-qLi-cdby tle !L. knB" o[rl'( ,ection. It is edier to lnite rrvo sectiomwhidr are thin than two which are thick od tle cutting spee.lpla)s an imloltdr pad. Thus if rhc cutriog speedwere abor ro per minute ud this gavegood but not exceptiorDllysood ribboning h the givd cooditions, dd this speedfrs reduftd to 30 per nnrre litde nore rhd nve sectiotuor so would .dhde to one aotlcr lcfore tle rilbon broLe. In such ciromtoces r dtting of r2o FctioD per lninute would give ex@ller nbboning. The hd.1d the innlEatior mcdium rhe thnuer n$r the sccriod be to lroduce a rioboo. \ de", .Mp c ol rln\ La b Lhebeh n .u. of ncrlil polyr-i"cryLe. Tlt reiadvel).hdd. clcar, morphots plstic will not ribbon at all wcj1 at r a, but if the thickaes is reducedto roo A a fair ribbon mav bc obtained. Ior good ribloing tluefore the rin ofthc block; the cuttilg spftdi the thiclnes ofthe section;ud the innltatioD nediln lurdnc$ relativc to the room temperature TUi Jlbe c 'riuUy col..de ed. r,odirior to the si:,-pbe ofth. Llife.

This is a sinsle sliceorbiological naterial gen*rlly produ@dfor subrqhr stdy undd the ni.ros.ofe. tr/ho scctiom* producedin w* or in vd-lile iniltotioa mediathey adhercto onc motltr and forn a long d1ah or ribbon. The tems 'thicL, thin, ultra thiD' de vagft leaue drey may be Nd qidr difcrenr ne&iigs b), di{: ferot leople. su.h rems should,therefore,be avoidcdifpo$ible or they should be supplenotcd by r dhect statcnot of thc thicloes ofthe scctioasbeinq discussed, t l u s 8 p : o r r r m r bo " p : o r : o o A . Iive plmcs of k.iidnis of tisucs ae comonly ued: r. T.dsveac scction(T.S.) which is ;scctiof, nonldly cur rraseercly, tlut is ar iSh rgl . to +" toDse o ' c "'gu or mJ or pla| rcm.acd. 2. Sasinal, or Lorgitudinel Veirical SectioD(LV.S.) which is cnt par"llel ro the long dis of dr specimendd in r mmer suchthat it will show both tie vmtral dd dr dosal regionsin rhc secrion.Ih a rar sucha sccrionrvotld pas letwear &e eyes,tuough tne nose,backbonemd tail to thc venffal surfacethus bisedine tle uimJ rero,J1. 3. Frontal, or Longitudinal Horizontal Secriotr(L.H.S.) which n at right mgles to the egitt l section,bui is alsolongitudinel.

GhssatYof Taw

165

4. Tdgotial. This is a sectior ot alorg 6e lorgti of a crlindrical spedmenin such. vay rhat the linc of tu crt foms a togeit with tir sortua ln uy cylhddcal specinm such c r Pldt stem tdgenrL.l sectiom may te few ir rrn'b- lcciue th. deper tlc crttirg goesthe greater the tddency lor the seaior to beconn radial. s it is whd the cut finxlly dchs tlre centt oftle srd. In sectios of tinler it is d esy oatt t to oblaiD tangential sectios beeusemost pieccsofwood for sectioningare derivedfron hrge ttees. 5. Radral. Theseue longitudinal seaiom ofq'lindrical specinm and dre line of dre cut is alongthe radiusofthe cyloder' In seneralit is esi€r for t]1e6nal udertmdiag or redtug ofsectionsifthe direction of tle ottlng plue lr tdy in the dte.tion requi!€d, becauseif obliqre sectionsde or dotirer grcai diliculty nay be ea?crielced cut rhich arc not traly on one lle in codtructing tlE tr@ sfatial aragencnt of the required orgds or regiols.

