03 Chap01 Instrumentation-e

8/13/2019 03 Chap01 Instrumentation-e

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CHAPTER 1

InstrumentationAutomatic tissue changers

Autotechnicon Mono and DuoUse of automatic tissue changersAutotechnicon UltraUpshaw automatic tissue processorsFisher TissuematonHistomatic TM Tissue ProcessorTissue-Tek III V.I.P.Use of clinical freezing microtomeMethod of preparing sectionsAttaching frozen sections to slidesThe microtome cryostatThe instrumentCutting technicMicrotome cryostat knivesSheehan rapid hematoxylin and eosin stain forfrozen sectionsThe compound microscopeThe light sourceCondenserThe stageObjectivesNosepieceBody tubeEyepieces ocularsFluorescence microscopyPhase microscopyPolarizing microscopyCare of the microscope

Microtomes

AUTOMATIC TISSUE CHANGERSAutotechnicon Mono and Duohe A u t o t e c h n i c o n ~ th e oldest of t he com-mercial t issue changers is a valuable ins tru-

ment in the histopathology laboratory. It is par-ticularly so in large hospitals where an abundantvolume of tissue is processed daily. heAutotechnicon consists of a timing clock

that is cu t to determine immersion periods ofthe tissues; reagent beakers of glass or plasticwhich contain the reagents required by the par-ticular technique being used; a beaker platformfor precise alignment of beakers; a master shiftcarriage to automatically transfer tissues fromone fluid to the next in order at time intervalsTechnicon Instrument s Corp. Tarrytown Y2

Microtome knivesKnife sharpenersSharpening by hand

American Optical Automatic Knife Sharpenero rse honing operationFine honing procedureRedressing the platesCleaning and lubricationHelpful hintsHacker Perma-Sharp Knife Sharpener

Instructions for the Perma-Sharp MK-4MaintenanceHelpful hintsShandon-Elliott SharpenersMK I and MK Autosharp IIIAutosharp IVA modified Autosharp IV Knife sharpening tech-nicReconditioningPolishing procedureWhat do with a gouged plateShandon-Elliott IV Automatic Knife sharpenerReconditioning of copper platesCare of the microtome knifeBuehler Isomet Low Speed SawFume-Gard

Honeywell GLS 360 Slide Stainer formerly SKIstainer

predetermined by the timing clock; individualbeaker covers or a centra l cover to preventevaporation of fluids; a displacer rotor whichprovides constant rotation of the tissue basketdUring immersion in the fluids; a receptaclebasket of stainless steel; stainless steel recepta-cles or cassettes for carrying the tissue duringthe processing and paraffin baths. hepathologist cuts the pieces of t issue andplaces them in the t issue receptacle with an

identifying number he receptacle basket is at-tached to the displacer rotor on an arm of themaster shift carriage. hetissue basket rotatesslowly dUring immersion and travels clockwisein an orbit moving progressively from one re-agent beaker to another through the various pro-cessing stages. he sequence and duration of


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immersion periods are determined by the pathologist and are precisely maintained by theclock mechanism. The timing mechanism is an l t e r n t e ~ u r r e n t electric clock controlled by atiming disc, which permits a definite sequenceofvarying time intervals to be preset. The disc iscalibrated over 24 hours and further subdividedinto 5-minute intervals. When operating, revolves on the clock face unti l a timing notch isencountered. tthis point, a timing lever fallsinto the notch, s ta rting the mechani sm thatraises the tissue basket from one fluid, shifts it,and immerses it into thenext in line. The basketis of stainless steel, with die-cut perforations.The firm closure of the cassette guards againstthe possibility of specimen loss or mix up. Thesnap-action opening facilitates removal of tissuewhen the receptacle is coated with paraffin fromthe paraffin bath. The cassettes are fully perforated at the top, bottom, and sides, permittingfree passage and draining of flUidsThe care of the Autotechnicon is extremelyimportant. Paraffin should be kept in the paraffin baths and removed from all other areas ofthe instrument with soft cloths soaked in xylene. The receptacles and basket should besoaked xylene and washed in very hot soapywater to remove all residual paraffin. Individual

INSTRUMENTATION 3lids or the large lid to cover beakers must bekept free of paraffin at all times.Lillie states to remove paraffin from metalembedding molds, Technicon tissue carriersincluding the nylon-plastic carriers furnishedby the Technicon Company), boil them for 5 to10minutes completely immersed in a tall metalvessel containing about 10 to 12 gm a level tablespoonful, or 16 ml) of powdered Oakite, Calgonite, or other technical sodium phosphate detergent in about a liter of water. Then cool untilthe paraffin can be removed as a solid cake;rinse and dry. The greasy film left by xylenecleaning is absent with this method, and thedanger of working with an inflammable solventis eliminated. Peers, J H.: m J Clin. Pathol. 21:794, 1951.)se of utom tic tissue ch ngers seeChapter 3 for additional precautionsI Instruments mus t be quality controlled: Monthly or bimonthly maintenancecheck by electricians depending on useof instrument.

B Wires and plugs must be dean and freefrom paraffin.C Total instrument must be dust free andfree from paraffin.

B

Fig. 1-1. Never cu t straight along a hne but rather angle back, as shown here. Timing notchesare easily cut with ordinary scissors.


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4 THEORY ND PR CTICE OF HISTOTECHNOLOGYai r and the tissue dries out, place the tissue in the following solution overnight:

utotechnicon ltraThe newest Autotechnicon is the Autotechnicon Ultra for automatic vacuum processing ofhistologic tissue specimens for pathologic interpretation (Fig. 1-2).The required reagents for the 12-station pro

cessing cycle are contained in stainless-steelbeakers that sit in a well of mineral oil arounda circular deck. As with preViously describedTechnicon t issue processors, the tissues areloaded into small perforated cassettes or receptacles, which are transported in a perforatedbasket. The loaded basket hangs from a vacu-

Process the following day on the standard 16-hour procedure, starting in 80alcohol.B I f the tissue goes back into the fixativeafter the paraffin:

Rinse in 95 alcohol.2 Rinse in absolute alcohol.3 Rinse in xylene.4 30 minutes of paraffin under vacuum.5 30 minutes of fresh paraffin undervacuum.6 30 minutes of fresh paraffin undervacuum.C the tissue goes back into the 80 al-cohol (first beaker on instrument) afterparaffin: Rinse in absolute alcohol.2 Rinse in absolute alcohol.3 Rinse in xylene.4 30 minutes of paraffin under vacuum.5 30 minutes of fresh paraffin undervacuum.6 30 minutes of fresh paraffin undervacuum.D the tissue is in 80 alcohol, or 95 al-cohol, instead of paraffin, in the morning: Check to see i the clock is properlyset.2 Check to see if the clock is tight.3 Check the master switch.4 Proceed through remaining alcohols,clearing agent, and paraffin.

D Clocks i f used) must be evenly cut. Cutting the 24-hour disc for Technicon (Fig. 1-1, black The timingdisc is divided by 24 radial lines representing I hour intervals. With ared pencil , mark off the desired timeintervals start ing from zero, whichshould represent the time at whichthe tissues are placed on the Autotechnicon. Make cuts 9 mm deep ateach ofthe red pencil marks. The cutshould not follow the radial line, bu tangle back. Now make the longerangle cut, remembering that theshallower the angle, the less wear onthe timing gears. A correctly cut discwill have one less notch than thenumber of steps used in the process 2 steps and cuts.2 Cutting the clocks for AutotechniconMono or Duo A clock cutter isfound at the rear of these instruments. Mark the clock and cut it withthe clock cutter. A correctly cut discwill have one less notch than thenumber of steps used in the process 2 steps and cuts.

Baskets and cassettes must be clean andfree of paraffin to allow for free exchange of fluids.F Baskets must be firmly attached to rotors to prevent spilling.G Smallpieces of paper may be used toidentify the specimen. Large pieces ofpaper hinder the penetration ofthe flUidH Tissues must not overlap in the receptacle, since this produces poor penetrationof fluid and paraffin.I The fluid level must always be higherthan the level of the receptacles.

J The tissues must never be more than 3mm in thickness.

K Always use clean tissue cassettes. Paraffin-coated cassettes inhibit fluid penetration.L h nge ll f luids on the instrument inaccordance with the work volume in thelaboratory. t is poor economy to skimp

on clean reagents for processing tissue.M When changing solutions, the beakersshould be thoroughly washed and driedand the instrument completely cleaned.II. Handl ing automatic t issue changer problems a basket of tissue is caught up in the

Sodium carbonateDistilled waterAbsolute alcohol0.6 gm

42ml18 ml


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INSTRUMENT TION

A

Fig. 1 2. Autotechnicon. Mono; B Duo; C Ultra. Courtesy echnicon nstrumentsCorp., Tarry-town, N.Y.


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INSTRUMENTATION 7

A

8 lcohol

bsolute lcohol

Fig. 1 4. Autotechnicon old black instrument . A 1 basket of tissue; B 2 baskets of tissue.


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8 THEORY ND PR CTICE OF HISTOTECHNOLOGY

Two baskets starting in80 alcohol at 3:30 P

Clearing agent

Absolute alcohol

Fig. 1 5. Autotechnicon Duo. A 1 basket upper 1 basket lower level; B 2 baskets upper 2 basketslower level.


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INSTRUM NT TION 9

Bouin s solution/ I \ P Q ~ ~ ~ Q f f i n80 alcohol \ Clearing agent95 alcohol Clearing agent95 alcohol Absolute alcoholAbsolute alcohol Absolute alcohol

Fig. 1-6. RUSH SPECIMENS Autotechnicon Mono or Duo 6-hour technic).

