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I |I fiecEruela Use and Proper Gare of the A0Microtome

AmericonOpficol 'i SCIENTIFICINSTRUMENT DIVISION 119a1o BUFFALO,NY 14215 EFFECTIVE USE AND PROPERCARE OF THE MICROTOME

@ AmericonOpticol SCIENTIFICINSTRUMENT DIVISION BUFFALO,NY 14215

@ TM Re& U.S; ?ar Off.

Copyisnro 1942,1949, 195 q I 971,I 9?5!y Areri{n OptimlCorporation "civen a substdrial nicrotone, sood resul.s ffe Idgel\ a matte ofpenonaJ.(il b .ha-peningxnle. dnd mmiprldrirg rhe mi..o'ome. LucA , lo27 "civen a substdrial nicrotone, sood resul.s ffe Idgel\ a matte ofpenonaJ.(il b .ha-peningxnle. dnd mmiprldrirg rhe mi..o'ome. LucA , lo27 5.- QA - n4* TABLE of CONTENTS 1q75 I. INTRODUCTION 4

II. CORRECTINGDIFFICULTIES ENCOUNTERED IN SECTIONING 4 Difficulties Common to All Methods 4 B. Dif{icultiesof SpecialMethods 1, Paraffin ErnbeddedMateriat 5 2. FrozcnSection Technic 7 3. CelloidinEmbedded Material 7

III. THE MICROTOMEKNIFE 8 Historical 8 B, Experjmenral 10 c. Sharpening

IV. MBTHODS A. Theory of Cutting I3 B. Positionins the Knife in thc Mjcroromc C. TableMiootome 14 D. FreezingTechnic 1. UnenclosedMicrotome 2. CryostatMicrotomy 18 E. CelloidinMethod 19 F, ParaffinMethod 20 StaticElectricity Elimi[ation 22 H, Temperatr.rreControl 23 L Kiv or Dtaoe llotders 23 J. Sectionir:gor SurfacingHard Materia.ls(Metals, Plasrics, etc.) K. lne Kateot uutlrng 25

V. MINIMIZING DISTORTION_ PARAFFIN METHOD 25 Precision of thc Microtome 26 B. MeasuringSection ThicknessandCutting Force 26 c. Materials 27 D. Disrortions 28 E, Floating Out 30 F. 30 G. Conclusions 30

VI. CONCLUSIONS 31

VII. HISTORY 3L

VIII. BIBLIOGRAPHY 35

uHtv {6

ot &ANFOEA

{a,tARli-$ I, INTRODUCTION

Microtomesare precisionin.rrumenrs designed precision instruments. Unless very old, dam- ior curriry mareiialsinro section'rhir en6ugh aged, or mistreated, the microtome is rarely Ior exarnlnatbn wlth a mrcroscope. the cause of poor sections.

Successfulsectioning requires: 4.A skilled operator. Most failures observedin microtomy could have been avoided by an ex- l.Properly preparedmrtelial. Somespecimen< pcriencedmiLroromist. Wirh perfe* marerjal, maybe secrionedas they arefoundr manyre- a sharp hnife. and propedyidjustcd rnicro- quire extensivepretreatment and embedding in tomc. auromaricsectioning wouJd be possible. i supporLingmedium. The supporLine medium Otherwi_scthe operr(or must be able to rccog- mustmarr h rhephysical character ofihe speci- nize and correci difficulriesas they arise. N-o men and have propertiessuitable for the cut- techniciar should be expcctedto secion im ting procedureto be ased, properlyprepared material. lor train,ngcour- 2. A sharpknife. Poorly preparedmateria.l can sessee Sreedman (1960). sometimesbe sectionedwith a goodknife, but a poor knife may fail to cut, or ruin the best The objectives of this manual are to provide materia.l. directions for the use and care of AO l.licto- tomes.call artenrionro somc of the specia.Jpro 3. The correctmicrotome. Different kinds of mi- blems of microromy. to share our research and crotomes are availablefor different uses and experienccon thc scctioningprocess, and ro co- the choice should deoend on the aoolica- ordinate and ourlinc thc bisii literature in this tion. Wirh prop". "".'" AO Microtomei will field, Detailed instructions may be found in the give many yearsof service,but abusewill ruin sepatatemanuals for the microtomes.

II. CORRECTINGDIT'FICULTIES ENCOUNTEREDIN SECTIONING

This check list shouldhelp the operatorto over- it. Corrcccby turning tle knifc holder ro eive come many of the common difficulties. When the proper cliaranceinsle (fre, fgt, berw-een the suggestedcorrecrion is not adequate,the the iucring facet of rhc [nife aid the specimen. correrpondinqpat of thc manuaJs|ould be readind. as;e;ded. or|er morecomplere sour Scored,qrooved, smca-red and deformedsecrion' ces(Chapter VlIl). are oftei causedby a dull knife. Regular, length- wise scr,tches.andano splitssPrrrs irm secrionisecrronsare usu-allusuaxy Do not return'rhe microtometo the factory un- causedby , defecr in rhe knife edge.aJthough les

4 e. Dip block into a softer paraflin and trim so t+l t- Lhara r\il layer of sofrerpraffir remains I on r\e upper and lower cdge of rhe olock. f. Unroll rhi secrionand holdlr lighrt) againsr the knife with a camel's hair biusli. if the ab+ b first few sections can be held down, the rib bon will often form and follow.

2. Crooked ribbons. a. When sections are wedEe-shapedthe sidesof A the block are not trim;d D;rllel. b. Edge of block not parallil to knife edge. c. In" a.norherp,rr of ihe knife somerinie' inegularities of the knife edgecause crooked

d, The panffins at one side of the block may be softer than at thc other side, especiallyif the materiJ has beenre-cmbedded in a par- affin of different hardness - re+mbed thc materialand srir rhe mclred paraffin. e. Onc .ide of rhe block may bc "armer than thc other, frorn a radiator, lamp or draft. Ler the block cool rnd placcllrc microlomc where thc temperature w l be uniform. KniteBock 3. Sections vary jn thickness or are skipped. ght Edse Stro a. Knife not tilted enough ro clear facet or be!el, or rilred roo much, and r;.uc isLom- presseduntil the inevitable expansion givcs rig. l. A.Di/grm s}'owingcl, d,n.e dgle Jnd inc,.."c a thrck sccion. u' \..rion rqi.tnc . lrom cohpr.$ion. B.Wedds.ir.cr b.Sornc of thc clampinqsel sLrewson rhc whcn thqc h no cledoce mgle. C.Howto se;th; cl"d- bloc\ o' Inife holder arc nor rrghror knirc holder block not clamped firmly. c. Microrome.wornthrough lacl of lubncarion. or nor rn adlustment, I}- DIFFICULTIESOF SPECIALMETHODS+ d. Very large blocks or blocks with hard re- gions nay spring tnife edgewhile secrio" l. ParaffinEmbedded i\4aterial ing soak block in water to soften, use (Seealso Chapter lV F) other methods for softcning the material or embed the celloidin. ThJblock will soak l.Ribbonfails to form. more quickly if the paraffin is trirnmed off a. Roon ls too cold of pataffin too hard. o"e "ide ro expo'e.iheri.sue. Verl lirrle wrtrr rs,b\orEed, bur rhts proces.some- (1) Use softer (lower melting polnt) p,raf flmes.make\pos\'blc (urring hdrd o. rough tln. r 2 \\ arm knife \righrly by blowirg rhe e. Paraffin is too soft - use higher melting brearh or ir. or immersein wJJm (nol point paraffin, or cool blocL and knife. hot) water. 'J Place_adesl lamp .o rhaLrhe lighr and 4. Sections compressed, wrinlcled, and jammed heat fall on the knife and block together. b. Tilt thc knife less. a. Knife too dull. c. Cut thinner sections. b. Room too warm cool trimmed block and d. Knife may be too du1l resharpen. knife in very cold, or ice water inmediately +seealso steednan (1960) before sectioning, or by rubbing surfaces (3) Calcareous or silicious pa:rticles m ma- with arr ice cube, or re-embed in harder terials - decalcify or desilicify. paraffin. c. Knife tilt too slight, so facetbevel rubs over 7. Knife rings on up "r.oLe and sectionsare block increase tilt. scratched. d. xrrile edge gummed whh paraffin - wipe borh srdeswrth ruger or cot(onmorstened a. Change knife tilt to greater or less degree - with xylene. tilt must be sufficient to clear facet bevel, e. Soakblock, before cutttng,from an hour or but not enough to scrape instead of cut. two to over night, in water, or 10%glycerin b- Materirl is too hard. n 60% alcohol (Baker, 1941). Lendrum (1) Soak in water to soften (Cl. 4e, 3d). (1944) adds aniliae. (z) clearing may be at fault (cf.4b). f. Cuttins too rapidly - very thin sections snoulo oe cut slowly. Increasesection thick- c. A thicker or wedge-shapedknife may pre- ness. vent spinging of the edge when cuttiag. g. When the specmenand not the peraf{inis d. Marerial may be too tough for paraffin compressed,infiltration is incompleteor the -.11' ^.1 - r-, ..11-iJ;. specimenis softer than the paraf{in. 8. Sectionslifted from knife on upstroke, 5. Sectionscrumble and specimenmay tear out. a. lncreaseLnife tilt. a. Material incompletelv dehv&ated or not b. Room too warm or paraffin too sofr - ry ptoperlv cleatei. harder -(Cf.paraffin - c6oler room; or cool b. ivhin s6ft aadmushy, material incompletely block. 4b,) infilteredlnr tered - reinfilterfelrlt{te! and embed.embec!. (salvage(Salvage c, Knife may be dull - resha.rpen, rarely possible if material was incompletely dehydrited,) 9. Sectionsstick to knife. (Cf, also 4 and 5). Alcoholnot completelyrcmoved by clearirg a. Knife edee'knifedirry - (Cf. 4d.) b. lncrease tilt. d. Object too long in paraffin bath or paraffin . T^, I .h..naf Lnifa too not, d. Kiife edgj f4cets rnay be corroded - re- e. Subjecthard and brittle becauseof cleariog polish, fluii. Trv toluene in place of xvlene' or a e, Llean.knife edgeand rub a very lirde lighr mixture o'f tolueneand'cedar oil. machhe orl on tacets. f, \Mlen the specimenshatters aad falls out of rhe wax, it-is too hard for the 'paraf{in.Use l0,Undulations in the surface of the section, a harderwax or wax mixture. 'e. Trv celloidin embeddins.or a rubber or a-r- a, Tighten a1l set screwson knife and blocl philr mlxrure with patiifin for fragile ma- holdersand seethat knife holderis clamped teria.l. fast to microtomebase. h.Try dioxan method for dehydrating. b. Lesscnexcessive knife tilt to preventvibra- tion. (Cf. 7c,3.) 6, Split ribbon or lengthwisescratches in ribbon. 11.Scratching noise during cutting. a. Nicks iri lcnife use another part of knife or rehsarpenknife, a. Material may be too hard, or small regions of b. Useless tilt ofknife so it will cut ratherthan materialmay be hard. (Ct 5c,d,6e.)