whfl . sectionis bciry cut the block psse the LnG edgedd n lien retuEedto a position aLovedre knili readyfor the rert section lf a nrcloiso is enploved bv *inch d'c block on its "p*rd jouney s c*ricd sidewrys ud uP{dds in such a r l 1 f u + t h r " - q : i t ec L : o i ? k i i ' c " o g " r b d n P . 6 r l r r n l c r o r o n - k n i f e e d g e o " l , o n . ep . r , , ' i . l r i . - i n s F J o u r l c )P r r f i e e o g c d d r h e r c l o rre d i . r o l o m e *\r." mbodi- rhs qpe o dov.rtut i' .-c l J "r.le px" nicrorom :odrh(\ni]-eFJgenl\,r t h e r d v f l " e e o f d r e i e . 1 : o u m e 1 b e i n *m ' d c - q , ] rhe e.no" yin! o. the loile ig' cmor bi "nied up on $e eru ils-block Thr o f ; o u r d o o J o oA b e - " s e I i , o r v J r $ l - . " nL i r r r n i n n r o n ' u e b e i r g o n e q .o d v h a p p o .r b " rs e n " t l J n c . r i o n . R t i t d ' , h , K " b , c - o f r b eb o c t o D Lhi' L l , eb l . o o o k . . l i g l ' t , s e " d o . - h n g ir "; r'.u;-,.".,,"r.,hee dd i' .e-rion rhe ldle edge Lhtop of m.y tcs ,c,oa roe Lbd'de of rLiebr , ! ; ' " I - . " " " g r ' ;b y 6 . " l * k @ , , d h j d r e , i I t | e n i c r o r o m e i , o ' d o u b l e

Thn is ar abbriatior for drc cLuge ofsatic dectriciry which is indued whet "irg Lhe.utring o'a a urrffi qa block pr.e rhecdceof- ni.roton e knJ'J o chargcd;Lh m"y becone oi cnmg Jr ribbo' rftion. A. " re'Jr ofir ilcoon it. tighdy !o object ed sticks to nemst 1t flies tle eledricity dnt Thc f;lloFing hedlods tuy rc&.e t]le sevcrity of fie cotrdltion: r. 2. 3. 4.

Breatheon tle blo&. Placca steming loql ofwate! nearthe midotone. C o m e ( tr h . d . r o r o r e r o , u r e ro r p cr n ' l a g i e e o l c o P P e"r i r c . P L c c . N c ' , F - s t u tc l o , e! o l h e l n i l e . l l [ i o n j / 6 J r . j r r d d t r . h , r B ed' e

The simplestsoiution is to use ' wx which ho bea designed!o re'i5t static

r66

Sec:tiorCrtting h Miaoro

RIFBRX.NCE

SOOKS

arJE,J. R. Ptincill es o.fbialogi.dl fl i il ot..hti q" e k..tqe Nt6 M the ue of the

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Chudil oxford Medicd Pub.

CiJrdoN, H. M. ind I&cE. !. H. coN, H. J.

ColrDi$ion for lhe stddardiation of biG Ptu ti cal elecran bi d oyop y Mioosoti. te*niqte ir biak

aid

Willius

dd WilLins

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C. D. and

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I'he handlins of chrMoenq Hi tol ogicd h ctho t1s Jor ehtah aIosistt

IateBcioce PlblishcB EannbookaJb6h nbatethfliq,e me bidat8itt'sJotmulftt Andsuide Prdai&l nMral o.f nedidl tud Hill biolagicdlnatuk! tthiit .t

G@, E.

Univdirt H{]MGE

and Haws

Inttodtctiot ta elerttunhidasap! Hinologl Jot netlndl tt4dets

of Chi.ago

McGraw-Hill Orford nediol prliMcGa*-H

LE-6, R. D.

Pari! (cautlitr-Viilart Mrsrov,

A. A ard Midosa?nd k.hni qaeJot z aologish

cambridge U,nvdsity

Hittochehishy, theary tud apllied Histolagial techdq"s fu ekftM

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Lt4

Bridg@d,

rn

clanorg&,

Im!6ia1 Chetuical hdutdes Lrd, DystuJE Division, Mdchestd, DiD.thyl hyalantoinfomaldehyde Rex Cm?te]l & Co Ltd, 7 Idol I!ne. Londoo, Glyco ?rcd!.$

Hons, Arkaas rbite Carlotudb, and ar@inim oxideftsin

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The Brirish Ddg Hoses, Poole, Dorser, lngled. Norton Abrasives Co, Ohio, USA. crtoed@ Co Ltd, Tefold Pdk, Mm.Istd,

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The Biitish Dtug Hou6. Poole, Doset, EngLdl Flatid & Gdett ltd, Oford Ro.d, Mdcheler Edw. [email protected]