With this bath, t he reagents remain at aconstant temperature of 38 to 40 C andthe paraffin bath remains at 58 to 60 CThe device for holding the basket containin g the tissue cassettes is suspendedunder a vacuum head that provides 15inches 380 mm of m er cur y pre ss ur ethroughout the processing cycle.3. The instrument can be operated manuallyor automatically and the constant temperature of the mineral oil can be changed tosuit the operator, or i t can be shut off completely. In addition to this, t he v ac uu mhead can be adjusted for varying pressure,or one deems advisable, the instrumentmay be operated without vacuum.

Upshaw automatic tissue processorsThere are five models of automatic tissue processors, the two most popular being the Trimat

ic and the 1000 model. The T ri mat ic may beused for conventional overnight processing, fasttissue processing under vacuum and heat, orautomatic staining. The basket continually agit at es a nd t he re is a 4 5- se co nd p au se over e ac hb ea ke r to allow for good d ra in ag e a nd p re ve ntfluid carry-over. There are four thermal baths

supplied as standard equipment for heating paraffin or solutions with e ac h i ns tr um ent . T heTrimatic will accommodate up to three baskets,each holding approximately 36 capsules. Theinstrument may be used for automatic stainingwith slide carriers holding 42 slides. The instrument may be mounted on a portable floor cabinet or used on a laboratory bench.The standard Lipshaw automatic tissue processor is 4 feet, 9 inches long and may bem ou nt ed on a laboratory s he lf or placed on aworktable or mounted on a special processingstand Fig. 1-8).

isher TissuematonThe Model 60 Fisher Tissuematon Fig. 1-9)carries 44 tissue specimens through a preprog ra mm ed s eq ue nc e of pr ep ar at or y steps. T hetissues are automatically fixed, dehydrated, infiltrated with embedding medium, and ready for

sectioning and examination. istomatic TM tissue processor

This model automatically fixes, dehydrates,clears, and infiltrates tissue specimens in an enclosed system. Model 166 Histomatic Fig. 1-10)automatically processes specimens in up to 120


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THEORY AND PRACTICE OF HISTOTECHNOLOGY

95 0 alcohol

95 0 alcohol

Absolute alcohol

Absolute alcohol

Fig. 1 7. RUSH SPECIMENS Autotechnicon Ultra 4 hour technic; B hour technic; C 2 hourtechnic; D I hour technic


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Paraffin

c

INSTRUMENT TION

bsolute alcohol

bsolute alcohol

Paraffin Y ~ C l e a r i n g agent

Clearing agent

61 .,....,.. Clearing agent

< l bsolute alcohol

: bsolute alcohol

bsolute alcoholJ> bsolute alcohol q J\ ~ 9 alcohol.4 /: 9~ ~ ~ ~ 9 alcoholFig. 1-7 cant d. For legend see opposite page


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THEORY ND PR TI E OF HISTOTE HNOLOGY

AB

Fig. 1-8. A Lipshaw Model 1000. Lipshaw Trimatic model. Courtesy Lipshaw ManufacturingCorp., Detroit, Mich.

Fig. 1-9. Fisher Tissuematon. Courtesy Fisher Scientific Co., Pittsburgh, Pa.

tissue cassettes at one time, immerses them in aser ies of solvents for fixing, dehydra ting, andclearing, and finally immerses them in paraffinfor infiltration. The 120-cassette capacity of theHistomatic is double that of most traditional processors.

Th e Histomatic concept is a totally enclosedsystem with minimum air exposure. Histomaticintroduces a truly major innovat ion in tissueprocessors: ll processing takes place in an enclosed system. Tissue, solvents, and infiltrating

media are inside the Histomatic system not inopen containers exposed to the air.Th e laboratory. No noxious fumes escape,and there is no chance offume buildup or explo

sions. ll solvent fumes are channeled to oexhaust port, which can be easily vented to theoutside.Solvents for fixing, dehydrating, and clearingplus media for infiltration automatically lowinto and out of a stationary tissue compartment.The Histomatic features a 2-minute drain period


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INSTRUMENTATION

Fig 1 10 Fisher Model 166 Histomatic Tissue Processor.

after each solvent cycle to assure minimum contamination of subsequent solvents. nceyou pu t tissues into the Histomatic tis

sue compartment, they are not fully exposed toair until the completely processed specimens areremoved from the final infiltration bath. thepower fails, tissues remain safe inside the tissuecompartment. As added protection, valveswill automatically close. When power is restored, thesystem will automatically resume theprogram sequence where it was interrupted.Programming for operating conditions Theoperator programs sequence and time period foreach solventlinfiltrating-medium stage by presetting controls on the control panel. Conventional timing discs are not needed. A clear plastic window may then be locked over the controlpanel to prevent accidental or unauthorized program changes.

The following operating conditions may be selected:Processing time Can be set for anywherefrom 10 minutes to 4 hours per cycle by 12 individual control timers, one for each of 10 solvent and 2 paraffin cycles. Tissues can be processed with or without vacuum vacuum withall 12 sta tions or vacuum only on paraffin.um er of solvent cycles Use up to 10 solvents for fixing, dehydrating and clearing cy-

cles. To skip any solvent cycle, just set its -dividual control time to OFF.Solvents are contained in ten 2-liter poly-ethylene reservoir containers. They are frontloaded and attached to the system by a series ofpermanently mounted screw caps. Bottles canbe easily removed, refilled, or replaced. They areenclosed behind a transparent plastic slidingdoor. Solutions may be changed while machineis processing. Machine may be partially cleanedwithout disturbing processing. Additional tissuemay be processed during the day on a shortcycle. umberof infiltration cycles Use one or twoinfiltrating cycles with media from two paraffinwells preheated to 63 CParaffin wells, contained in a slide-out drawer, can be easily drained and filled in place.Temperature Use the solvent heat control toapply gentle heat directly to the tissue compartment during all solvent cycles. ryou may optto apply no heat at all. Thermostatically controlled system is factory-set to 40 1C but isscrewdriver adjustable over a 30 to 45 C range.Infiltrating media already preheated in paraffin wells are automatically heated during infiltration in tissue compartment to factory-set temperature of 59 1 C; screwdriver adjustableover 50 to 60 C range.


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THEORY ND PR TI E OF HISTOTE HNOLOGY

Fig. 1-11. Tissue-Tek III V.I.P. Vacuum Infiltration Processor).

Tissue-Tek III VJ.P. Vacuum InfiltrationProcessor

he new Tissue-Tek III V.J.P. Fig. 1-11) operates on the fluid-exchange principle. The tissue specimens are retained in onelocation whilethe processing fluids, paraffin, and cleaningagent are exchanged by l ~ m t i o n of vacuumand pJ;essure i n the proper sequence. This procedufe ensures quality-processed tissue specimens and permits faster, safer, and more efficient tissue processing.Light-emitting diode displays on the controlpanel indicate exact instrument status such astime remaining in delay cycle, processing cycle,and temperatures.

heV. . P is designed to accommodate mostprocessing fluids and paraffins used in processing tissues. here are ten I-gallon 3.8liter) containers for processing fluids and an additional I-gallon 3.B-liter) container for cleaning fluids. he oven holds two I-gallon 3.8liter) paraffin reservoirs. A consistent oven temperature is maintained to assure proper paraffinconditions. Fluid and paraffin are sequentiallydrawn from the container s in to a cent ral processing retor t by vacuum and are returned tothe ir respec tive conta iners by pressure. hevacuum pressure cycle ensures thorough fluidand paraffin penetration during processing.

iris blown through the transfer lines aftereach fluid transfer to assure clean, dry tubing.A charcoal filter is available to provide closedsystem and to eliminate f umes and noxiousodors. n audible and visible alarm system isprovided to alert operators to power loss, instrument malfunction, processing problems, andsystem interruptions.

A printed c ircuit board can be programmedfor the des ired t ime sequence; the processingcycle can be delayed up to 99.9 hours. A specialprogrammable cleaning cycle prepares the unitfor the next processing cycle.

he retort chamber can process over 300specimens simultaneously on a regular long cycle or single immediate biopsy on a rapid cycle. has two locking l atches and an interlock forsealed security during processing.USE OF CLINICAL FREEZING MICROTOME

Fresh or fixed tissues may be placed directlyon the freez ing chamber of a clinical freezingmicrotome, frozen with carbon dioxide, and sectioned with little damage to the tissue. Themethod is rapid and its usage can avoid theuse of fixatives, especially when the fixativeswould affect using the tissue later for fluorescence microscopy or microincineration. Thereare several disadvantages to this method:


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1 Distortion from both the cutting and freezing2 Difficulty in cutting sections much largerthan 2 x 2 cm3. Considerable skill required to prepare sections thinner than 15 / tm

Method of preparing sectionsFresh material may be cut, bu t better sectionsare obtained after tissue has been fixed in formalin and washed before freezing. tissues are

in alcohol, they should be hydrated to waterand washed before freezing. Trim the tissue to2 x 2 cm and 3 to 5 mmin thickness. Cut a circle of filter paper, wet it, and place it on thefreezing chamber. Lay tissue on paper and addenough water to surround it. Proceed in the fol-lowing manner:1 Freeze with a rapid on-and-off gas flow ofCO2 by holding a small beaker over the tis

sue and object holder to aid in even hardening and freeZing.2 The knife should enter a point of the tissueon a square block, rather than the fullwidth of the tissue. a capsule, skin, ormucous membrane is present, it shouldface the knife, either on the shorter width,o r on the lef t of the longer width, so thatthe cut is made from the capsule into thetissue to prevent the capsule from tearingaway from the tissue.3. The knife must be cool to prevent the sections from sticking to it and the tissuemust be held firmly against the chamberuntil freezing begins.4 Freeze the tissue hard and cut the sectionswhen i t has thawed down to the right consistency. As the block warms up andreaches the proper temperature, make therequired number of sections by drawingthe knife slowly and evenly through thetissue. Cut the required number of sections with an even and slow stroke. Remove from the knife with a finger moistened with distilled water and place thesections in distilled water at room temperature (250 C). If the block is frozen toohard, the sections crumble; if is not frozen hard enough, the t issues are injuredand jammed together.5 The average thickness o f sections is 15Lm Very hard or dense tissue may not allow cutting at less than 18 to 20 Lm Considerable skill is required to cut thinnersections. The rate of cutting is very impor-

INSTRUM NT TION tant. Sections, when cu t too fast, tend tocurl or break into shreds. Slow, even cutting permits warming of the cut surface ofthe block, a controlling factor in procuringa pliable section, free from undulations.This pliability prevents disintegration ofthe section when it is placed in water.Uneven sect ions are usually the result offreezing the block too hard and applyingtoo much speed and unnecessary pressure, particularly when firm tissue is beingcut. Sections that float rigidly are too thick,and when selecting sections for staining,choose complete sections that fold and unfold freely in the water.6 All set screws holding the freezing equipment and the knife must be tight to avoidvibration when frozen sections are beingcut.