c. Knile edgeditty. (Cf. 4d.) 12.Sections fly and stick to partsof microtomeor d. Object may coolarge for paraffinmethod other nea.rbyobjects because of static electri- ,be city formed from the friction of cutting. This e. Hard patticlesin block may causescratching. unially occurs only ia winter when thi air is very dry. (1)Dirt in paraffin - filter or decant melted pararnn. a. lncreasehumidity of room by boilmg warer 12)Crystals from killirlq fluid (mercuric ln an open pan, or burn a Bunsenburner in chlo.ide) when washiirgwas insufficient. b. Ground mictotomc to a water pipe with a The microtome is used muchthe same as ar room wne or a chain. temperature. Seesection lV, lVB. c. lonize rhe ;ir b) ar e ecrric.rlme,hod, Cf. ChapterIV, G). 3. Celloidin Embedded Material The knife should dice through the material with a slant angle of about 10' - 35" to the direction of 2. Frozen Section Technic the cut,-and the knife should be tilted more tharr required for paraffin embeddedmaterial. UnenclosedMicrotomes (Clinical or S1idhg)

Fresh rnaterial may be cut as soon as frozen, bnt 1. The chief difficulty comes from t.ying to cut better sections may be obtained after the tissue imoroperlv preparedmarerial. Adequatein- 'i-kc has been killed, washed, and so:Jblade. Lold ir aga'nsr was incomplete. the chest or body to braie the arms and makc tJre a. Dehydration incomplete reinfiltcr,re-em- cut by swaying the body b.Infihation bcd, and harden. Verv hard or densc tissue may not be cut at less c. Harden block if too soft, in chloroform, or a tha; 18 20pm. The average thickness is often mixture of equal parts of 95% alcohol and l5pm, Con'iderab'cskill '. requiredfor curring glycerine. dlinner scctions. 4, Variation in thickness of sections. Al' ,er s.rew. holdinerhe frcezineeq.ripnent dnd knifc mLr"rbe ugl'r io avoid vib'ar'on w'ren cur- a. Loose scrcws on knife or block holdcrs ting frozen sections. tlghtcn all set screws. Avoid strain on the knifc frorn unequal tightness of the knife holding screws. b. Knife holder depressedor raised by thc hand CRYOSTAT MICROTOMES while sectioning - hold knife block so as not to move it vertically while cuttins. not tilted enough to clear Iacet of cut Sectioning-when is easier and thinner sections may be c. Knife obained the microtome and nssue are tinq bcvel, maintained at the same temperaturc by refrigera- d. t(mte too durL, rion. For optrmalrcrioning rhc cryo.rrr ,hould e. Microtome worn and out of adjustmenr. bc .cr ro rhe p'op.r rempeia',r'ealJotrng suff'' f. Material not hatdened properly - cl 3c. cient tirne for iemperature to stabillzebefoic use. g. S EL, d./ing of bloc( berween "ecriop.. III. THE MICROTOMEKNIFE

A. HISTORICAL More critical tests utilize multiple beam interfer- The cutting edge of an ideal microtome knife ence microscopy'6uli.(Ciunrini arld Edlinger, 1c54; would be the straightline formedbv the mrersec- Hallen. le54r r9s8). whiJeshariening the lion of Ewoplanes. rhe cunirg facers.The rngle knife, or at failure to section, examination o? the- berreen the-planesis calledth-e bevel angle arJis condition ofthe edgewith a microscopemay save greaterthan the wedqeanele between thi sidesof both tirne and mat"iial ihe [nife. Such,n l-aedide" is nor Dossiblebe causethe inhomoseneousstricture ofihe steelre- The sDe and general shape of de microrome sultsin a sJighrlyrounded edse. The radrusof knjfe havebecome esrabl;shed by use, The stand curvatureoflhij edgewas meisured from paraf- ard microtome knife has a -edee a@le of 15o to iin impressionsof th-eknife by Kisser(1927), He l8o and the be\el anglc berieen"rhe curring considereda ndius of curvatureof 0.3 to 0.35pm facets for hnives of Amirican manufacrure r,aries a good approachto geometricalsha-rpnes",2 between 27-32', The width of the two facers Schmericz(1932) recommended rhat cheradius which make the curring edge of the knife has of curvaturebe between0.1 and 1,0pm. Ardenne been recommendedfrom O.t ro about 0.6 milli- mecer (Malone, 1922; Nageotre.l92bi Fran7. (1939) would limit the radius of curvature to 'fhe 0.1pm. 1.929), early double concave knife is rarely used now, even for hand sectioninq, and th'e The cutrinq edgeof r very sharpknife, when ex- modern microtome knife is either we-dge-shaped wirh sliehtlv hollow eround srde or pla"o-con- amined by reflected lighi _undir 100 magnific+ tions, will appeara as a very fine discontinuousline cave. T-he blarlo-conc-aveknife is used'prirnarily varyingveryins slightlyslishrly ia width. HighermagnificationsmaEnifications for celloidiri seciioning. Aparhy \1,91,2. lSgi) of io-und-SOtixaround 500X wiJlwill eivegive thiithis edgeedeeifinelya finelv ser- recommends facers be-of "niqual lingrh and his recommcndarionis ratedappearance. von'Uohl ltASTy re"o-rirend- supported-by Kiiser t1926) eclsharpenine the microtomeknife until the two and Ldw {1932,, Such'asymmeiricaledges have generallv pJanespianes ofoI rhetne cuttingcuttlng facerstacets come togethertosether to giveerve nor been required nor entered commer- iuch a miaimum refliction; his crireiion of shtp- cial prac[ice becau-sethe'incrcase in cosr is not nessis still used. Most microtomistsrecommerid justified, Tfie r.rseralso obiecrs to the inconven- a magnificationof 100 diametersfor this purpose. ience of having to ser rhe" knife in accordance Exact recommendationsvarv widelv from 40X with which sidi is toward the block. (Chamberlain,1925) ro 7OO - 1000 drameters (Funk, 1910). Julian (1903)recommended the The natureof the edgeis irnporranrt for curtine samecriterion of sharpness,and pointed out fur- hard,dense marerial, iuch aswood. a uerv'visible smoori' rherrhat rheactual cutrinq edge musr be rhinner edge is required with no fine serrations at thanthe marerialro becui Ifrhe currirgedge i, 200 diamerers(Bailey. 1937). The edsedepends, rhickerthan the cells,for instance,rhey wili be of course. on rhe finenessof rhe abrasivemiL.ri"l destroyedrather than sectioned.Ssobolew (1909) used for forming it, Julian (1903) callcd atten- emphasi.zed rhe importanceofproper hardness of rion ro rhis limicarionof sharpenirg,-Sinci and ir war rheknife temperaird thar a fin'eeige couldonJy emphasizedby Funk (t910). then ir has be obtainedb! usingfine honingmaierial been customaryro choosefire erahed Loncs.r abrasivesseparited bv decaaraliJn so as ro retain The proof of a sharpknife, accordingto Apathy only rhe firer sizedparricles for knife sharpenhg, (1912),was the ability to cut a pataffinribbon at 2pmwith no compres'ion.Thisiricerion deoends A glass plate seemsto have had the longest and on rhe paraffin a'swell as rhe knife (Cf. Chiaprer most successful use as a surface on which to lV and V, sectionD). Bensley and Benslev .harpen the microtome knife (von Mohl. 1857J, (1q38)also recommend polishing the edgeunriJ Bishop (1954) vibrates the glassplate with a mo- no rell(ctroncan be seenfrom rherclual (utlrns tor as an aid ro sharoenins. Inscead of usine a edge. They test with paraffin at 3pm and advisi large plate Lendvai illoli recornmended thiee the ooeratornot to trv the hnife on hair or skin pieces of plate glassabout the sizeof an ordinary as thaserather diflicult t""t" -ry spoil the edge. hone for use with different grades of abrasives. zonu -i"."-"t"" 1p-; = 0.001 nillimeier (fome'ly ca.llednicon).

8 Apathy (1912) and Krause (1926)used glass-Nageorrewith and when sharpeningthe knife. The composition Vrenna chalI a* a polishing ogenr. of rhe steelil rhe cJtt,nq edqeis rerv rmporranr (1920 prefenedr horizon rai roiaLinqelass lunu- and. unless rlie righr blJn;e oI rhc different Ius (l revolution per second).and F-r;nz(lq2q) phascsof sreelis obtainedir rempc'rns,rhe kniie developeda sharpeningmachine wirh a rorarins * ill ne'rher rale nor keep a good edge,-rrnits AO gla.sdisc a-nda clarnpio hold, o.ciJJ,{e,and rur; Mirro,ome Krive! Jre rinirhed io clo,;e of rhe kqife. A rertica.lrorrtins slasswheel was rec- hardnessand each knife is etched and examined ommendedb1 Long 1a,d,,. rhe method wasde- wirh ; rlicro"copebefor. Flal sharpcningr. rnake veloped turrlier bltaJldnd / leJ5,. (,rber 9J6l surelhdt rhecomponen|\ ol (ie ,reelarc prescnl and Hillier (1951). Hallen (1954) prefersa cast in proper proportions. iron lrp and Bcll , rc58) a bron e lap irnpreg- nared wirh fine abrasive ro glass platei. while pa.t experienceha. raughr ccrtair basic principles.ir t clear lrom the "bove hLtorica] A water motor and, later, an electric moror were summaiq rhar rhere ir no sbgle generJly ac usedby Funl' r l9l0r ro drivea reciprocaringmo- cepted procedurefor rakins ca;e oT rh. ,""'"- tron ror mo\rng thc stone to and lrom rhe opera- rohe knife. "on Mohl's 11"857;early shrrpness tor about 3 st;kes a minute to lighten the ivork criterion, utilizing the light re{lected from the of sharpening the knife. Malone (1922) advo- cuttrng cdge.is onc of rhe besr. Arcally smoorh, cated wide leather strops with specially preparcd *elJ-polishcdedge shou' oniv a 'liehr reflecrion: surfaces and used with incrcasiigly fi;e; e;aded a narrow."traigh1. and unbrolen Ur"gtrrline. nny abrasives.Weller (1924), Chamb;l;in (192"5)and imperfecrionsin rhc edgegrve an iriegular,inrei- Evenden(.l9J8) reporrhavine u'ed Carborundum mpr-edlire. rnd unlessihc-knife is pr;pcrly sharp- rlones roriowco Dy,l|ner galned honesor strop- ened to a true thin edge, a broad reflection ap Plng on canvas or lcather, pears.