&

Co,

48 ?etonvile

Ro.d,

Iishu Sci@ti6c Co Ltd, 7o9 Folbs Strtrt, Pittslag, Pemrylrarir, USA; md 898 Stjdee street, Montred, PQ, Coad.. Germl Biologiol sulp1y Houc Inc., 82@ Sourh HolaE Avdue, Chi.ago, Ilinoj5, USA. Edw. Gn Ltd, 42 Ulptr Ricbmond Road, G. T. G@ & Co, 136 New KiDgs Road, London,

sw6. Howrds of tford, rss*, Englald. Ga(lner Labomtory Inc., Berhesd. 14, Mich., USA. Ni?a labb.aroiies, Treforest Ponrytridd,

Polyethylene glycols dd 6tds

The BriGh Drug Houses, Poole, Dotri, Etrglad. waford Chemical Co Lt4 22 Coff€rfeld Rcd, cm.l Ro.d, London, E3. Glyco Prcducts Co hc., Edpne stare Buildirs, G. ?. Gu

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U.@-fomaldehlde

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INDEX Choie ofin

lfttion

Dediun

Alcid blue wdoline re.l

roh@di: (lerrinC rgMB)

Broct nakins, rruesiye tlpe codpresior

of *ions

43 hrdelins

tine ofblocL

43

!ye, v.fr cbnte, inilErlion

C{latos librte low yi*ority ?3

wit! ua-fomddchy&

3,8r,34,88 33

SectiokCuftingi Mi.ro{afl Pbrccovary Fl*tding,

aeaE$6

wax sion. r.a-fom:ldehyd€

lotul

dlcium

hildrion

c,dniDn

r6ia

without trtion

Jig, for cuttins slas knivcs for ihi+ening szor blads

I]ddns

Hvdronl

bfltution

ol annoco*e in w* ,es in wu or s&in ir w* pm6! w wd lotye*r

muD!

i! Ex$

Bedinn,

eeech

Mi@scope Jides, clanins

34

33

Polrster wr-.dr. dlying enpeaiw ?olyetlylde sircok lntedtrg sctios

mtL sa-fomaldeLyde Porosity ofprc.eins

33

3S coml,Bion

of &dioa

43

Rizor bladeLoldsl tronbc R$in inilhtion with

werght

ua-formaldehy.le brdflilg

d@ of blo.&

37 33 Scdion .&esion to slide eondar/ viiboui e.lL$ives

lgrcEw* 73

@npr*io!

Folyetlyl* gly@k lF-fomrl.lLyde wEr loluue w.g w, frurs duina dting bdel di$tion one otr.he sL&

of stiols

Sdios, wd, 6dt5

5,1

S€dioB, wr,

si-'ilbn68

UFr-folnrld.htd.

fad!e.d,,.

f&tiotr

Sodiu.brcn ti$6 Soabi4

rnd

33

gle@b in wx block

Tisu6, io softer in w Tnining of nifttonnb

Ur6-fofraldehydc

r6in-@rtd-

6in

block

witL G-tom.ldehyd€ wd inilftrior vi!ho!! 6*tio!

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Cutting

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The Cutting

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Metal Cutting

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Acoustic Microscopy

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The Cutting

Cutting Edge

Cutting Edge

Cutting Room

The Cutting

Cutting Cords

Pharmaceutical Microscopy

Electron Microscopy

Atomic Force Microscopy Scanning Tunneling Microscopy

Sea-Section

Section 8

Section 8

Section 8

Section 8

Section 8

Section 8

Video Microscopy

Confocal Microscopy

Sea-Section

Section 8

Section 8

Section cutting in Microscopy

Guide to Thin Section Microscopy

Cutting

Cutting Edge

The Cutting

Cutting Edge

Metal Cutting

Cutting Edge

Acoustic Microscopy

Cutting Edge

The Cutting

Cutting Edge

Cutting Edge

Cutting Room

The Cutting

Cutting Cords

Pharmaceutical Microscopy

Electron Microscopy

Atomic Force Microscopy Scanning Tunneling Microscopy

Sea-Section

Section 8

Section 8

Section 8

Section 8

Section 8

Section 8

Video Microscopy

Confocal Microscopy

Sea-Section

Section 8

Section 8

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