Attaching frozen sections to slideselloi in metho1 Float the section on slide from water.Spread it out smoothly with camel s hairbrush. Blot with bibulous paper.2. Cover section with 95 alcohol for seconds and blot with bibulous paper.3. Flow 0.5 celloidin solution over tissueand slide and drain off the excess fluid atonce.4 Blow briskly on the section and immediately immerse the slide in distilled water.5 The section is now attached to the slide bythe thin layer of celloidin and may bestained by most of the usual stainingmethods, since celloidin does not preventthe penetration of stains and does not interfere with the visibility of the section.Avoid drying at any stage by proceedingquickly. the tissue was not properlyfixed before sectioning, or if containsmucoid material, the seconds in 95alcohol in the first step prevents the tissuefrom sticking to the bibulous paper.6 For clearing after staining and dehydrating, use oil of origanum or oil of thymefollowed by xylene and mounting.

THE MICROTOME CRYOSTATThe preparat ion of frozen sections by themethod previously described (ifone uses a clini- .cal or standard freezing microtome, freezes thetissue with carbon dioxide, and cu ts the sectionsat room temperature) has many disadvantages.1 the tissue has not been fixed, it is ex-


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THEORY ND PR TI E OF HISTOTE HNOLOGYtremely difficult to cut even when a freez-ing attachment to keep the knife cool isused.2 Thin sections of unfixed tissue are limitedin thinness since most standard or clinicalfreezing microtomes can only be set atmultiples of 5 Jl m and the tissue is too fri-able to produce good sections at this thick-ness.3 The limit of thinness of fixed t issue is 10to 15 jLm and most tissues are cu t be-tween 15 and 25 jLm4 Under these conditions therefore fixationis necessary but provides multiple barriersto procedures involving enzyme histo-chemistry fluorescence microscopy auto-radiography and protein and inorganichistochemistry.The cryostat is a refrigerated cabinet con-taining a microtome cooled by a mechanical re-frigeration unit. It has been described as a rap-id and easy means of preparing large thin un-wrinkled sections of single or multiple pieces offresh frozen tissue. We believe however that itis an ins trument requiring an acquired skillbefore it can be operated efficiently. Since noskill requiring manual dexterity can developfully without practice over a reasonable period

of time previous experience of section cuttingwith conventional microtomes is invaluable tothe technologist using the cryostat since thequality of the final section will depend on his orhe r experience and skill.The instrument

There are many good cryostats sold in thelaboratory market today. In preparing each ofthese for use however one should lubricate allmoving surfaces with a silicone lubricant pro-vided for this purpose. there is insufficientlubricant the microtome is stiff and difficult tooperate. This problem may be alleviated bya fewdrops of absolute alcohol being placed on themoving surfaces to remove ice crystal forma-tion followed by drying and oiling of the sur-faces with the silicone lubricant.The microtome is enclosed in a deep f reezebox and in most instances is mounted in thefreezing compartment at a 45 degree angle.When originally plugged in to the electricalsocket the microtome will take approximately 4hours to reach the required temperature of 1C Place the microtome knife in the knife holderand keep an extra knife in the freeZing compart-ment for immediate use. is essential to use a

cold knife. If the knife temperature is higherthan 50 C the temperature ofthe tissue blockmust be lower than 1 C In our laboratoryour cryostats are used at 2 0C The efficiencyof the cryostat depends on the tissue tempera-ture chamber temperature and knife tempera-ture and is leas t dependent on cutting roomtemperature.Knife angles are not critical and an angle ofapproximately 3 degrees provides a base linefor cutting the majority of tissue. This angle isfrom knife tilt only and not the angle of the cut-ting facet. If the knife angle is too shallowthe tissue will pass over the knife without pro-ducing a section and give off a rubbing sound.Too steep an angle gives rise to compressionand although there is obviously an optimal knifeangle for each type of tissue deviation from the3D degree angle is undesirable unless difficul-ties arise in cutting the tissue.Cutting technic1 Set the knife angle and the thickness scale.Each division on the scale is equal to 2 jLm;therefore i f the scale is set at 2 the tissuewill be cut at 4 Jl m2 Place sufficient Lab Tek OCT an embed-ding medium for freezing tissue on theobject holder. Freeze it slightly in liquidnitrogen which is kept in a widemouthedthermos container. Place the tissue in theslightly frozen OCT add more OCT aroundand overthe tissue and freeze solid in liquidnitrogen.3 A device may be made in the followingmanner for freeZing tissue in liquid nitro

.Fig. 1 12. Chuck holderfor freezing tissue for the cryo-stat technic.


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gen: We take two strands of heavy wireabout 50 cm in length and continuallycurve the strands together until they reach23 to 25 cm in length . Bend and curve theremaining wire into a circle to hold the object holder. This holder is indispensable forsuspending the tissue down into the liquidnitrogen for rapid freezing of t issue fromsurgical cases (Fig. 1-12).

4 Transfer the frozen tissue on the objectholder to the microtome head. Be sure thehead is in the upper position and the drivewheel locked.

5 Tighten the clamp to hold the object holderfirmly in place. Pull the knife holding clampback so that there is 6 mm clearance between the subsequent down-travel of theblock and the knife.

6 Release the lock of the drive wheel andbring the knife to within 1 or 2 mm of thetissue and adjus t the block to the knife sothat it is precisely parallel.

7 Bring the t issue almost into contact withthe knife edge, release the ratchet from themicrometer wheel at the rear of the microtome, and advance the wheel clockwise byhand until the tissue begins to section.Trim off the face of the tissue until the desired cutting plane is reached.

8 Return the ratchet to t he teeth of the micrometer wheel, clear the knife entirely freeof tissue debris, and turn the drive wheelslowly until the leading edge of the sectionbegins to cut.

9 With a fine camel s hair brush, gentlystroke the section onto the microtome knifeas the tissue moves down over the knife. Al-ternately, an antirol l plate device may beused.

10 A clean slide, at room temperature, isplaced a tiny distance over the frozen section and the section will be attracted directly to the warm slide. Do not use pressure,since this will cause distortion or stretchingartifacts. The slide may be held in the handor attached to a suction-pickup device.Once mounted, the tissue slides may bestained or stored in a Deepfreeze for futureuse.

Microtome cryostat knivesEither a wedge-shaped knife or a slightly hol

low ground knife should be used. The wedgeshaped knife holds an edge longer under constant use, whereas the hollow-ground knife can

INSTRUMENTATION be made sharper . A knife 185 mm in length ismost suitable, but the ends should be coveredwith a slotted piece of plastic tubing to preventaccidents.When paraffin-embedded sections are beingcut, the soft tissues such as liver, kidney, spleen,and so forth usually a re cut more easily thanare hard tissues such as the uterus, skin, breast,and so on, which are normally more difficult tocut. With the cryostat, the reverse is true. Thehard tissues cu t easily and the soft tissues aremore difficult to cut. may be necessary to cu tthe soft tissue at a temperature of - 50 C Theexception is brain, which will cut well at 2 0C

All frozen sections from surgery are cu t on thecryostat and stained in the following manner toprovide a rapid section for a permanent frozensection record.Sheehan rapid hematoxylin and eosinstain for frozen sections1 Acetone for 30 seconds.2 Xylene. Agitate until slide clears.3 Absolute alcohol. Agitate until slide clears.4 95 alcohol. Agitate until slide clears.5 70 alcohol. Agitate until slide clears.6 Tap water. Agitate until slide clears.7 Delafield s hematoxylin for 1 minute.8 Tap water. Rinse well.9 Tap water. Rinse well.

10 Ammonia water until blue.11 Tap water. Rinse well.12 95 alcohol. Agitate until slide clears.13 Alcoholic eosin for 10 seconds.14 95 alcohol, 4 dips.15. 95 alcohol, 4 dips.16. Absolute alcohol, 4 dips.17 Absolute alcohol, 4 dips.18 Absolute alcohol and xylene (equal parts).Agitate until slide clears.19 Absolute alcohol and xylene (equal parts).Agitate until slide clears.20. Xylene.21. Xylene.22. Mount in synthetic resin in xylene.olut onsAmmoniawater: 25 drops ofconcentrated ammo

nium hydroxide in 500 ml of tap waterAlcoholic eosin ( th is formula is well used inthe routine H E stain as well as this rapidstain)to k solutions

1 aqueous eosin Y1 aqueous phloxine


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Fig. 1-13. The compound microscope.

ondenserfocusing

The light sourceIn modern instruments the light source issupplied by low-voltage electric bulbs operatedby a transformer that can be adjusted to the intensity of light required.Of great importance to microscopy is the behavior of refraction. In fact, the refractive behavior of light brings about the formation ofimages; therefore, refraction might be considered the single most important underlying concept in the functioning of the microscope. Refraction is the bending of a ray of l ight when it

1.3301.3361.49 to 1.501.50 to 1.511.515 to 1.521.51

Refractive index1.000

Optical mediai rImmersion substancesEthanolWaterXyleneCedar oilImmersion oil

Glass (slideand cover glass)

strikes a new optical medium at any angle otherthan the 'normal.' When one discusses microscopy, the new optical medium will consist of aninterface of glass and air, air and glass, glass andglass, and other interfaces such as optical cements, mount ing media, and immersion oilsThe 'normal,' when one refers to a lens, is aline perpendicular to the tangent of the curvedsurface (Wilson, M B.).