Thcrc has been no agreement on the advisability A convenicnrmcrhod for rourinc useis to place of using a strop. Many of rhe experrs protest a- rhc knife or . block li(e Frg. 2 and .rminare gainsrsrropping while orhersrccommcnd thar. ir with .r l:mp, fie, lA. The polisl,of rheracet c.r, be done, Whcn the edseis honedrcallv sharpno bc cxamjnid *iih the t

-l-l\llluminotion" fWt B. EXPERIMENTAL Angte . ^lw "/ The microtome koife must be made of a qood r-rnrsh-durrDroc( t___:l gradeof.rccl of proper hardnes,. A .ofr Inite lails ro hold an loo edge. hard a knife is brirrle. Fig.2. Block for sppofing ni.rotorne knife (dinen- .o rhat tiny piece. of de edeeare li-kelvro be sions in mmJ, A. Posirjor fo, exoinhg 5h"rpne"s. broken out'during rhe .ecrionGgof hard material B. Position for ob*warion of poJls}. As previously mentioned in this publication, the elasiplare technique h,s h,d 'he lonqesrrnd mos. iucces'ful use "s a su.Faceon whiih to sharpen the microrome kniie, The more popular ir.rru- ments in r.rsetodav all use the glass plate or a A variation of that method. one-of tliese rs tne Am

In preparinqrhe knrfe for:harpenirs it shoLldbc .*".i".d J"d"' rhe microscopito dererminerne e"tent of damageto the edge. Severc nicks may requre rhar rhi kn;febe riturned ro rhc manu- faiturer for rccondrtioning. The alrernatrveto this is considcrablyexten-ded sharpening rimes. B bL'r expericnccwilL enablerhc rcchnicicl to de- TFANSMITTEDLIGHT termini what action must be taker. The initial sharpeninq of a new or reconditioned knife on atrv shntoiner must follow the cornplete process as oudded in the referencc manual for rhat sharpener. lt is unlikely that the facet anglespro- ducJd by f.crory merhodscan bc exactly dupli- Fig' 3' cared.and ir ls imDerariverhat rhe aneleswhich are ch$acteristic ol that sharpenerbe e-stablishcd on the cdgc of the knife, This will-faciliute sub- A knife for usc in rhe averaqebiological or medi- sequentsharpeninps gswhich will be of shorrerdura- cal laborarory should be shaipcnedintil rhe edge tron. and wtll makckc it oossiblepossiblc underr:ndcr certain con- appearsflee llom sefiations ;t 100 diametersaid ditions to simply polislilisli therhe curtingcurtine facet wirhourwirhout tfii reflection from the edgeshows only a narrow goingthrough the full process, and almost unbroken line.

C, SHARPENING PROCEDURE tn the modern laboratoty the practice of sharPen- ins microlome lnives bv hand has been larselv 1. Foliow setup instructionsas describedin the dis'cornnucd. This is piimarrly due ro t\c Taci refefencemAnuai to instali all necessarycom- that the technician's time is at a pr€mium, and ponentsof the sharpener. second'vbecause the rechnrquers rapidly bccom- ine a lo.r a't. lt is ro lonqciLaushr in 'ihools of 2. lnstallhone pLre in rheposirion as prescribed midica' re.hnology.""d oni c.' o"ly acquirepro- tor the coafsehoning proceclure. f'ciency in rhrs merhod urdcr thc direcr super vision of a competent technician. 3. Aoplvabrasivc ro rhehone plare in a quanriry .uffiii"n, ro e'rend beyoni ,h. ."d; .r,h; Most laboratories, rherefore, will either send their knifewhict' is ro besharpened. lfrhcabr,.ivc knives to an outside firm for resharpening or if is oil based rather than a paste, it should be the volume iustifies it, wili procure one of the first stirred or shaken until thoroughly ml-xed severalautomatic knife sharpeningdevices availa- to pr"ride thc p'oper nixru-c ot lubricanr ble in todav's market. The maiority of these and abrasive. instruments'have been designed to eliminare the necessity for a $eat deal of technique on the part 4. Install the knife into the knife holder follow- of the technician, re

10 5. Set the timer for minimum of thirty minutes, 9. Install the knifc in the knife holder again fol' close the cover and turn the instrument on. lowing the manufacturer's instructions. The sharpencr should never be operated with the cover open, and in some nrstanceswill 10. Set the timer for a minimum of fifree" -r" have an interlock on the covet to prevent this. utcs, closethc cover and start the instrument.

At the end ofthirty minutes remove the knife 1 l. After fifteer minutes rcmovc thc knife, care' from the instrument, clean with xylere or fu11yclean with xylcne or other suitable agent .lorh orhersuirrLle agcnt. a-d cram nernicro* opic- . 'i g " '.fr .o orerelt darnager. rl'. ally to determine the progrcssof the sharpen- cdgc. Al":1' uipc rouardstl'e edgerrrh"r ing. ligL-c. 5 ar'd o shuu .hr rypi,:l rh,-nfollo" ing rl'c Iengrh oi r.rekni.e. appe"rancc'ofrl'e Frcttunder rhc mi.roscope polis| when illuminated as illustrated nr F{, 3A. If 12. The facct should show a high and be necessarycontinue coarselroning afier clean, relatively cvcn for the full lengtli of the knife. ingthe honcpl;tc ard applyrngfrc.h abrasi'e. It is possiblethat rhe facets may be of differ- As the abrasivediscolors, indicating thc pres- ent widths on opposing sidcsof the knifc, and ence of metal it should be removcd snrcc the rhis will be explrined Lrter in this texr. metal particles could damagcthe facer. | 3. FieurcsI rhrouelr ' 2 .h ,w r he prosre* of rhe dciclopmert"fihe poh'hcdL.cton rhc "nifc. 7. Whenir isderermrned by rn:.ro.copr.er,rn; nation rhet rhe coarse sharpcning procedure 14. Examin€ thc knilc microscopically to deter- has been completed,rhe hone plate should minc rh;r rLc faccrhas l.cen prope.l1 deu.l"p bc removcd from rhe instrument, thoroughly ed and po i.hed. rnd a..y finr rricksrcmair. 'g cleaned of the coatse abrasive,and teinstalled aftcr coirsc honing have been removed. as orescribedbv rhe manuf.cturer for frnc honlJrg. 15. lf further finc honine is necessaryfollow the stcp".s or.rrhnedabo-ve a{rer firsr'c)eaning rh* 8. Afrc' insralhngt\e horreplate apply an ;rmount honc plare ard applvirrsfrc:h .brasrvc. The of rhe fine .6rasrvcjn iuffrcieiL'quantity to pr.*n'.. uf ,,c.al'p,itrc'icsi1 rhc flrc h.ni-g ertcnd bcvond rhe .nds o[ the krifc ro be comoorrnd is nrorc crui.al than durrng rhe coarscnonrng Proccoure.

Fig.5, Illusluted ab.vc is dn 'ntcmre_ diate riagc in the .oese honnrgcycle.

FB 4. AO 925 AulonaLlc Knifesndtencr Fig. 6, Coase noning comllote.

1l Fig.9, Appedance of an AO knife edgeaJter proper codse honing Nole thll lhere is just a singl€ facet, the surfae of which is evenly ground and unifoin in

Fig, lO. luusEaredhere h M mirial sbee in lhe tine lLoninscy.le. fhc kcond otling iaFt is beginnrnglo become eyid€nt near the cdse of ihe lnile Fig. 9 Fig. 10

Fig. 11. The fine facei is now clearlyappdentin this ''ad!rnce slage thotocrapn ln honrns,lhe fDe honinerchon proceds flon thelront edge of the knjt€ ro fte back.

Fig,12. Fi@ honing comlrlcte, Note the eveneidth oi the fine facet (from end to end oi knifc), SmaU nicki I'a\c been ,arrfdcrorily rernovcd Mrcrorcopr 's - "n.ped ljner on rine lalet arc a no-ncl re.ulr of pofisningaction dnd do noi aliecl ctrtting qualities Fig.

The procedure for reconditioninE microtome advise you of this fact when your knives are knivei is eenerally identical to the 6risinal manu' arereturned for .econditioning, fact urrnq ;roce si Repcatcd sh.rpcnlnqof thc knife will iend to exrend the *rdth of tTrefacet, After using the knife it should be cleanedand and alsoincrease the facetangle appreciably. As wioed drv bcfore beinepur away. ln manylabo- srated elscwhcrc in tiris rcxt.-the faccr anglc on rar'oriestie knifc will iciv in -puteo6d condirion until lnives ofAmerican manufactr:re can vary beiween it is neededasair when it is back in rts oox 27 and 32 degccs, To reestablish the reiation- afrer rhoroug[ drying. ln'orhcr ]aborarories ship bctwecn ihe faccr ansle and wedee anele of whercrhe armorpbereis morecorrosive. it may thi knife. the manufacrurirwill grind'rheicdge be necessaryto oij the knife in order to prcvcnt anqle,drereby reducinqrlre rhickness of rhe knilc co osion, A good qrade ol lidtt, neutral oi ar-the hecl.'and also-decreasirrgdre dimensron shouldbe used.-Undeiumrsual c6ndirions ofhu- from the heel to the edge. Thc re-ductionin thick- midity or corrosiveatmosphere some of $c regu- ness of rhc knife at the heel on somc sharpcnets lar antj+usringgteases may be requircd ro prorecr will have thc effcct of presenting the knifc at a r'iehrlv different anqlero rhe honinqplare. when th-k oicurs a wider lacer will be oroiducedon one Glassand diamond knives are used for ultrathin side of the kni{e thau on the otfrer. This will in .ecrioningand glassknives are u'ed for fine cur- no way affccrrhe currirgqual'rics of rhc knife. tingon r6raryriictoco.e" rBehnke& Rosrgaard. but simply mears that so"re srighradjustmert. 1963;Clevenger, 1964). will h"vc ro bc madc wirh rhc lnifc in rhe mi.ro- "Of tomc knifc holderto establishthe proper clearancc Marsh (1878) observed not less importance angle for cuning. Some vendors will recondition than the microtome is the sectionknife, to be Inivcsbv erindjneon y a quayrrerro onehalF'rch used in coniunctionwith it. How perfect soever b,ck f-;; rhe idgc.' This is nor a gcnerally the former, and whatever the dexterity of the acceptedpractice, and causesproblems in resharp- operator,unless he be providedwith a suitable, well madeknife, he will neversucceed in obtain- ening knivcs. When sharpening metho.ls are ' changedwithin a laboratory, it is generallyrccom- irg satisfacroryresulrs. lt is poor economyro mended that the knivesbe reconditioned ro manu- skimpon keepinea m;crorome[ni[e in goodcon- facrurer'.spccif;carion' before arrempr,ngro dirion. No lr-iu"t "f diecrion can-cal

