.-- 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<it is damaged.or old and.obvioLrslyworir and they may rcsult from dirt or hard material in noneor rnesesuggestrons work. rhe specimen. \4oving the (nife ro an unu"ed area. or replacing.wirha s\,rpe' l.nife .nay re srore gooct sectlonmg. A. DIFFICULTIESCOMMON TO ALL Sections that fall out of the matrlx or show a METHODS differen( amounr of .o.nprersion than rhe em beddhs medium frame - indica(e rhar rhe lrregularsecrions, skipped sections. or LIrickand \upporting. embeddin€ medium is irrdequare. rhinsections. are usualiy the rezulc of irsufficienr Mushy apperiry secrions indicare insufllcicnr tilt (fig. 18) of the knife, thar compressesrhe dehyd,ation or clearing. Re-embedrhe .peci- block on the return stroke, or of too much tilt men in a morc suitable material for better which scrapesoff the sectioninstead of cutting sectiors. (See Gray, 1958; Chapter VIII). 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.