Condenserhe substage condenseri s the first part of thelens system. The functions of the substage condenser are threefold: To concentrate light upon the tissue speci

men2 To produce an adequate area of illumination upon the tissue specimen3 To furnish strongly convergent light to thetissue so that full resolving power ofobjectives may be used.To give even illumination, the condensermust be accurately centered with respect to theaxis of the objective. The common mechanismprovided on the complete substage for the centering of the condenser is a pair of centeringscrews.The regulation of light illuminating an objectis the function of the diaphragm. The intensityofillumination is, in part, regulated by the fielddiaphragm of the condenser. The aperture diaphragm regulates the convergence of the coneoflight rays from the condenser. The purpose isto match the numerical aperture of the condenser to the numerical aperture of the objectivethat will give optimal image quality.

The stageThe s tage of the microscope sits above thecondenser with an opening through which the.light passes. One of the most useful accessorieson a microscope is a mechanical stage. t is amechanism for moving the slide by rack andpinion or screw movement slowly in either oftwo mutually perpendicular directions. Me-chanical stages are bui lt into microscopes or

100 ml10 ml780 mlml

orking solutionStock eosinStock phloxine95 ethanolGlacial acetic acidDelafield's hematoxylin (p. 142)

THE COMPOUND MICROSCOPE Fig. 1-13The compound microscope is one of the veryimportant instruments used in the histopathology laboratory. Knowledge of the instrumentand its use and care is fundamental . The component parts ofthe microscope are as follows:A light source ondenserStageObjectivesNosepieceBody tubeEyepiece (or ocular)

Substagecondenser


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added on. hemechanical stage is particularlyhel pf ul w he n o ne ne ed s to se ar ch a spe cim ento make certain no part has been missed. Onsome mechanical stages a Vernier is located tomake it possible to note the position of the slidein each direction. fthe position of the field oneach scale is noted, t he slide c an be immediately replaced in the same location to relocatethe same field.Objectives heobjectives are the second lens system ofthe compound microscope. heobjective is thelen s a t the lower end o f t he body t ube and h as amajor responsibility for the magnification andresolution of the image. t is the most importantcomponent of the microscope.Either achromatic or apochromatic objectionsmay be chosen. F or . r ou ti ne microscopy i n ahistopathology laboratory, the less expensive

achromatic objectives are most commonly used. heseare corrected for two colors, red and blue. hemost highly corrected are the apochromaticobjectives, which are corrected for three colors.Plan apochromats provide a perfectly flat fieldof view and are ideally suited for photomicrography. he numerical aperture N.A.) of the objective is fundamentally important, since the microscope s ability to resolve is entirely depen

dent on the N.A., which is engraved on the sideof the objective. Numerical aperture is the sine sineu of the half-aperture angle multiplied bythe refractive index n of the med iu m fillingthe space between the cover glass and the frontlens.

N.A. n x sin uNosepiece he nosepiece on the microscope revolves

and can handle multiple objectives. The objectives, when possible, should remain screwedinto the nosepiece to avoid damage.ody tube he body t ub e is monoc ular or binocular.Bette r vision results whe n it is possible to useboth eyes simultaneously. A binocular body on

the microscope permits the use ofboth eyes andgives some appearance ofdepth. The standardbody tub e is 6 cm in length.Eyepieces oculars

he eyep ie ces a re the third len s sys te m on acompound microscope. heprimary function of

INSTRUMENT TION 19the eyepiece is to magnify the image of thespecimen produced at the rear of the objectiveso that the eye can come closer to the image. hebinocular bodies usually have inclined eyepiece tubes for greater comfort of the user. hebinocular body has two adjustments, onechanges the distance between the eyepieces until both eyes see a single field. This is an interpupillary adjustment that sets the centers of thelenses in the two eyepieces at exactly the samedistance apart as t he c en te rs o f the observer seyes. he other adjustment compensates forany difference between the observer s eyes. Tomake this adjustment the microscope is focusedsharp to the rig ht eye. hen the r ight eye isclosed a nd the a djustmen t o n the le ft eyepieceturned until the ima ge is sha rp for the left eyeBoth eyes s houl d t he n s ee t he im age equallywell.FLUORESCENCE MICROSCOPY

An object fluoresces when absorbs ultraviolet light reflected on it or transmitted throughit a nd then emits the energy in visible lights ofa specific violet, blue, green, yellow, orange, orred color. These substances, present in some tissues, are called fluorophors Secondary fluorescence can b e ind uc ed by the use of fluoro-chromes which are strongly fluorescent dyes orchemicals that a re applied to the tissu e specimen.See p 311 for information on the equipmentand reagents used in fluorescence procedures.PHASE MICROSCOPY

Phase microscopy is the preferred microscopemethod for the study ofunstained cells. Apparatus for phase microscopy is simple, relatively inexpensive, and easy to use and can be added toany conventional microscope. It may be used tostudy living material, cytoplasm, nucleus, cellinclusions, and the action ofphysical and chemical agents on tissue. However, theus e of phasemicroscopy is not limited to the study of livingcells or to unstained fixed tissue sections. Themethod is particularly useful to visualize tissuecomponents that are essentially transparentand cannot be studied with bright-field microscopy.A standard binocular microscope can be converted to a phase microscope by replacement ofthe sta nd ard c on de nser and objectives withspecial p ha se e qu ip me nt. A p ha se -telesco peeyepiece is also available.t is possible to determine the approximate


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THEORY ND PR TI E HISTOTE HNOLOGY

refractive index of living and fixed tissue components with the aid of the phase-contrast microscope.POL RIZING MI ROS OPY

The use of polarized light in the histopathology laboratory is distinctly valuable in the examination of doubly refractile particles such ascrystals and some lipids, and it may also be usedto study myelinated nervous tissue, collagen,and cross-striated muscle.Lillie writes: Polarized light may be producedby use of discs of Polaroid material, by interposition of a series of obliquely placed thin coverglasses, or by use of a Nicol prism. The polarizing device is set at any convenient place between the light source and the study slide. Alsorequired is a second polarizing device, called ananalyzer, that is usually placed over the microscope ocular.

When a ray of plane-polarized light, whichvibrates in one plane, falls on the object , it issplit into two rays, one ray obeying the law ofrefraction and the other passing through at adifferent velo ity After emerging from the object, the two rays are recombined.Bright apple-green birefringence of amyloid iseasily seen after Congo red and Sirius red staining. RE MI ROS OPE The microscope is a precis ion instrument

that must be handled skillfully and carefully.2 t must be kept scrupulously clean in everydetail.3 Aside from cleaning the outer surface of thelenses, the objectives should remain in thenosepiece. A soft camel's hair brush shouldbe used to remove dust from the objectives,and they can then be polished with lenspaper or soft old linen.4 The top lens of the eyepiece can be dustedwith a camel 's hai r b rush and then polishedwith lens paper or soft old linen.5 Prisms should never be touched, and cleaning should be confined to blowing off thedust with a rubber bulb fitted with a smallbore metal tube, since the slightest misalignment of the prisms will cause enormous eyefatigue. (Culling)6 Never use facial tissues to clean opticalglass. Use lens paper only7 it is necessary to use a l iquid to clean thedry objectives, Mallinckrodt's lens cleaner isexcellent for this purpose.

8 When immersion oil is used, it may becleaned off with a smal l amount of xylenefollowed by polishing with lens paper.MI ROTOMESThe first microtome, called a cutting engine,

was made by Cummings in 1770, followed byAdam's cutting engine in 1798, which was essentially the first sliding microtome. Pritchard,in 1835, fastened Cumming's model to the edgeof the table with a clamp and used a separatetwo-handled knife for cutting sections. n 1839,Chevalier introduced the word microtome.Rotary n'licrotomes were invented independently by Pfeifer at the ohns Hopkins Universityin 1883 and by Minot at Harvard University in1886. Bausch and Lomb Optical Companymanufactured the sliding microtome in 1882,and Spencer Lens Company produced the Clinical Microtome in 1901 followed by the largeSpencer Rotary Microtome in 1910 (Richards)(American Optical Co., Buffalo, N.Y.).

The follOwing microtomes are used in histopathology laboratories: Standard rotary microtome for paraffinsectioning2 Rustproof rotary microtome for cryostatsectioning3 Clinical freezing microtome for cuttingfresh and fixed tissues; primarily used forcutting tissue for fat stains4 Sliding microtome for cutting celloidinembedded material5 Ultrathin sectioning microtome used with

either a diamond or glass knife in electronmicroscopy6 The JB-4 Porter-Blum microtome forplastic and paraffin sectioning van Sorvall, Inc., Newtown, Conn.)With good care, a microtome will have a longuseful life. The following maintenance shouldbe carefully carried out and recorded: Thorough daily cleaning of rotary microtomes used for paraffin sectioning. Softcloths moistened with xylene, followed bythorough oiling of the knife-holder slides,will keep the instrument free of paraffinand rust. Do not use xylene on the painted

surfaces, since it will remove the finish.2 Use two drops of Bear Brand oil (NortonCompany, Troy, N.Y., formerly Pike Oil),on inner oil pits (check manufacturer'sdiagram) requiring oil This should bedone every week or so on instruments thatreceive average use daily.