72 IV. METHODS

A. THEORY OF CUTTING The mechanism of separation ofthe section from the block and the effect of thc embedding medi- The 'harp Inife wirl a nanor edge probably um will be different for various materials and wedges.rather than shears off rhe secrions. offcr many opportunities for further nvestl Splirring i. posible although ir probably does gation. nor occur with hord gelr, Tearing ma1 be a rersonableerplanation for curriry. Molccular .pritring from rl'e uedging of the recondary valcnces and crushing on a fine scale may occur with some materials, according to Bailey B, POSTTIONING THE KNIFE IN THE " \1937), who concludcs . . . in colloida.l material lile MTCROTOME wood, the mechanism of cutting is sub-micro- scopic tearing, crushing or a cornbination ofboth, The actual cutting facets ofa knife are very small rather than of thc splitting of molecular scpara- in proportion to the knife as shown by a scale tron types." Bailey'founJ that wood secnoonq dr"r ins.Fig. 13. Thearrqlebcrwecnrher$ocuL requiriil a very sinooth knifc edgc, free froin ring facit. ric.rabli"hedbi rhe"h:rpening merhod. serrations and highly polishcd. When thc knifc is set on the microtomeit must be tiltcd so that therc is clearance between the von Ardenne (1939) analyzed cutting and statcd cutting facet next to the block of tissue and the that the thinness of the scction was limited by surface of the block of tissue, Fig. 1A. If this deform.rtionrnd inner de"trucoon. Re.isrance tilr is nor 3dcqua(e,as shown in Fje. lB, rhe to cutting depends on 1) the adhcsion which surface of the llock L forced doun fro:n thc varies with temperatule and che cutting angle; wcdgingcffcct ofthe cutt:ng facerand no section 2) resistance to deformation and 3) shcaring results. The next time that the knife passesover pressurc. The cocfficient of friction dependi the tissue this compression is incrcaseiland a par- upon lr the qualiry of rhe object cut: 2; the tial section or no siction may be obtained, Hbw- o;igin, qualirf o{ rhe f.cer surlaceof rhe knife cver the tissueis soon so compressedrhar lr sud- and 3) the pollution of the facet surface. Bv denJy expands "nd rle nexr sc.rion is very combinins E;ler s formula for brcalias srrensri, rhicli. S[iDoine of a section.or dre cLrrrins-dueof wjth a formul" for inerrjahe w,' ablc io preJc' alterrare rhick ind rhrn secrion' is usually that about lpm wi.li be the approximate limir to insufficient or to excessiveknife tilc. The tilt of thinness for ordinary sectioning, can be obtained by trial and error.

HoninsBock

Knife 0I234 5nm

1/8 in.

CuriingFocet

Fig. I 3. Knife dd honing back drawn to scale to show ertent ud fomatior of the curting bevet a facets.

13 Not only is the clea.ranceangle impotant but also the angle between the outer facet and the line perpendicularto thc block at the point of cutting, This is called the rake anglc,F!. 14C. E C,upperlocet onsle uo"'4/, The use of kniveswich unequalcucring bevels. Roke90'-E wich djflercnt and perhapsmore aduarrageou. G=D+l-l rake angles,have beeo idvocated by apat\ F=A+G.A+D+H (1912), Walsam(1916), Kisser (1926) aIL[ Litw (1932J. The qain from thesespecial edees. dc- spirethe extra-difficulryof usniqrhem,las nor bien enoughro brhq'rhem inio eeneraluse. The tilt anglesar sho-*non Fiq. I?Cwhenun- equalbevel-angles are used.

Sometimesbecause of sharpeninemethod or re- w58' 25' condicioning,the facet on one sideis iongerthan that on the-other. If tLis is true, asin Fig. 14A, which showsthe crosssection of the edqi after the knifehasbeen pushed into a pieceof thin lead 65" and examinedunder the microscope,it is then necessaryro rilt rhe knrfe for the properclearance anglein accordancewirh whjchside of rheknife is towa.rdthe specimen,Fig. 14C.

The standard holders on rotary microtomes maintain the edgeof the knife at right aagles-micro-to the direction of che cut. with sliding tomes,it is possiblero set rhe edgcof tle knife at an anglc io rhe direction of crir. Thc slicing cut is advantageousfor celloidin embeddedand for hard mateials. When the sliceangle is small, then the wedgingof the knife in the tissueis less, as shown rn Fig. 15. This smallereffective cut- rirg angle is helpful when cucring tough and 14. brirrle materials. The proper Fig. A.lmp.ession nadc bv lnife currine edseshow- serrinqsof the irginc uieoualunequal shdDenino.shdpening, B. Geonet.yceometryileonet- of"it krife"ii" edee.d,. knife for differenr spccir;neriswiJJ be-discussed ddes.dd4. c.C R,Le md rilt uglmusles fofor a clea;anceclea' ansle of 5% with the methodsfoi cutting them, Al1 adjust- for proper plring of knlves;t}l unequJ f&ers, mentsonthe hnife holder ani block adjustmeits musrbc righrenedby hand to prevenivibrarion duringcutring. Tooli arenot re{uiredand should

C. TABLE MICROTOME Soft tissuescar be sectioned with a knife having a small included angle between the facets; harder The table rnicrotome is a hand microtome with tissues require stronger edges. A uscful com- a clamp for fasreninsir to rhc edee of a table. promi

I4 I

K // ,/t/ ,/ /z z/,2 !N

I - Efrectivecuttins onsle

e= Nornol cutting onsle sin / =sinO sind

Fig.- l5. A.Shosi,F siiceanslc o r for .qree andrcctangula blocks, B.Decrea'cd scdgihg wirh tmrllsliccrgies. C. rie rlari;" l;' **" ih"^ pl.*"d wiri pernis"io' from dara of Prcs;ni 19J3r. shaped knife. A plane bit sharpened to e kcen arc rapidity (as neededwhen a tissue diagnosis edge is satisfacrorywler very rlrrn 5ecriousarc determincsan operarion in progress),lcss djs- not required. ln cutting hard material the handle tortion and frecdom from the adverseaffects of o{ the knife is heJd against the hip and the cut heacand solventson the tissue. made by .waying rhe body. This fi,- hoid ,ni frozen sccri6n, m"y 5e cur. rhc table mictorome, Fig. 16, is arranged to take thc ordinary specimen holdcr and it is possiblc to cut paraffin and cclloidin embedded material, aldrough this is more difficult than with the rargef mrcroromes,

D. FREEZING TECHNIC Freczinernd sccnoninsr ri,sucis a "apid mcrhod rl'arauJid. jrering Ll'i ri.,ueby solvint,.kiJl'ng and fir ng fluid". and distorrionfrom embedd'"g nedia. Boyle cur frozen eyes in 1603. SLilliqg "ecLionedfrozen .pinJ co'd in 1842.and Ruther ford used the method in the 1870's. At first, cooling uas a.compli.hed by ou'door i'eezing in cold wearheror *nh rolatile chemicalssuch as ether and rigolene. In this century carbon dioxide ard Freon have replaced less efficient m,re iJr :.d rhermoelecrricrooling ;r now coming into use in microtomy. The chief advantagesof freezing over embedding methods Fig. 16. Ao Table Mi6otomc.

15 l UnenclosedMiclotomes harden the material and wash it well before freezirq. tJnlesskillbq fluids are wa'hed Freezing chambers are usually mounted on our or-unle'saJcoholicaliy dehydrated specr- sliding,F{. 17, or shortstroke, Fig. 18 micro mens are brousht back io ab6ut 207, ilco- tomes, Dry ice hoiders can als" be usedfor hol, it is difficult to freeze them properly. freezing tissues(Smith, 1940).

Fresh tissue is frozen directly, or in a few Tissue damagefrom ice cr1'rals formec drop. of eum-"rrup,3 5% melred ,gar. or therne cellsce[s may beDe reducedreouceo byDy embeddinemDeoamg inm 10% albu;en or eilarin. Killed and fixed gon wga;3,' 5% melted agai, ot loV' Jb.i- tissucsshould be *ashed choro.rghlybefo'e men or gelatin. Spongy tissue should be freezins and dehydratedspecimen' should embedded before cutting. b" ru""d"wn an icohol 'eriesro 207oor le"s alcohol before freezing. The samole to be frozen on a standard freez- While no prepararionis required For somc ins cha;ber should be trirnmed to about tis.ues. in general ir is preferable to kill and 2 i 2 ccntimeters or less and 3-5 mm thick.

Fig. 17. AO 860 SlidingMidotone.

lcum sgr is prep*ed by dissolvinSin lO0 cc. of warer, lo0 srms cde sg&, 35 gr@s of gun &&id, and 0.1 grm rhymol. Agr is reconmendedby Evendenmd Schu$er(.19J8).

16 when the block is about two-thirds froze., level off the top. of the section and then complerc rhe ircezirg. lt is preferableto freeze rhe block , lirtle hardci rh,n c,n bc c11t,then as it warrns up aad rear:hesthe r$hr stage.make rhe required number of secrion. rrpidll. lf the block is frozen too hard, rhe secrionscrumble. lf rhe block rs too soft, the tissucs are injured and smeared together.

A medium slicing stroke is desirable. The knife should be tilted so that there is at least5' clearanceangle. Cur rhe sectionby drawine rhe Inife s]owl', throush thc tis- s.re, T"o cur tiozcn "ecrionssuciessf.rlly ir is necessarythat the knife be very sharp and the edge fiee from nicks and other impirfec- tions. As soon as lhe section is cut, ir shouLdbc rcmo"ed from the knife by rhe rip of thc finger or with a camel's hair brush, and sha[en into a dish of water or isotonic salt solution, The latter reducescytolysis of the cells. On the other hand, with a cool knifc and rapid cutting, three or four sec- tions may be cut and tnnsferred at one dme from the knife to the sroraEe dish. Alginate gel is used for serial sections by Lcwis & Shutc, 1963. Fig. 18. AO 888 AutomalicClinicrl Mic.otomes and 930 FreezingAttachmenx. In attachine the tissue to the freezins charr ber one sh"ould be careful not to lut too much water around thc tissue as shown ar Place a few drops ofwater, or better, ofgum B in Fig. 19. Watcr {brms hard ice which sugar, on tlle fteezing chamber; place the is liable to deflect the knife, causinguneven sections. Rather, keep thc ice lcvel below tissue or this and add iust enoueh-slowly fluid to sunound it. Freeze tire tissue by the surface of the tissues as shown at A in Fig. tuming on the CO2 for a moment or two 19. If the tissue is spongy and is and turning it off. A series of successrve embedded only in water, hollow spots, as jets freeze bcttcr and waste less gas than shown at C and D in Fis. 19, aie liable to runninq it conrinuouslv. The tissue should form hard ice crystals wiiich tcar the tissue be helJ flat against thJ chambcr until frcez- ing begin5. 1f the freezingchamber i. pro- vided with a knife cooling attachment, the knife should be moved so that it is in the path of fie cooling gas and deflects the gas onto the top of the block. This cools the knile arrd also assistsirr uniform cooling of the block. Some techniciansfacilitate freez- irg by Lolding an r-.vened,nedcine gl"ss over rhe cpecrmenwhen a del'ecrar r\ anr available. Staggand Tappen (1963) insulated the holder from the microtome and used a Fig. 19. Diagran lllustratins Difficultics with Freezing perforated paper cup to facilitate freezing. Technique. cl texr.