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3 Grease inner surfa ces with a good lightn eu tr al g re as e every 3 m on th s if th e instrument is u se d daily, or every 6 mo nth sif is u se d l es s than daily.

4 Keep the instrument covered and freefrom dust when not in use.MI ROTOME KNIVES

icrotomeknives must be kept in perfectcondition if the technologist is to produce goodhistologic preparations for patient diagnosis. hesharpening of microtome knives is a skilledtechnology a nd may be done in the laboratorywit h t he many adequat e and good i nstr umentssold today see discussion on knife sharpening,below), or, if technical time is costly, may bedone commercially by many companies supplying laboratory needs. C Sturkey P.O. Box59, Per ki omenvil le, Pa. 18074) manuf acturesmicrotome knives and does an excellent job ofboth reconditioning and sharpening microtomeknives. They are picked up and del iver ed i n t hemetropolitan Philadelphia area, but may be serviced by mail an ywh ere in the country. Wehighly recommend this service. is economicaland expedient i n a laboratory where t echnicaltime is expensive and at a premi um. he wedge-shaped knife is used for cut ti ngparaffin-embedded material on a rotary microtome, for cryo stat s ectio nin g, a nd for c utt in gfresh and fixed ti ss ue s on a clin ica l freezingmicrotome. heplano-concave knife is used forcelloidin sectioning.The standard microtome knife has a wedge

angle of about 15 degrees. A bevel angle between the cutting facets for knives of Americanmanufacture varies between 27 and 32 degrees.For t he best possible secti ons of a given specimen, the kni fe shoul d be adj usted to t he propertilt for the particular specimen. For an averages pe ci me n th is tilt s ho ul d be e no ug h to give aclearance angle of 3 to 8 degrees Richards).KNIFE SH RPENERSLyn Richardson Margaret S JUdgeand Maria Sugulas with permission of The merican Society for Medical TechnologySharpening hand hesingle most important step in the laboratory is to produce a knife edge that will cu tt hi n secti ons wit hout knife mar ks or compression. An adequate tissue section may still be obtained wit h a shar p knife despi te poor fixationand processing. heimplications are obvious.

INSTRUMENT TION a section can be obtained, a diagnosis is possible

he t echniques for produci ng sharp kniveswere applied by h an d for h un dr ed s of years.Stone and leather were used to grind and polishsteel to a k ee n edge. Un til t he de ve lop men t ofau toma tic knife s ha rp en er s, s ha rp en in g byhand was considered a vital skill of the histologist.Hand- shar peni ng tools commonly used arehones with coarse to fine abrasive surfaces andstrops. A hone is e it he r a n at ur al or s yn the ticabrasive stone or glass plate that is used to forma bevel or wedge tip on the microtome knifeand to remove nicks. A h on e s hou ld be at l ea st2.5 x 20 x 30 cm long to prevent uneven wearof the knife.Excellent oil stones are produced in theUnited States and may be p ur ch as ed as a unit*consisting of three stone grades that rotatethrough an oil bath to which they are attached.The t hr ee stone grades are 1) coarse synthetic for grinding a new bevel and removing largenicks, 2) medium synthetic) for removing theserrated edge resulting from coarse honing and, 3) fine Arkansas oil stone natural) for finishing the edge.Some t echnol ogists prefer t he wat er stonesbecause a stream of water can be used whileone i s honing to wash away met al and abrasiveparticles that might damage the knife edge.fa glass p la te is to be u se d as a hone, it mustbe ground with a car borundum paste, oil or wa-ter mixture, and another sheet of glass to pro

duce an abrasi ve sur face. The ground plate isused with an abrasive or soap mixture to honethe knife. A carborundum paste is recomme nd ed for fas t grin din g a nd Di ama nt in e forfinishing. A neutr al soap solution may also beused for finishing. he hones must b e k ep t flat to prevent uneven gri nding along t he knife edge. Diamondr ubbi ng blockst are available for r esur faci ngand finishing all worn hones. hehone, regardless of type, must be washed and dried with alint-free cloth after resurfacing or honing.Strops are used to finish the knife edge. Thisfinal pol ishi ng step forms a facet or secondary

bevel at the tip of the bevel Fig. 1-14). Th estrop ping p ro ced ure is t he a ct ua l s ha rp en in gstep. The strop gives under t he pressur e of t heknife and the bevel angle at the very tip in- Lipshaw Manufacturing Co., Detroit, MI 48210t Norton Co., Troy, NY 12181.


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when the knife is laid across the hone or strop.The bevel must lay flat on the surface in order togrind the bevel to the edge without changingthe angle. This is extremely important andshould be checked before honing. The honingback is fitted to the knife at the factory and thetwo should be returned for refitting if the bevelis not flush with a flat surface. Any movementof the knife back on the knife should be corrected. loose, the back may be closed slightlyby pressing in a vise. When the knife back becomes worn, it should be replaced, since a wornback will cause a change in the bevel angle.Although a variety of opinions exists amongtechnologists on the choice ofhones, strops, andabrasives and when or how to use them, thefollowing basic rules should be observed:

1 Choose the finest quality hones, strops,and abrasives available.2 Clean the knife thoroughly before sharpening.3 Check the honing back for wear and fit4 Always slide the indicated end ofthe honing back onto the knife. The AmericanOptical and Schmid honing backs arerounded at the inser tion end. The Upshaw back is not marked. One endshould be marked with a file or diamondpen to ensure proper placement whenthe insertion end is not indicated.5 Check the hone for uneven wear. Grindflat and resurface with a rubbing block, ifnecessary, and wash well with hot water.6 Check that the bevel lays flat on the honeafter the back is fitted to the knife.7 Lubricate the hone with light machine oilor soap and water depending on the typeof stone used.8 Hone and strop the knife with the leastamount of pressure. The weight of theknife is usually sufficient. The only timepressure should be applied is while honing a curved edge. In that case, applypressure to the knife back along the highareas.9 Use a microscope to check the progressof the knife edge at 100x magnification.A fine bright line should be visible. Thisis the secondary bevel.1 Remove any abrasive along the knifeedge after a change of hones and beforestropping.11 Examine the strop for nicks and replaceif damaged. Keep the strop free of foreignmatter. Lubricate the strop occasionally

\I \ I \ \ /t,

\\\

Fig. 1-14. 1 Primary bevel hone . 2, Bevel angle.3, Secondary bevel strop . From Judge, S., Richardson, L. and Sugulas, M : Knife sharpening madeeasy, Bellaire, Texas, 1978, The American Society forMedical Technology.

creases slightly to create a very small facet,which becomes the cutting surface. The facetappears as a fine white line when viewedthrough the microscope at 100x magnification.Excess stropping will round the edge and dullthe knife. Six to 12 strokes on either side ofthe knife are usually sufficient.Some strops are e m e ~ with diamond particles. These strops are excellent bu t should beused cautiously. Five strokes on either side ofthe knife are recommended.Strops are leather horsehide, calfskin, andpigskin or linen. There are three types ofstrops: 1 the hanging st rop razor strop ,2 the saddleback strop, a strop stretchedacross a heavy frame and made taut by turningan extending screw at one end, 3 the blockstrop, a strop mounted on a felt-padded woodblock. The block strop is recommended for better support of the knife.Linen cloth, when fastened to a stroppingframe and stretched to its maximum, serves asa fine finishing strop. The linen strop producesa sharp edge and is sometimes used alone tostrop a knife that is slightly dull.Whether honing, stropping or doing both, around metal ba ck must be slipped onto a wedgeor plano-concave knife. A handle is locked intoone end of the knife beforehand. The honingback lifts the knife to the correct bevel angle


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THEORY ND PR CTICE OF HISTOTECHNOLOGY

tant magnification of 100. The wooden knifeinspection block holds. the knife at the properangle for viewing. See Figs. 1-15 to 1-20.The following is the recommended procedurefor sharpening on the Model 925.

o rse honing oper tion Inspect the knife for the presence and sizeof nicks.2 Place the glass hone plate in the upper position. The proper positioning of the glassplate is essential for correct coarse honing,following the instructions of the manufacturer.

3 Attach the knife with the American Opticaltrademark to the right; this puts the handleslot to the left. Check to see that the knifeholder is correctly and securely fastened tothe shaft. Then with the two clamps facingup and the clamp screws loosened, installthe knife so that the end with the AO trademark is to the right. This places the slottedend of the knife to the left, while facingthe instrument. Tighten the two clampscrews until the knife is safely bu t temporarily fastened. ote Always sharpen thelongest knives first.

4 Center the knife in the holder using a ruler.

Surface of hone

oI 2I 3 5 mmI Iin

Fig. 1-16. Knife and honing back drawn to scale to show extent and formation of cutting bevel andfacets. From Richards, O W.: Effective use and proper care of the A.O. Microtome, Buffalo, N.Y.,American Optical Scientific Instrument Division.

Finish: dull black

B ~ > llumin tion ngle

Fig. 1-17. Block for supporting microtome knife dimensions in millimeters . A, Position for examining sharpness B, Position for observation of polish. From Richards, O W.: Effective use and propercare of the A.O. Microtome, Buffalo, N.Y., American Optical Scientific Instrument Division.


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INSTRUMENTATION 5

AFig. 1-18. A Impression made by knife s cutting edgeshowing unequal sharpening. B Geometry of knifeedge angles. C Rake and t il t angles for a clearanceangle of 5 degrees for proper placing of knives withunequal facets. From Richards, O W : Effective useand proper care of the A.a. Microtome, Buffalo, N.Y.,American Optical Scientific Instrument Division.)

ber to apply the coarse abrasive at least 2.5cm ins ide the front edge of the plate. It isimperative that the plate not become drywhile the sharpening procedure is in use.7 Firs t t ime set ting is 60 minutes . Close thePlexiglas cover and turn on the switch.The knife is then au tomatically strokedagainst the high-frequency vibrating glassplate. After the equivalent of three fullstrokes on one side, a cam follower automatically turns the knife and hones theother cutting facet with three strokes. Thiscycle is repeated continuously for 60 minutes.8 Remove the knife carefully. At the end ofthe cycle, the knife holder will stop in araised horizontal position. Note: If theholder should stop upside down, with theknife c lamps facing downward, turn theautomatic timer knob beyond the 10-minute setting. Wait until the knife starts tomove upward and then go through a halfcycle and stop it in the correct raised position.

9 Clean the knife and inspect its condition.It will be necessary to wipe the knife witha clean cloth moistened with a solvent suchas xylene. Then inspect the cutting facetunder the microscope at 100x .1 Clean the glass plate-continue coarsehoning. To clean the glass plate, merelywash it under hot running tap water usinga detergent to remove the abrasive and finemetal particles. Then wipe the plate so i t iscompletely dry. Now apply fresh coarseabrasive and continue honing as required. Periodically inspect to check theprogress of the knife. Remember to addabrasive as needed and to wash the platewhen the abrasive becomes nearly black incolor.After the coarse honing procedure has beencompleted, it is then necessary to pu t a fine finished edge on the knife. This is accomplishedby the use of the fine honing procedure.

ine honing pro edure1 Check the knife for cleanliness. It is essen

tial that all t races of the coarse abrasive areremoved from the knife, the knife holder,and the glass honing plate before beginningthe fine honing. Be sure to check and cleanthem thoroughly. If the same glass honingplate is to be used, remove it and wash theplate under hot running t ap water us ing an

r Bevel

B

A Wedge angleE G B Bevef angleC Upper facet angle 50 D Lower facet angleI E Cutt ing angleJ G Tilt lower side i / H Clearance ang Ie E Block620 / 13 o f = A G = A D H j e d g e n ~ ~

/ ../ / / /

Rake //;

FU

This is to ensure proper balance of the knifeduring the honing procedure. Using a ruleradjus t the knife s posi tion until the samedistance is measured from the outside edgeof each c lamp to each end of the knife.Gradually tighten the clamp screws, alternating from one to the other until the knifeis held firmly in place.5 Thoroughly shake the coarse abrasive no937. Shake the abrasive thoroughly until allof the particles are in suspension.6 Apply the coarse abrasive to the plate. Thisis accomplished by squeezing a narrow ribbon, about the width of a pencil, of thecoarse abrasive across the plate. The ribbonshould be approximately equal in length tothe knife that is being sharpened. Remem-


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90 ~ r ~ c = 45 or less 30 or less II

5 = / / 0 ~ 45 / / ~ V D0 ~5 10 15 20 25 30 35 A5 c

Z ~ ~ ~ Effective cutting angleU = Slice angleObject 8 = Normal cutting anglesin l = sin sin c

Fig. 1-19. A Showing slice angle, c for square and rectangular blocks. Decreased wedging withsmall slice angles. C Relations between them plotted from data of Preston (1933). From Richards,O W.: Effective use and proper care ofthe A D Microtome, Buffalo, N.Y., American Optical Scientific Instrument Division.)

ordinary detergent and carefully dry theplate.2. Place the glass honing plate in the lowerposition. After inspecting the glass plate forevidence of wear, place in the lower position.3. Attach and center the knife. Install and center the knife the same way as the coarsehoning procedure. Always double check tomake sure tha t the clamp screws are tight.A loose knife might cause a serious accident.

4 Shake the fine abrasive no. 938.5 Apply the fine abrasive to the glass plate.6 Set the timer for 30 minutes when beginning

the fine honing operation. Be sure to keepthe Plexiglas cover closed while the instrument is running.

7 Clean the knife and inspect the condition ofthe edge. t is imperative that all abrasive beremoved from the edge of the knife before tis inspected under the microscope at 100x.Look careful ly for any small nicks that arestill evident in the knife edge. The removal ofsuch nicks, and not the width of the finefacets, determines the progress of the sharpening procedure.

8 Continue the fine honing procedure. Experience with models 925 and 935 will soonenable the technologist or student to estimate how much more time is needed to

achieve the desired results. Remember thatmicroscopic examination is still the final determining factor. Add the abrasive as required and periodically check that the pla teis not a grayish color from the buildup ofmetal particles. Should this occur, the plateshould be washed and fresh abrasive applied.9 After fine honing is complete, the knife

should be cleaned and carefully wiped dry.Where the atmosphere is corrosive and theknife is to be stored for any length of time,lubricate the knife with a good grade oflight,neutral oil The knife should never bestropped.The honing action of the knife against thefrosted glass plate will eventually cause ashiny path to be worn on the face of the plateas wide as the length of the knife.

edressing the platesFollow manufacturer s instructions.

leaning and lubrication1 leaning The Plexiglas cover and the outside enameled surfaces should be kept clean.Use warm water and detergent to keep theseareas clean. Then sponge out and wipe drythe catch-basin on the hone table after eachsharpening session. The knife holder, knife-


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A

c

E

F ig. 1-20. A , Microtome knife-edge from th e Shandon-Elliott Microsharp technic. B, Microtomeknife-edge using 6 m diamond compound. C, Microtome knife-edge finished with 1 m diamondcompound. D, Microtome knife-edge before sharpening. E, Microtome knife-edge during sharpening.F, Microtome knife-edge at completion of sharpening procedure. FromJudge, S., Richardson, L andSugulas, M.: Knife sharpening made easy, Bellaire, Texas, 1978, Th e American Society for MedicalTechnology.

B

o

F

holder shaft, a nd t he exposed fittings are of anoncorrosive material an d require no a ttention other than normal cleaning. ubrication Lubrica te the instrument approximately once each month, more usedextensively. Lifetime Oilite bearings are used

throughout th e instrument; therefore only afew points require lubrica tion. Pla ce just adrop or two of Bear Brand oil on the fol-lowing:a Round felt pad beneath t he wo rm g ear o fth e motor.b Th e two bra ss bea rings of th e pivot pointsbetween the carrier arm assembly and theslide castings.c Th e bra ss bea ring a t the e nd of t he mo to r

crank arm.

d Th e two slide rods. For this area, useMolykote spray graphite lubricant, whichis usually available locally.A modified p ro ce du re u si ng d ia mo nd compound ha s also been developed for us e on theAmerican Optical sharpeners, as follows.Richardson s modified coarse honing Place th e frosted glass plate in th e coarse honing

position.2 Apply a small amount of 6 otm diamond compound

to th e plate.3. Lubricate th e diamond compound with lubricatingflUid4. Ru b th e lubricated compound over th e plate.

5 Allow th e knife to recondition for 20 minutes.6 Remove th e knife from the sharpener and clean itwith xylene.


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to sharpen microtome knives has made thePerma-Sharp knife sharpener a widely acceptedtool for obtaining good tissue sections.he mai nt enance of t his i nst rument i s minimal. he Perma-Sharp does not involve lappingb ars to fla tten w or n plates, or messy abrasive

mixtures to gri nd and poli sh knives sharpenedon rotating plates. On the Perma-Sharp, theknife is passed through low-speed circularhones a nd s trops to achieve a fine edge. hecircular hones withstand months, or even years,of use before the honing surfaces need redressing. A simple redressing device can be purchased from the m an uf ac tu re r to re dre ss t hehones o n all P er ma -S ha rp models. T he life ofthe stropping wheels is determined by frequency of use. Even with frequent use, the stropswill last for years.An i nexpensi ve abrasive wax sti ck is used onthe cir cular str ops for t he final pol ishi ng step.All that is necessary to mai nt ai n t hem i s an occasional removal of the wax buildup.he Per ma-Sharp knife sharpener i s a relatively simple and effective means for producingexcellent c ut ti ng surfaces on knives of standard shapes that are less than 25 cm 250 mm)i n l engt h and a minimum of 2 cm in width.he operation of the Perma-Sharp sharpenerdoes r equi re some dexterity, since t he kni fe i smoved along the carrier b ar by th e operatorwh o h as also s et the knife s position for the correct bevel.For Perma-Sharp, there are several importantpoints that must be covered:1. Examine the knife to be sharpened beforeproceeding.2. t he kni fe is badly damaged, i t will needadditional honing to we ar away th e d amage.3. the knife is new, or being sharpenedo n th e Pe rma-Sharp for the first time, itwill need additional honing to establish a

new bevel.4. A felt ma rking pen may be used to markthe knife bevel to the edge of the knifeapproximately 2.5 cm from either end ofthe knife. W hen t he mar ks are ground offby the hon es, the n ew bevel is completeI t is re ad y to pass t hr ou gh t he strops.5. Always slide the knife into the holder withthe same end to the left. he knife may bemarke d with a diamond marking pen, orus e the drill hole found on most knives.6. Always clean the knife with a wipe soakedin a solvent, such as xylene, to remove paraffin and debris.

INSTRUMENT TION Instruction for the Perma Sharp MK

he Model MK 4 Hacker Perma-Sharp has anumber of i mprovement s t hat make t hi s knifesharpener easier to operate than previous models MK-I to MK 3. n the MK 1 to MK 3 models, rapi d on-off swi tchi ng is r equi red to runt he strops at slow speed whil e t he final polishing s tep of stropping is done. he end r esul tdepends on the dexterity of the operator.n the MK 4 the addition of t he variableslow-speed control allows an inexperiencedoperator to produce a highly-poli shed edge.When properly adjusted and operated, the MK 4will sharpen k niv es t ha t c u t s ec tions as th in as

f mhe MK 4 is a well-constructed, quality mi-crotome knife sharpener th at can produceexcellent knives when properly used.

he sharpening i nstr ucti ons for t he HackerPerma-Sharp MK 4 are as follows: urn switch to N or full-speed position.Apply abr asive compound sti ck lightlyacross each rotating strop once only.