17 as the knifc qoes through it. With such tis- sues it is mu-ch better io embed ir one of the media advised, or at least to soak the tissue for a time in gum sugar. The gum susar will not freeze-hard and will leisen this type of tissue damage.

Rapid methods havebeen developedfor hos- oitil use.a whoie anrnals can be scctioned hozen with large microromes (Patschke, 1968). Me*odl lor staining, dehydrating ard mounting frozen sections ate also cov- ered in the siandard tcxtbooks. Bush aad Hewen (1954) collcct the sectionson a presrainedfilm .,,ip. Chaptcr ll grvp.3 it'..t 1i"t "f comm;n difficilties wiih this ,nethod.

Skill and experience are requred to mahe scctions thinner rhan 15 pm with this

2. Cryostrt Microtonly

Errclosingtle microtomein a constanrrcin- pcraturc chamberholds the tissueand knife Fig.20- AOc,yocut at tllc samc tcrnpcratlrre,w|ich avoidsdis- tortjorr frorn unc'vcn freczine ard rr*cs for casicr recovcry of thc scction. I-indcrsrroni bc sprayd, as nccclecl, Lrrg J,d laorg,n-on lq lx, rc r.,rl v thc tissoc should (rcdr(cd or tLr5Jd\Jrtrc rrr lrozcn,uLtrol- with r rcleasinscompound to prcvcntstick 1,1gtcchnnttrc. irrel'. The tis;c should bc triinmcd bcfo.e o" thc microtcmc. Tlc knife is 'l\' moun6'g r'yosrrr. Iig. )n. l,:r' r bu'lr r" ru'o po'rrioncd,nd r lrd ro p ovidc rd.qu"r. mairc retngcfallon unrt tllat cai be ser ro clcrrarc

For histochemistry, the section can be Tissues can bc frozen quickly with a CO2 moved into lhe desired container, or onto a device or on cold holdets on the heat sink cold slide. with a camel's hair brush. When in the cryosrat. Holding a cold nctal heat a wmm slide or cover glass (fron room e\r'rcro'on rop of tl'e-'i*..re.id.f"c.zrng ,(flpe,arureis brouql'rclo c ro the.ecrio" :nd gives, nrre r:-u( .rrtacc. f\e .rr on r\c kn'ie. Lhev. r o- u'll r.raJy 1uno face of the I'eat e'tractor in conra* with onto the glass, thaw and srick well enough acf. Bvendenind schuster (1938), ceschickter et al (1931). cradwohland Krajid (19a0),Hjort dd Mollton (1931), or Manhal(1940). *E.g. Acrosol zinc Ste&atePlastic Mold Spray,chicago, or similarreleasing agent.

18 for direct stajrring. When permanent mounts E. CELLOIDIN I\IETHOD "re r

19 mareh of the section with a commercial num- rheoperaror,5 A briefcheck lisL of thecommon bering machine as each one is cut (Rasmussen, difficulties found ia the useof rhecelloidin merh- 1940). od will be found in Chapter ttB, Meansfor counting a.rrdnumbering the sectionsare available The knife for celJoidinsectioning musi be very (Duddy & Cunan 1962; }rody lr Tanski 1964). .harp becauseany irregularitiesin the edge wiJJ lere markson the section.Lil

5ror methods cf. the chapre. by wesbotrom in Bensley and Bensley (1938), Fol distortion cf. Dempster (1942t).

6Ca reher {r93al eivesr rerptra(urcrabh on p. 228. lor a 45'C pualtu, room .empefuturesshould be 70"ro sO'F f.' section's&o- I 3 roi4 um : for52"C p*rfff n. 62ito ?5'F for 3 ro I J r; secrions.

20 ln addition to matchingthe hardnessof the paraf- the tempcature was maiatained at the mehing 0n ro rheremperarurei. w\ic\ ,r . ro be.ccrion pont oi the wa-\. ed, it is necessaryto match the hardnessof the paraffin to the hardnessof the tissue. The hard- The actual process of sectioning leads to distor ness ol the tissue.will depend upon the prepara- tion so that the sectionswill be a little bit thicker tory treatment and its sttucture, A Penerromeier and a little shorter than the block itself, This dis, was made, Fq. 21, ro study thesevariations. Thc tortion results from the nature of rhe paraffin, material to be tested is placed undcr the standard the tissue, and the action of rhe knifc. Large (A.S.T.\.a.r.phdlrj nccdlcand thc hekhr of rhe crystals are sectioned or pushed aoarr bv tie inde\ reado; rhe millimerersc.rre. I h-eorsronce r.rcrlgingof {l'e Injfe. e.Dc.iall\ -,"" ,t. which rhi.,tardard nccdlepoirt penerraresis i,*,f- jin is brirle and.ha.a higl,pl"su. polnr. Tl,is up- I proportional to the hardness.T \elrrngoi cr)staloglves rhe !el\et) appedran(cro

When the crystais are not of propcr size to fit .losclyro rhe ri.,ue.Lr'ey cr .or ,rppo" rner,. ,rc ade,luat'lyond localdefornranon will o..ur. givinga sectionwhich cannotbe completelyflar- tened out. Small folds show when the rhin parts were smaller than the size of the paraffin crystals and comprcsslcss than e paraffin.

I hc crysr,Js show clearlyin polarrzcd lQhr. Pho tomicrosraphsin bl& l, ;nd whitc do n-orrecord a.ll the ;l;r differences and losc clearness. All Fig.2I. Pen€troheterfor measuringhudness of microtomists should examine a section from cach brtch of paraffrrrw'rh a.polanzing,nicroscooe.' A rtler .r\5ld trnc prr.1,rngr!c. Jdcqu.'c sup por'. Wl,c- rhe . rv.r:l ..rucrurc;s roo large"nd A paraf{in uscd for some cxpcrimcntal work (Tis inhomogeneous,that barch should be repctcd. suemal 56 ) gave a penetration of 1.7mm with a 100gram wciql,r. A 'rardencdpa'rifin8 gave : re,di;s of 2.fim. K rdneyri,sui i"orn , rluin.a pig wis used to find out ihc cffect of the-usual Warm paraffin shrinks as it cools and compresses procedurcs on the tissue. The fresh tissue failed the tissue in the block. Tissue that is harder than to support the weight of the needie and pan (2 the paraffin withstands rhis pressute,but soft or grrnr'. A{ier lardcnirg "'< ri.,ue rr Bouurs spongy tissue may bc,:nder considerable strain, frr-rid the oe"errrrion w,. 5.4,nn. Afrer de\l- WLen sccrioncdt\e r'.sL

/ Hrdnes ud testirg nethods h avenot bcen extensivelydeveloped fot praffin cl wilims ( 1940). steedtuan( 1960) used

8F.on Wat"rnu (1939) Pdaffin (Pdowd) 80%, stefic &id 16%, sperm&erti 3%, baybeny 17o. Melring poinr 46'C, 9r:or turther detailsthe teaderis referred ro Denpsrer (1941, 1942a). I am sreairy indebted ro Professoroempster for a previewofhis excellert work ud for his friendll .;itici,. d,.i^s th" proses oiny .*pe.i-e"ts. *A binoculr biobjective (creenough) Midoscope witl POLARoID dbove dd below the scction is suitablefor this use.

21 counc is givento ir in currentpublic,rions,l0 would damagerhe Inife edqcwirh rhjs tvpe of Srowell( ls4 l) reporredrhar Parowar.hrinir.s jn accidenr.Seiial secrions ma! be cur "n , 'iid;g volume 14.3 per cent on cooling from 59' to microtomebut lessconvenientlv. as the hand suo- 20'C, Shrinkingcan also vary #ith the attach- portinq the end of the ribbon'must mou" ba& ment to the slide and the drying temperature ;d fo-rthwith the knife. (Seepage 2.) (Smith 1962). Since the pataffin method is used more than UnJessthe hardnessof rhe paraffinis rdapredLo any other at the present time, other problems the temperarurert uhich rlie currinejs done rnd of sec- tioning will be considered in tlie the natureof the ri

The microtomeknife shouldbe set at 90' to the directionof the cur (no sliceangle) and lihed to aslirde clearanceas will gile qoo=dsec(ions (2' ro 6' cf. V.41. rhe knifeeige s."hould nor show any pronouncedserrations when examinedat 100 dia- meters,althouqh the edqeneed nor be sosmoorh for_paraff-inas-for somiorher methods.Thc pos- sibiJityof cutcingwith only a fair edgeprevenrs many techniciansfrom getringexcellenr tect,ons, beciusethey do nor keip rhe*microromeknife ln th€ bestco;dirion.

The block must be trimmed so that the edg€s parallcl ro the knife are srraiqhtand parallel-to Fig.22. AO OLjectclamp. each other; orherwjse,the r-ibbonwill nor bc straQht and the disrortion will be increased.A camels hair brush lpenciJrype) is used ro handlerhe ribbon. Ir-maybe nicessaryro hold G. STATICELECTRICTTY ELIMINATION the first few sectionsonto the knife with gentle One greatinconvenience of pataffinsectioning, pressureof the brush until the ribbon forms. prtti"-"lrrly in winteror at o$er timeswhen th; Then the end ofthe ribbon is raisedwith a dissec- atmosphere is dry, is the flying of sections and ting needle and placedover the brush. This is their sticking to parts ofthe microtome and other withdrawn from the knife asthe ribbon lensthens nearby objects,-by due to the static electrical charee until it becomestoo lonq to handle, The ;ibbon gene.ired rhe friccionofcurring. Rai"ingLiie lengthsarc placedln ordir on rmoorlrpaper or in humidlry by boiJingwarer in the room or burnrng a shallowbox untiJtl'ey arc mountedon slide.. a Bunsen burner near the microtome may allevi- Someexperience is necessaryto curserial secrions are the diFficLrlty.,Sometimes.groundingthe m; without the loss of parts of the ribbon. Should croLometo a nea-rbywater PrPewrth r wrre or the brush be carelesslyallowed in contact with chain will carry the electricity away. A wet belt the knife, it will be spoiledrather than the knife. is used by Church aad Kroeger (1970) to reduce The use of a disseccingneedle in placeofa brush static charge on ribbons or sections.

l0Kbser(1927) gi'es fomxlae {or miting praftus md givtr the folowingfigues for volumeshri*age to 18'C roomrem- perarure: 36', a.8q., 40", ro.3E"; 45,;IO.6%t 50',fl.r%t 55.,1,2.6%t60.,13.8%, Seealso S;id1 1962! Steedaa (1960).

22 H. TEMPERATURE CONTROL dry ice container and the ca.rton,to control the amount of carbon dioxide passing into the box, An air conditioned room mairtained at the opti the temperature is regulated, The front end of murn..rcmperarLrewoLld be ideal for secrioni,,g. rhe bo:.-wa. lefr open for accessto rhc ir.rru. Pamffin .e.tion. cooline. A coolinq channelwa. buiJrinro a saTeryrazo"r blade holdei by Crargand wilsonI lel5). Duflield1le4l7lra. propo_sed-" bor to {ir rh< knife clo,cly. whiclr may be 611edwith warm or cold water as re- quired. A box surtoundins the knjfe was used to iool the knife with dry ice by Huepcr (1933). Fig.21. AO R3/o, qlJdp.o Fo.. y Mi.,. on, .