Note Apply abrasive once for every sixto eight kni ves sharpened. Thi s will depend on the number of stropping strokesused on each knife. he wax i n t he abrasive tends to build up on t he str ops if applied too frequently. he abrasive becomes embedded i n t he wax, t hereby reducing abrasive polishing action.) Switchthe instrument off.2. Clamp the knife in the holder.3. urn t he ver tical control knob at t he topfront of the panel from the left to theright until the knife edge clears the intersection of the hones. Do t hi s before t heknife is moved between the hones to prevent jamming.4. Slide t he knife b etw ee n t he h on es untilthe left end of the knife holder is approximately 6 mm from the sharpener s side.he gauge reading will then be madealong the a ct ua l c ut ti ng s urfa ce of t heknife. the center of t he kni fe i s higherthan the outer edges, additional honingwill produce a straight edge.5. Attach the centrality gauge to the knife.Keep your hands away from t he i nstr ument or table during the next steps, sincea false gauge reading could result.6. Face the sharpener in line with thegauge. View t he ga uge from a positionabove the center line for accuracy.7. Center the knife between the hones by


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THEORY AND PRACTICE OF HISTOTECHNOLOGYturning thehorizontal control knob frontside p an el ) wtth the r ig ht ha nd . Afterevery l eft o r r ig ht turn o f t he knob, tiltthe knife lightly back and forward againstt he hones. W hen the bubble positions onthe gauge are an equal distance from t hecenter line proceed to the next step.8. Position the kni fe bet ween t he hones forthe correct bevel by turning the verticalcontrol knob, top front panel. Tilt th eknife lightly backward and forwardagainst the hones after every right or leftturn. When the knife is positioned for thecorrect bevel, the bubble of the gaugewill to uch t he outside e dg e of t he blacklines on either side of the center linewhen t he knife is tilted. Repeat s teps 7and 8 unt il a cor rect set ti ng is obt ai ned.

9. Remove t he c ent ra li ty g au ge a nd movethe kni fe away from t he hones.

10. Switch the ins trument to N position.Allow h on es to reach maximum speed.Slide knife edge just inside rotatinghones. Tilt knife forward a nd quicklymove knife ac ros s b ac k h on e with li gh tpressure. Do n ot p au se o r apply u ne ve npressur e. Til t knif e back with knife edgejust inside hones. Move the knife quicklyacross front hone.11. nspect theknife. nicks are still visible,repeat the honing step. fknife is beings ha rpen ed on this instrument for thefirst time, repeat the honing step to complete new bevel. Do not h on e mo re t ha nnecessaryon pass on eit her side of t heknife is usually sufficient. heknife willha ve a r ou gh b ur r or s er ra te d e dg e w he nthe bevel is complete.12. Move the knife along the carrier bar tothe strops. Do n ot s tri ke t he knife e dg eagainst the hones.13. Move the knife evenly along th e backstr op, usi ng moder at e pressur e. Beforethe knife leaves the strop, tilt t he knifeb ac k a nd move it a cross t he front strop.Repeat this process 10 times. The knifeedge will h av e a fine burr at t hi s point. ote f t he knife does leave t he strops,there is a dange r of strop damage. Thekni fe edge may str ike the side o f a stropon subsequent passes.14. Switch the ins trumen t off. Continues tr op pin g u nti l s tro ps slow to a stop.Strop the knife twice o n t he stationarystrops. This step furthe r removes theburr.

15. Switch the instrument to P. urn thespeed-control knob, at the left of t he toppanel, to slow the strops down to approximately one revolution per minute. Strop10 times gn either side of the knife.When the strops rotate at very slowspeeds, t hey r emove the fine b urr andpolish the final facet o f the knife. Uselight pressure. Too much pressure maycau se the strops to stall. Increase thespeed of the strops slightly if stalling occurs with light pressure.16. Switch the instrument off. Move theknife to t he r ight end of t he car ri er bar.Carefully move the knife holder to an upright position. Grip the knife holder. Turnthe Allen screw counterclockwise toloosen t he clamp. U se great c are in removing the knife.17. Remove abrasive from the knife edgewith Scott s Micro Assembly Wipes No.5 31 0 p. 4 44 ) d ip pe d in xyl ene or othersolvent. Wipe toward the edge with shortstrokes to prevent damage to the edge. is important to remember th at once theknife is removed from the holder, it mustbe recentered and repositioned if furthersharpening is necessary.An alternate stropping method:

1. Strop 10 tim es on each side at maximumspeed.2. R ed uc e s pee d to m ed iu m. Strop 10 timeson each side.3. Reduce speed to slow approximately 60

RPM). Strop 10 t imes on each side.T he edg e will be sh arp ; so h and le the knifewith care. inten nce

Maintenance is essential for the proper operation of t he equipment . he follOwing mainten an ce ins truc tion s are for models MK 1 toMK 4

1. Remove the cabinet and vacuum-clean itat le as t o nc e a yea r to avoid creating a fireh az ar d from a cc um ul at ed dust. Removean d e mp ty the dus t tray at least once amonth. he handle for the tray is at theleft base of the cabinet.2. Remove abrasive buildup on strops. Clampan old knife in the h old er a nd a djus t acco rdin g to s ta nd ar d pro ced ure s. Bypassthe hones and strop hard against stropsw he n the y a re ro ta ti ng at full speed. Orremove the rod from an American Opticalknife handl e and run the r ound grip end


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along the strops. Hold the rod firmly andapply moderate pressure along each strop.3 Apply a few drop s o f l ig ht m ac hi ne oil tothe carrier bar once a week. Do not drip onhon es or strops. Spread along bar withScott s Micro Assembly Wipes No 5310.Remove excess. Slide holder along the barseveral times.4 heck t he d ia me te r o f t he h on es with t hespecial gauge supplied with the sharpener.a Move t he c ar ri er b ar fully forward byturning the horizontal control.b I ns er t g au ge b et we en the h on es a nd

the motors so that the base of the gaugerests on both shafts with the gauge pen_across both wheels.c Rock the gauge back and forth. thereis no movement of the handle, theh on es a re l es s than 14.83 cm in diameter and must be replaced.5 To remove small irregularities, hones mayneed redressing i they are new or rough.A s imp le r ed re ss in g devi ce i s now availabl e from H ac ke r I ns tr um en ts . I ns tr uc tions are included or you may have theHacker representative redress the honesfor a fee.6 Replace the synchronization belt at the lefto f s tr op s i f it be com es worn. U np lu g t heinstrument, remove the cover, and cut theold belt. S tr et ch the new belt over onestrop. Twist the belt into a figure 8 andstretch the end over the other strop. Makecertain the center rod is between the belt.7 Remove t he ho ri zo nt al a nd vert ical controls a nd oil the threads. Do not applygrease. Remove a ny g re as e f ou nd on therod ends b ec au se gr ea se will c au se slippage of the cont rol rods dUring s ha rpening.8 Make sure that your Perma-Sharp distributor stocks hones, strops, and belts so

that you h av e qUick ac ces s to pa rt s w he nthey are needed.elpful hints Impossible to center the knife. Make certainthe knife is positioned in the V groove on thelower clamp of the centrality gauge.2 Wavy knife edge. Uneven pressure appliedto hones. Hone again using light, even pres

sure until the knife has a straight edge. Stropas usual.International Micro Optical, Inc., 5 Daniel Rd., Fairfield,N.]. 07006.

INSTRUMENT TION 3 Too much pressure has b ee n appli ed a t t heends of the knife during honing step. Thiswill not interfere with the actual cutting surface o f the knife. The knife may be honeduntil the edge is st rai ght from end to end.Strop at least 20 times to remove rough burr.

Polish on slow-moving strops.4 Knife edge is dull or leaves knife marks aftersharpening.a Make c er ta in knife is firmly cl am ped i nholder. Any movement of the knife couldaff ect t he s ha rp en in g pr oce ss as well asthe sharpness.b The new bevel may not extend to theknife edge. Mark bevel with a felt-tippedpen from top of bevel to knife edge 2.5 cmfrom either end. When honing step grindsaway the m ar k compl etely, t he bevel iscomplete. Proceed to stropping steps.c Excess stropping may dull the knife. Re

sharpen, or use the solutions given above.d B ui ldup of w ax on st ro ps will r ed uc e th epolishing action. See m ai nt en an ce instructions for method of removal. Applyabrasive lightly to the strops before resharpening.5 Knife holder does not slide smoothly alongcarrier bar.a See maintenance instructions.

b nspect t he c ar ri er bar. it is bent ordamaged, it must be replaced.6 The controls turn during sharpening procedure. Unscrew control knobs and pull themout completely. Remove grease and clean rodthreads in xylene. Dry and apply oil tothreads as described in maintenance procedures.

Shandon Elliott SharpenersMK n MK

The Shandon-Elliott MK I was o ne o f t he firsta ut om at ic knife s ha rp en er s o f t hi s type to besold in the United States. The MK II model,however, was the first sharpener to catch onThe MK II was to revolutionize knife sharpeni ng wi thi n a s ho rt period o f t i me a ft er i t was onthe market. This instrument has a simple construction, is easy to repair, and easy to operate.The instrument consists ofa round glass plate36.83 cm in diameter. also has a knife pressure adjustment, which adjusts the amount ofpressure with which the knife rests on thesharpening surface. With this particular instrument, t he plate rotates in a ci rc ula r m an ne rwhile the knife changes from side to side to assure t hat ea ch edge is sha rpene d evenly. A


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THEORY AND PRACTICE OF HISTOTECHNOLOGYspeed control is-built into the instrument so thatthe knife will change back and forth at a fast orslow setting, depending on the desired sharpening procedure.

is the technologist s responsibility to choosewhich abrasive is best sui ted to current needs.There are, however, two main considerationsthat govern the choice of abrasives:1 Straightness of cutting edge2 Condition of edge

The four recommended grades of abrasivethat come with the knife sharpener ar e as fol-lows:

1 Aluminum Oxide 2F, or coarse abrasiveto be used only when the edge is badlynicked or for the reconditioning of theglass plate.2 Aluminum Oxide Optical 50, or mediumabrasive-this is used until a straight edgeand new facets are obtained.