Since it is not always feasibleto cool the knife or the entire room, a cabinet can be used to cool the microtome. Foot and Strobel (1905) built a box The thickcr types of flat safety razor biades are complctcly around their microtome with hand .nore .arisfa rory rh,n rhe *afer-th n type. lh. holes for manipulation and cooled by ice. Grave blade.hould bc ir.erred so thar the cut, ng facet and Glaser (1910) placed an ice tray over a hollow isjust beyond the cdge ofthe holder. truncated pyramid so that cold air flowed over (he mrcroro'ne. A laree cardboard carron is pl"cedover r\e microromcby Hance( lqJTl wirh As soon as the edge is wotn, a ncw blade should a .onrai-t'erot d11icc orer a hole ir rhe rop of rhe be. used. lr,i. rare'1 worrr.while ro rerharpen carton. By adjusting the apertures betwlen thc sarctl razor blades.

23 Disposable Microtome Blades are availablewhich J. SECTIONING OR SURF'ACING HARD use a spe,iallv desgned holder replating rhe MATERIALS (METALS, PLASTICS, ETC.) srandardholder on rhe microtome{Fiq. 24). The holder clampsthe blade in a manner'wfiictr rnsures Soft metals. olasticsand other matedals not hard- rigidity and eliminates vibration. Sincethe blade er than abor;t 18 Brinnell may be sectioned or is always clanped in the sameposition, and at the sufaced with a microtome. The uooer limit de- pendson the bntdenessor plasricitv propercleararre arqle to the specimen,Inife tllr .].he of the ma- idjrisrment. a.e noi neces"arl. rhe de.gn also ierial. chicltnessof the "ecrion is lirnited br mikes ir porsiblero chanecknives durins secrron rhe nature of the m;reria-land rhe size of thc lng without the lossofa siction. specimen. Som€times thinner 5llm sectronscan be cut and with others thicker 15um sections may cut better. The specimenshould be no larger than the area to be examined.

Some materials can be clamped in the holdcr and seccjoned, Other specimcnsrequire sorrresup- Dot(. One useful method is ro olace the m"r..i"l bet*een t*o pieces of pith. Piper and fiim are cut when only a millimeier or so exrendsbeyond a smoorh. iaw clamp. A slJdingmicrotome is pre- fetable foi sectionihg plastics-and a sljcing cur may succeedwhen i iguare cur fails. Fig:.|7. Plasticswith glassor mineralfibers or other hard materialsusually cannot be sectionedwith a mi-

Rubber is a diflicult material. Somerubbers can be sectionedafter freezinghard and allowingto Fig.24. DisposableBladc Holder thaw enouehfor cuttinq, Somespongv and hard rubberscainot be secri6nedand 6nlvlhc surfacc can be examinedwith a verticelilldminrr"t - . A elassknife holder (Fis. 25) allows rhe useof fresh can sometimesbe obtained by glaisknives in rhe rotaty"micotome, fhe holder surface the specimen. is clamped in the standard microrome knife breaking afrer freezing holder. It is recommendedfor usewith the ultra Leadalloys thin sectionadapter (829) on the AO Model S2O and other soft metalsmav be surfaced smoothenouqh for erchingand micr6scopicsrudv with a microiornemore rioidlu rh"n b,, ih. '.'"'ril grinding andpolishingmerhod;. 12 Otfier meta.ls may requireonly polishilg after surfacing..Great cate ls necesserywttn magnesiumallovs and other mcralswhich surface harien by cold'workJng,ro stop before rhe surfacebecomes so hard thir ir suddenlydestroys the knife edge.

Metalsmay be embeddedin methacrylate,other sofr plasriisor Bal<.elite.If che Iarrer, i pureresin shouldbe usedrather than onerntued with asbes- tos or other fib€rs that will unduly wear tLe knife edge. SmalJpieces may be clampeddnecdy il thi jaws of it . t'"ta.i Fig.22. The currug shouldbe moderatelvslo- ind onlv oneor rwo microns removedat a trme. For siurfacing,the largeRotary Microtomeis preferableto a Sliding Microtomebecause of the very solidconstruction Fig 25. GlassKnife Holder of the knife holderand the feedmechanisn. 12cl.t tc.s lezt1

24 Hard rubber, wa11board, pressedwood, soldered K. THE RATE OF CUTTING joints, horn, bone, and similar substanceshave The rate at which the section is cut influences rrs been sectioned with microtomes. The materials quality. Frozen sections cut better slowly. A embedded in hard paraffin, soft plastics, or cven slow even cut is desirablewhcn cutting celloidin melted sulphur are lield in a clamp, When rhe sec, embedded material. Any hesistanceduring the tion is desired rarher than smoothing the surface, cut may leave knile marki on the seoion. Some r largeSlid ng Microtome,Fre. I i. prefcrred,A paraffin biocks of tissuemust be cut slowly to ob- slicrns\,'oke r'.h a.on.iderrble ilr.e,ne'c i. tain the best section, while others give a better often advantageous, Fig. 15. The resour'ceful ribbon ifthe microtome is run at a slightly operator will soon find from experience thc best greater .p.eed, t ndue speedinrarr"oly re'ulis in section. meansof hrndlinga givenmarerra-. Pre'oaling'n or poor qualltv. water or ol ta,ditatc,curring *L.en.r.h rrear ment wjll not inrerfere with rhe subsequentuse to More time is savedby cutting slowly and carefully be made wirh the mat€rial (Lendrun, 1944). rhan ru.hine rhrouehsevcrul vard. of se.rionsrn llood hrs been'reamed during cuttrng by K.sscr rhe hope rh-a'n goJd on".",rle found in rhe lor. . lc)20. lr i. L,ua1l1 ea.ier ro cur thin"ecrion" of a hard matcrial; e.g. Harlow (1940) recornmends The motor drive tbr AO Rotary Microtomes that wood be cut at 3-5pm. Other speciJ meth- should not be run faster than si-xty cuts pcr ods are dcscribed in the tcchnical booirs listed in the bibliography. Each matcrial sectioned on the microtome has an oprimLmc!rtins 'peed uhich ,hould be dere' mrnedcxpcri-enrilv; r' ] dependson the n.r When a long slicing type of stroke is not rcquired, ,.,e of thcma,e,i.l. ihe."r'rngedgc.'hc."g1" oi 'c.rion. rhc razor blade holder and thicker type of bladcs tnc knifc "nd rnc rhicl'nessof thc W'rh ate uscd for sectioning various hard ind diffictlr further rcsear.h and control it may be possiblcul- matcrials, becausea few cuts can dLrll the knifc timarcly to predictthese rates rather than depend and it may be nrorc economicairo replaccblades on trlal irsat Present. rhan to resharpcna standardkuifc. Thc AO Oscillating or vibrating modifications oI rnicro- H.ldcr. fr-. ,, {, is .rro, s en"uqh ,o I.old z fl,r tomes have been used for sectioning bonc, tecth tl.d, ,luri-g,\. |,roc.r iuL 'c- rrr rl,ccdgr ol and nineralized rissucs (Southern & Thomas, thc blade-ill-lrl .r,n,t. 1962; Burgstedt1964; Eschcnbrenner& Eggle- ston, 1964). Baud a Morgenthaler used a rnodi- {ied dentrl technique and rncthacrylate embed Phillips (1961) uses a diamond knifc when sec ding proved useful for Siglitz (1964). see also tioning metals. Brah, 1966.

V. MINIMIZING DISTORTION WITH SPECIALREFERENCE TO THE PARAFFIN METHOD

An ideal section would bc of thc thickness indi- derablc source of error in three dimensiona.ire' cated by thc microtome feed settinq and of the same size and shape as the tissue in thc block. A furrh

Fig. 27. rntcrference frinees seen with th€ rnterferomcterofFigure 26. B. MEASURINCSECTION TTIICKNESS AND CUTTINGFORCE For srudyins variarionson rhc surfacc,a c.v

13rh. b.se piecc -.s of thick slus, 26 x 75mn, with tLe un&r side groud to avoid refiections from the non-flat surfae. The 25 x 38;n cover g1*s wai ground -edge-sl.ped to avoid ioterfeirg reflectioos. A pleceofblak photographi. Ea!- pingpaperwas placedurder the inrtrument. WiliMs (1930) dGcnbesthe ur ofinterfarometers. The resistanceto cutting was determiaed by at- small. When the knife was tilted too little or too taching a tuning fork beside a sliding microtome, rru.h. r\e force u.ed u..rsirsufl. cnr ro p';l . e Fb. /,8. ,"j re.ordirg it. vibrarior. on a..movirg knife through the block. Bctween these exrremes record surlice as a constant we&ht pulled the the.method gave intercsting and useful infot- knifc through the block. The diflerence in time required ro puli the knife the length of thc block, wher rhe in.trumenrran free, rnd wlen a,e.rion C, MATERIAL was cul, is a direct measurc ofthe resistanceto cutting, or of the forcc required, since the force Driferent spccimcnsvary ir rheir hornogeneiri. equals the mass times thc accelcration. The force (onseqJentlyin all of rhcir currinepropeiries. lr ':l gra' L1 dr'ving weShr and tirema,, rcmaincon- ii dirfi..,lr io f.nd mare .ufiic"ienrlyconsrant ,trnr.o rhedelal ir rime i' " mcasureoirhcac- for a reference standard. To obtain a reasonably celeration. Permanent rccords were obtained by lomogeneous reference material, one of the hatd- fastening a platcn to tlre knife block of thc mrcro- ened paraffins made accordineto Waterman s me tome and rccording rhe runirg forl rrccc on thodt was uscd. This was Tairly homogcreous wd\aa R.rr,,l;nR 1'.tp{, Record. $cre madc and satisfactory, but less dense than paraffin with the microtome feed ser to 10pm. The posi blocks. Cutting was done with blocks trimmed to rion. of thc beginningand cnd of rheblock arrd 15mn square, While results from this matcrla-lat of trre rissuccm-bedde-d in tlre block weremorked Lhe.ame rpmper.rtu,rc ucrc r omparablefrom trmc on the paper before it was removed from tbe cat, to. ti,ne, it nowise represented actua-iconditions riage. Ihc runins forl was adlu.redso {hJ( rhc or cur.mg. record'rartcd crch timc from the.a.e po'','on. The rcsistantcof rhe parrffrn and of rr'e ri,,uc Thrce tissues were chosen for the experimental could bc determined separatelyfrom the records, {ork. and larseblock" prcparcdso rhar. numbe, | .B.2oA. The rime inrcnaL \0.01 .e(, were of rcsrscouLd bc madc on rhc b ock. A typjcal cornred with rl'c ,id oi a low powersrcrco)coprc blocl of Luman lc4rr musclcwirh sone rrtcrro microscope, and the difference between the fice sclerosiswas obtained frora a locaJhospital. This run and - cuttins run was exoressedbv A . For was rrimmed to 15 x l Bmm with rhe actual tissuc cach experimenr"thrc" to fiuJ sepa.atc'cuts wcrc areaof 11,5x 13mm, The Peneiromcter(100 ^cugcd-,dependinq on rhc amounrot vr|lanon gram weiglr7 | ig. .'| . show.d rhar rhe ri:.ue was sl .rwD. a Wirh go-odcu,rin" co-d;r,,rrr5A wr5 much harderlhan rhe paraffinbecau,e rhe peue- tration into the tissuewas only 0,4mm,whiG t|e paraffin wa, l.smm. lt was difiiculr ro ger fl.rr secrron..Lecau"e the paraffin frame. orevenrcd expansion of the wrinkled secrions. Thc sccond spccimcn was a piccc of cat testis, killcd by eouin s fluid, dchydrated in dioxan, and cmbedl ded in 55"C paraffln. A block was rrimmcd to 12 x 12mm and the tissue was an oval 9mm in the direction of the cut and 10mm at rght angles to thc direction ofthc cot. The hardness of-the tissuewas O.7mm and of the block 1.2 mm Penetration. The third section was ofhuman '.rterus,killed in formalin, dehydrated in aicohol, :nd enbedded'" 60' p"r:ffin. The blocl,"a. rrirnmed ro 14 x 21mm and th. rL'uc wa, o x Io.5mn. I he pcn.rranonof rhet.'ue wa' 0.o ard of r\c pai;ffrn O.omn. rhc.e 'pccirncn. Fig. 28. SlidingMicrotonc dranged for measuringthe were used for the comparative work and others fe$s.nce oi cu!fug. were used from time to time for general study.

14the decten,. i. .ate of novement is duc to 1) the resistanceto rupture ofthe naterial beingsectioned, 2) the friction be tween ihe knife and the paraffin and tissueand 3) rhe fri.rion bctw;cn rhc pans ofthe nicrotone. The larter arerelatively constantso tlat standar&ing againsra freerun Ms as satisfactoryas a crr ofbloch contatuingro rissuc. LfLl,lfll AB ll,ll,'fll'l,tllil' --f- I B ___t_ N I

F.-- A------.1

MM.

- SkipCur AB B-4-{ llfliliftliir!liirlllt,l'rt,nwt t wwt't tvi

Fig. 29, A. Reshtanceto cutting record. B. Diaglan illusb.ting dtutortion.

D. DISTORTIONS meter usuallv demonstrated greater thickness than this comouted value. whicliwill be ca-llede*- wjth asdes- Paraffin chargesin size temperature- cessthicknesi in this report. With poor cutting IV. Consequently, cribed in sect'ionF, Chapcer conditions this €xcess ;hickness mav brine Loth dn thi the thicknessof the secti;nscut depends total thickness of the section 2ll tim;s the ihick- at which rhey were the temperatureof the block ness to which the microtome feed was set. The The interfero- cur and'the rhicknessmeasured €xcessthickness was little influenced by tcmpera- meter showedthat the averagesection decreases 'C.(3 ture. aooroximatelv0.9mm jn thicknessper fiirses). Un[.ssa differenctemperarure is srated, The increasedthickness of the section, computed rhe?o[owing discussionis basedon recordsat or from the distortion of the section. deoendson the corrected t; ao averageroom temperatureof nature of the knife edee, the tilt of the kniG 25'C. Sectionswere nirtened for mountingwith (clearanceand rake anglis), the tempefature, and water at 45oCr and measurementsmade afrer the material sectioned, The compression and d.yi"g. thickness distortion decreaseas the knife is sharp- ened with firer abrasives. L{htly stropping the The sectionis usuallycompressed in the direction knife improved ir" appearanceunder rhe micro of cuttine. so that iibecomesshorter and propor- scope.but compressionand di:rorrion. particu- tionatelv";hicker while retainins the sam6width larly the excessthichness, was increased. as rhe 6lock. Takins jnto accorinrche change in proportion,Fig. 298. the predicredthickness car SecEioningdssue is quite differenr from section- Le cornpuredfrom the formulagiven. Thb is true ine oarafl-mblocks *iih no rissue.The relationof onJvfoi t-o dimcnsionalchanqi with theknife at exi6ss thickness, A in Fig. 30, and the computed rie(t .nslesto chedirection oi cur. Whenaslice increasedthicLness, B in Fi,q.10. from distortion .u-. l" 'i-rd", rhe sectionwill be di*orted ir all ate shown with .e"pect to th" tilt of the knife. three dimensions (Dempster, 1947, 1942a.). The tilt is expressedas the clearance ansle and Mer$rem€nt of the thicliness with the interfero- th.." r.sults were obtained with standatd lactorv.

28 sharpenedknives. A11sections were cut ar 10!m en.e in rirne des.ribcd jn the prerioussecrion, onthe sliding microtome with no slice angle(knife ;s illu.trzred by C on Fig. 30. Le,s re.i.rr-ncei, at 90o). With knife tilt up to 10o clcararce angle, encountered as the clea.anceangle is increasedto dle hearr lissue thickness was very close to ;he aborr.4' . fu' rhercha-ge occurs up ro . on- rc_r. .ehi-g .Lrnle J of rle B in I g.J0. rtre s derablerilr. when rhe rc.i.ra:cc aga,. ,'ncrea.e,, o L,e-ris5 re- wcrc rror L i.(rr r rr" qrirr.r com_ The heart tissuc resisrancc to cutiing increased p-e sio in le-qrh; r0..ho;. A in F,q. rnccrces, rapidy ar i0' clearanceangle. -d- "irl, r:. !hrc(n",s.or iLe d fr.'e ic Lerweerrh. rotJ rlearunrc angle skippirg oc.urred. prod.rcJngal- thicknessmeasured with the inrerferorneterand ternarellthir and rhick se,rions. Arlo{rhe mate- the thicknesscomputed from compression.For r;.11.o far inre.r$",ed suggesr.thar lre oprimurn all three tissues, riris decreasedrajtdly to clear, tdr rngle r!nger bcrweenJ' rnd 8. knife cle;r- ance angles of 4' s" and sho;ed a dei.inite ance, Wrrhin rhi. limired range rlre re.i.rance mlnllnum tot each t1sstrc. ro,Lutring i, mbim"l.,nd rhe rneasuredrhickne.. The forces required for cutting, or conversely,the ot the sertion. approrchera value rerv clo.e ro resntance to curting as measured by thc differ_ that calculated fr_ommeasurement of the desree of section shortcning; this is considered is a 14 nccessarlcondirion lor optimal .ecrionirg. 12 With the knife set at the optimum ciearanceanqle for r givenrrssue, it is posiiblero studr t0 A rhecffJc r of diflerenrkrnd. of_e;gc,. A ,l*.",(.,t,-p.," 8 ._-_J!_., stroppededgc Bave r]lc leasrrerjsranc. ro currrng ( A=0.3ro 1). 6f Tl,e rcsrsrJnrcto curringdccreascs wirh lcmperJ turc. and th. rarc ol rhe 4 detrcasc is cbour rhc same as thc change in thickness of individua.lsec tlons wrih temperaturc, Fig, 31. Thc forcc rc_ 2 14

Ub t3 22 2A 12 2A 1t r6 -'-V::\',r'l t8 t0 14 16 l?\

I0,i c

8, .,...- 6^ --...:._,1I llr

2 T "Knife 0 Titl 2 6 I l0 12 14 t6 t8 20

Fig,J0. Curins r.nrJn.c rd cohp,e$ionwi.h -espect ,o knile ril-. A.t\.c* rhicln.r,. B.Thicknes..oneared Fig.31. Effec,ofrempe%rue on LA)'Lickn..sorsecdon for conprcssion. C. Resistanccto cuffing. Cl texr. dd on (B) tesisruce ro cnftins.

29 quired.o. conversely.rhe resistancero curtingof for errorsfrom distortion,or actualknowledge of chetissue itself, is aboutthe same as ofthe encire the true thicknessof the sections. Recentwork block and tissue.but the curveis slishdvc".c.".. giresdue consideration to correcfionor minimiza- Thesedata were obtainedwith st;d;d factory- tion of sucherrors; e.g, Dean and Magnum, 1945; sharpenedknives and set to a dearanceangle of Eliasetal,1951-71;Hennig, 1954, 1956; Mer- 6' usingthe uterustissue. A few scatteredexperi- rian, 1957; Shieldsand Dean, 1949; Wiistenfeld, menrsrirh remperatu,ega'e essenrialiy rne \ame 1957. He*d (le5l) de.clbesa phouographic slopeof curvetor Ini'es.fdifferenr bevel.How- method for the rect;Ficarionof compression. ever,whenthe bevelwasreduced to 17' the curve was muchsteeper. With sucha knife it is difficuit Lison (1937) and Pusey(1939) may be consulted to cut in cool laboratories. t^. ^-- -,- l --,L^,.1"

E. FLOATING OUT Exceptunder the most perfectconditions for cut- G. CONCLUSIONS ting, ihe ribbon is formid with someirregularity, whichmusr be flartenedbefore the sectionsmay Distotion is any changein the form from the be attachedhrmly arrdevenly to the slide. Two rissuein rheblock, or in rhicknessof thesccrion generalmethods are in use, The ribbon may be different from the settingof the microtomefeed, ir.rt into convenientlcngths. and pJaccdon the Distortion hasbeen shownto alisefrom the pre- slide.a fe* dropsof wateradded to the slidc.and liminaryprcparation of rhe rissue,shrinkage, -of Lrn- plasticproperries the wholc gcntly -armed until the sectionsex- favorablecrystallinc or rhe pand to their full size. The other mcthod is to embeddingriredium, the-sharpnissbf rhc micro- position place the piecesof the ribbon onto warm water tome knife and its in the holder,and the and, afterixpansion, to recoverrhem bv placine temperatureat which the cutting is done, The de- a slideunderneath the sections.n uery ihin layei formation may involve one. two or ail threc di' mensrons of albumen fi-xativesmay be rubbed onto the ot the speclmen. slide or included in the water used for floatias out by the first merhod. The floatinqout should To reducedistortion, care shouldbe usedin the be done at as low a temperatureas-possible or Preparationof the tissueand in choosingan em- about five to s€vendegre;s -A less than the melting Leddingmedium. which shouldhave rhe a-ppropri- point of the paraffin. smallamount of oneoT arc plas(ic properties for the temperature at wettinq agentsadded ao the warer ihe commercial which the seitioninesecrionine is to be done. The hardness in assisrsmarerially in r;ducing the differences of the paraffin,oaraffin. wh]enn it is used.used,should corres- flarrer surfacerension'and obtahjng a section pondwith that of the tissue, The knife must be (croat, 1941; Medawax,1941). Thc floatingout sha.ro.arp, with a smooth cdsecdge and well-oolisheiwell-polished temperaturemust not be too waJmor the paraf- facets.:ets. It should.hould be placedpiacedinir therhe holder toro therhe fb irself may be disinregraredand chesec(ions position (tilt and sliceingles) which givesthc bcst unduly stretched. Likewise they must not be sections. The edge should be kept clean durine strerchedwirh needlesor by graricyfrom coo the cutting of th; block, especialy if serial seci great tilt of the slideas the water is poured off, tions ar€ requiJed. The rate of cuttins should be Some tis$resabsorb more water than othersand mainrJred at the besr rpeed for rhe rpecirnen. becomeswollen even though cmbedded in paraf- and the rcmperarure'hould be adjustedco mair- fin. Coarselvcrvstallinc ;araffins do "". "". tain the optimum relations between the above round tiss'ueciosely and take up considerable the factors, which control the excellence of the sec With floatingout does water by capillarity, care, tions. The mounting Drocedure must not add not contributeto distortion,but ca.relesshandling distortion and should r!l;""" as much of the de- may easily deform thc section, Lin and Collet formation due to cuttinq as possibl€. The abilitv (1969) recornmenda xylene sgrayfor flattcning to do a.ll of this is lear"Jd fr"m study .nd "*pe.i ence by the skillful technician.

F. RECONSTRUCTIONS For additional irformation seethe recent work of Steedman (1960) and for other aspectsofdistor- Earlier three dimensionalreconstruction models tions see: Gough, (1968); Lehmann, (1969); were madefrom serialsections with litde concern Tomasch,(1969).

30 VI. CONCLUSIONS

Microtomes are precision instruments and require of the specimen to prevent undue shrinkage,and carc to kecp them in proper condition. To insure to the temperature of the room at which the sec- oprimJ performarLe,nd long liig of rhe ir'rnr tlonng 1sto Deoone. ment, the slideways should bc cleanedwith a soft cloth when cutting is finished or at the end of the The typesof distortion commonly found in sec- day. A11bearings should be ptoperly oiled. The rion cutting have becn discussedand recommen- instrument must be kept clean, free from bns of dations frorn experimental work given, so that embcdding m.rtcri.rlJnd. *hen nor in u'e. ir these distortions may be minimized, should be covered or placed in a cabinet to pre- vent damage. This rnanual has been written to cover the use of the microtomc and to give you the benefit of thc Unless the microtome knife is kept in excellent rxperien.c of orr .*" ,*.j" h laborrrory. Sec ,ondirron.it i' rot po.sibleto cur good.ecrion.. rion curting depend' on no 'bglc irem. rl,e lor Delicarc and dense objects requlre the bcst, muL for obra;ning

VII. HISTORY'

Microtomcs were callcd cutring engires r-rntil Thc sliding rricrotomc with a vertical {ied screw 1839, whcn Chevalierintroduccd rhe word micro- was developcd in 1798 by Ada,r's, who obtained rom.. Cummrnq, ru,ri.s cneirF,1770. I'cld rh. a slicing cui by the use of a knifc with a slanring .pc.im.n w'rhrn, .ylindc' ;d forc.d rr up ior "dn". iu "t u". ?astettcd thc straieht ra.or ar an a"- sectionine by rneans of a fine screw. The knife gie"in 1799 to atta.rn rhe same-result, Qreckett r.livorcdibour r fir.d pivor. HiJrprepaed cr' el- pivoted the knife at one end and supported thc L", """d *-,,.", r.r:r\ rhi. b'irumerr. P ir- other on a *ay in 1s48. The knife holder, adjust- ra. cFard. lbJ5, fa.ren.drhc spc.,mencontain rg ab e fo. borL.rilr and slrc. angl<.. bcen arrri- cylindcr and feed mcchanism to the edgc of the buted to Schanze about the middle of the nine- teenth century. rable with a clamp-cutting and used a separate two handled knife for sections. Later thc clanp was omi*ed. and the instrument was hcld The sliding microtome whercin the specjmen ls in on. l"nd uhilc rheot|er rard cut the s(c(ions r.i,cd by pu.hr"g rhe 'pc.incn holdcr up m in- with a heavy razor. The popularity of the hand c irc *r, developedb1 Capnemo m 1848. Ri- modei was due larecly to the work of Ranvier vet's improved design was man0facturcd in wood about I880. BakerlLlndon) manufactured hand by Vcrick (Paris)in 1863, and in metal by Leyser- and table microtomcs as early as 1840. A table Brandt (Leipsig) in 1870. model with a danring top was developedby His in 1866 ard manufacturcd by rhe Societ6 Gencvois The rocking microtome was invented by Caldwell in 1870. and Trefa1l in 1881, and improved by Darwrn rn

1Cf. lehrer. "t,1 (1889). Gatenbyud Painter(1937), Minot (1903),Roneis (1932), Snith (r915),Cowles &Ricl&ds (1947).

31 The Filst Americu Microiome. Adm's Cutting Engine,1798 Pfeifer'sRotary Microtome.

1885. The disadvantaeeof thjr designwas the TABLEt currinqof a curvedcyliirdricat block sirfare, and led rolhe developmentof the flar-cutcingtypc of I. With movableknife rockingmiootome in 1888, A. Object feed vertica.l Rotary microtomeswere inventedindependendy 1. Knife is held in hand - table and hand by Pfeiferat the John Hoplins Universiryin 1883 models. and bv Minot in 1886. Minot's modelwas manu- 2. Knife movesaround a vertica.laxrs at one the f".tur'.d by Baltzer(Leipsig) in 1887.and by end, with the other end free or supported FranklinEducational co. {Boston)in 1895. by a slideway. 3. (nife holdei pivots on a parallelogram. wirh a triangu" ln America.rhe slidingmicrorome Two unequal supports provide a slicing bv Bauschand lar knife block was manufactured stroke. in 1882. arid an irnprov- Lomb Oprical Company 4. Knife holder moves on a horzontal yearslater, Their ftsr rotary mi- ed model three track, crotomewas manufacturedin 1901,and the hori- zontal Minot Automatic PrecisionMicrotome B. Objecrfecds up an incliaedplane lCapa- wirh starionaryknife and slidirg specimenholder nema'RivetmodeL). and feedin J909. SpencerLens Company pro- duced the ClinicalMicrotome in 1901, usingthe lI. With stationaryknife unequalparallelogram knife movementro glvea slicingstroke. In 1910 the larg€Spencer Rotaxy Microtome first appeared,with increasedpreci- 5. object moves alo_nga vertical guide sion of cutting obt-ainedby separatingthe feiding lMinot Rotaiv modell. mechadsm from the driving mechanism(Ott, 6. obiect holdei moves-arounda horrzontat I s | 1). The SpencerSliding Microtomes were de- a-'riitRoclire model). Sectionsare cut velooeddurirs the middleof rhenineteen twen- FromcylindrriaJly .urfaced block ties. Other Li-ndsof Spencermicroromes are also 7, Similar to o wirh a parallelogramobjecr m"nufactured.Instead-of catalogirg very la-rgein- supportLo give flar sictionsrflr""rti;ng strumentsit hasbeen the practiceof malufactur- Rockine"rotatesModel). ers in the United States to make speciallyde- 8. Object around a horizontal axis signedinstruments as required. paiallel to knife (Peelingmethod). D. Knife honzonta] Many kinds of microtomesha"e been designed and tried. The moreimportant typesare summa- 9. Objectmoves on hori/ontalway.. rized in TableI. All the principlesexcept those in I0.Objectrorares about a verricalaxis. the circularand rhe peelirg microtomeare in commoouse at the presenttime. The peelingmi I. w]TH MOVAALB KNIFX tI I I AI AI

A4

II. STATIONARYKNIFB I I c6 c8 c9 cl0

I Types of micotome mechdtuml t 33 crotomeha" been adopted by chewood veneer in- satisfactory for anirnal tissues. The latter could dustry. Circular, rotiting knives have been tried be sectionedwell only after they weree-b.dd.d with little success,It is not possibleto keep the ln a suppor.mgmearum. blade sharp and ro mainrain the precisionre- quired for this work, although they are practical Paraffin embedding was established by Klebs, for use in food stores. The last type (No. 10) is 1869, and Biitchli in 1881; gelatin by Klebs, no longermarketed. Krause(1926) also classifies 1869,and latet by Kaiser,1880. Soapembedding and descdbesearly European microtomes, was initiated by Flemminq- in 1873 ind has been used spor"dicilv s;nce. Duval oriejnatedthe The needfor very thin sections(<0.1pm)for the ce[oid;; method in rsz9. electton microscopehas producednew ultrami- crotomes(Richardi, 1956). An incline planefield The fundamentalmaterials have been modified in with lessslope is availablefor sectioningless than various ways to obtain more useful media. Paraf- 1pmwith rhe AO Rotary Microtome. fins have been haldenedwith ceresinard modi- fied by the additionofrubber, baylerry, or other Microtomeshave been modified for cutting hard wa-xes.Harder mixtures without highermelting materialsby the addition of a motor arrd saw pointswere prcpared by wa(erman(19J9). Hy- (Roofe,1949; Kwan, 1970), drocarboni'"-"b"' resi-ns*ii"i **ewere added to paroaraffin!vgryibv Croac (1941), The freezingmethod was usedin 1871. About 1839 Valentinedeveloped a doubleknife by Rutherford(1873); Berdeen(1901) attributes with two parallelblades with ihich he rezulated the methodto Stillingduring 1846,and Boylc cut the thickriessof the sectionbv adiustinqi slide, sectionsof frozen eyesin 1663. An inteiesting a screw,t; bring tli" blades recentcornbination oftechniques is the cutting of or in later ,models closertogetner. celloidin embeddedtissues on the freezingmicro- rome. warer solublewaxes and polyehtylene gJy- The earlv cutting enginesand the modern hand cols areuseful when dehvdrationshould be avoid- microromes*ed-ee tf,e -ate.ial to be sectioned ed, and epoxys. methyf/methacrylateand other into the feed cylinder of the microtome. Pith plasticsari usedin very thin sectjoning,(Fall and may be used to hold leavesand other softer tis- hawson.1955: Glaue;tel dl.1956i eray.1954; suci. This js a quick method usefulfor plant or- H.,le,1952t Lillie, 1954; Milesand Linder, 1952; gans.bur the zoologistsfound the merhod not Steedman,1957;1960).

34 VIII. BIBLIOGRAPHY

The items given are intended to suqqestcurent methods arrd to lead to detailed sourcesof information. Popular, no-n+echnical,and the oldJi less availablebooks are omitted. It is not the intent of this biblio- graphy ro gire information on rhe preparationof materialsor micro\copeslider, Cenair generalbook. markedbyanarrerisk1)andCrayandCray{le56)conrainreferencestothebibliographyirthisfieldand may be consulrcd. lF rhe referencesdo not leadtoan answer.you areinvited tosend your questionor pro- blem to American Optical Corporation, Scjentific Instrument Division, Buffalo, N.Y. 14215.

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