3. Aluminum Oxide 1200, or fine abrasivethis is for polishing the knife edge.4 Polishing Alumina 350-this grade is fordaily maintenance of the knife edge.The actual procedures to be followed can bevaried at the discretion of the user.

utosharpShandon-Elliott brought out a new model theAutosharp III. has all the same features, except that now instead of two glass plates, a glassand copper plate* are used. Also, instead ofpow

dered abrasives, a 3 Lm diamond compound isused, and this is called the Microsharp Technique. This was a revolutionary step in knifesharpening. Expectations were that an excellent edge could be achieved in 5 minutes. Again,technologists found disappointment in the timeframe. However, this procedure produced farsuperior knife edges than have ever been produced before.utosharp V

Since the development of the MK II and Autosharp III knife sharpeners, Shandon-Elliott hasb rough t ou t a new model called the AutosharpIVThis new instrument is basically the same asthe other two models except for the following:1 The change-over control is designed for 5-,10-, or 25-second intervals.2 The knife-damping device has been eliminated. Recently copper plates have been replaced byiron plates.

3 The bevel setting is now in degrees insteadof in numbers.4 The knife-alignment device has been eliminated.5 The use of the glass pla te has been eliminated. Instead of glass plates, two copper*microsharp plates are used.6 Powdered abrasives have been replacedby an 8 Lm diamond compound for reconditioning and a 3 m diamond compoundfor finishing.

modified utosharp V knifesharpening technicJohn Spair Jr.1 Clean knife thoroughly with xylene, check

under microscope lOX to determine condition of knife edge. If knife has a smooth edgewith no visible knicks, go directly to the finishing procedure. If knife has knicks orburrs, first take burrs off with a woodentongue depressor, and then go to the reconditioning procedure doing the follOwing finishing procedure.

2 a knife needs recondit ioning, you shalldetermine the t ime it takes to get out knickson a time scale o f5 to 30 minutes . a knifeneeds longer than 30 minutes, you mightwant to use a glass plate and a powderedabrasive (coarse) to grind your edge downand then go through the reconditioning procedure and finishing. You might also want tosend your knife out to a professional knifesharpening company if you are pressed fort ime or do not have the mater ials available.Under normal conditions you should neverhave any knife that is so badly knicked thatit needs this special treatment, unless, ofcourse, carelessness is involved by the technologist.

Reconditioning Apply a small amount (no more than 2.5 cm)of 8 Lm Shandon-Elliott diamond compoundto a red b cked copper* plate, or whateveryou have designated for this purpose.2 Lubricate the diamond compound with 25drops of Microsharp Lubricating Fluid, and

spread evenly over the plate.3 Set facet angle for whatever is indicated onthe knife box. (We have found a 35 degree tobe satisfactory for all our knives.)4 Set the pressure control to maximum. Recently copper plates have been replaced by iron plates.


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5 Set the change over to 25 seconds.6 Allow the knife to sharpen from 5 to 30 minutes. Remember that experience is the bestjudge for this step. n the beginning it maybe better to keep checking the knife microscopically before finishing the edge. t is alsoa wise idea to mark your knife box according to the procedure or time you used sothat you can always refer to it should theneed arise. I find that most knicked knivesusually need no longer than 15 minutesunless i t is a bone knife.7 When the time goes off stop the machine inthe forward position to remove your knife,and clean the knife off with xylene.

olishing pro edure1 Apply a small amount no more than 2.5 cmof Shandon-Elliott 3 p m diamond compound

to a green-backed iron plate, or whatever youhave designated for this purpose.2 Lubricate the diamond compound with Mi-crosharp Lubricating fluid, about 25 drops,and spread evenly over the plate.3. Set facet angle the same as you set i t for thereconditioning procedure.4. Set pressure control for m ium pressure.5 Set the change over control to 10 seconds.6 Set the t ime for 10 minutes and run.7 When machine has stopped, reset the pressure to minimum, the change-over to 5 seconds, and the timer for 15 minutes and letrun.8. Take knife off the machine when it is in theforward position, Clean it with xylene andtake knife clamp off knife.9 Check the knife under the microscope. Theedge should look completely straight if youhave done everything right. there are anysmall knicks because you did not give itenough time on the reconditioning procedure, mark these areas with a penci l so thatthe cut te r may avoid these areas. Spread athin film ofoil on the knife edge to preservethe knife and re turn to the box Mark theknife accordingly.Troubleshooting methods he troubleshooting methods are divided into three sections. he first section to be discussed will be theknife hol ers here are at present three typesof holders the standard holder that most laboratories use, the holder for drilled knives, andthe universal holder that is provided with theAutosharp IVhe main problem encountered with the

INSTRUM NT TION

knife holder is a technical error. he standardholder is placed on a knife without a set pattern. In otherwords, the same holder is not usedwith the same knife. This crea ted numerousproblems, such as double bevels, and evengouged plates. Since the holder is not centeredcorrectly each time, the same problems arose.This is solved easily when one numbers boththe knife holder and the knife, so that the sameholder will always be used with the same knife.The holder is scored and the knife is scored;doing so ensures that the knife is placed in theholder the same way each time.To ensure a correct bevel and not an unevenone, the holder with the knife in position andlocked down is measured on both sides to eliminate the chance of having the knife unevenlybalanced in the holder.The s tandard holder cannot be used on the,Autosharp IV without some difficulty. It tends to

b e ~ m e loose and let the knife slip out andgouge the plate. he holder can be modifiedby one of two ways. First, the notch in theholder can be scored deeper, or second, a smallhole can be drilled into the notch to lock theholder to the locking screw.The second holder is the one for drilledknives. This was believed to be the answer.However, not all companies manufacture drilledknives. heproblem that arose using the drilledknives was unusual. heknife tended to gougethe plate. the knife is placed in the same holder the same way each time, most gouging wouldbe eliminated. Therefore, the holder and knivesshould be marked to ensure uniformity. heuniversal holder, the third type, was introduced with the advent ofthe Autosharp IV thas been observed that this holder w ll not holda knife the same way twice. Because of theconstruction of the holder, knives can lean for-ward or backward, depending on the angle atwhich they are locked in. t is difficult to consistently t ighten the screws evenly on eitherside of the holder. To help eliminate this problem, the knife should be marked so i t faces thesame way every time it is placed in the holder.This does not eliminate the problem of the knifetilting differently each time.

Now that all the holders have been briefly dis-cussed, here is a simple check list to follow be-fore one starts to sharpen a knife:1 Place the numbered holder with the properly numbered knife.2 Make sure that the score on the holdermatches the score on the knife.


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THEORY ND PR TI E OF HISTOTE HNOLOGY3 Measure both sides of the knife to ensureequal distance. Also measure the distancefrom t he edge o f the holder to the edge of

the knife. This will ensure the equal dist an ce of the ho ld er a nd kni fe edge. T hi swill help eliminate placing the knife in theholder at an incorrect angle.4 Clamp the knife in the knife sharpener. Donot tighten excessively. Overtightening ofthe clamp might cause it to break.Now that the holder and knife are properlyaligned, there should be no more problems thatwould cause the M icr os har p pl at e to gouge.T his does not always hold true.

is also essenti al to w at ch for w ea r insi dethe device. One laboratory was repeatedly gouging the plate. T his did not occur when the knifefirst started, but after it had been sharpeningfor a while. The problem was simple bu t not obvious. Th e alignment device was too small. Thismade the screw in the knife holder loosen afterthe knife had been rotating. This, in tum allowed the knife to slip forward a nd go ug e t heplate. There are two ways to correct this. First,simply file the device so that the screw will notloosen and, second, have the company replacethe alignment device.

The second section to be discussed, the -crosh rp copper pl tes is of utmost importanceto the technologist.With proper care and maintenance o f t he seplates, excellent knives can be obtained.Many problems have been encountered withthe use of these plates. Ther e are two mainproblems that should be considered. First is thef lat nes s o f t he plat es; s ec on d is the ease withwhich the plate can be gouged.Flatness is one of the sel li ng p oi nt s for t heuse of the copper plate. The manufacturerstresses the fact tha t the copper plate will notwear like previously used glass plates. For themost part, this is a t rue claim. However, thecopper plate does and will wear. has been ob-served that some copper plates are shippedw ar pe d or n ot flat from the factory. The platesare either convex h ig h) so t ha t the middle oft he knife is gr oun d d ow n o r c on ca ve bell ied)so t ha t only the en ds s ha rp en. The interestingaspect of this problem is that the knife onlyshows these defects on pretreatment with eithert he gl ass p lat e a nd a br as ive or the new procedures using large-mesh diamond compoundsfor reconditioning on the copper plate.Now the question is raised that perhaps thefirst p la te was no t flat or the knife was warped.It is a proved fact that just one diamond com-

pound will not reveal bevel defects. Th e knife ismerely ground to fit the plate. Alignment device out of adjustment2 Screws in the knife holder loose3 Bevel setting not locked4 Incorrect bevel setting the knife should

always be sharpened on the same setting5 Knife without an established bevelh t o with gouged pl te

If the gouge in the plate is not of major damage, we recommend that you, the technologist,repair it. T here are several ways to accomplishthis. First, if t he gouge is relatively small, apiece of Scotch Brite may be used to flatten thearea. A se co nd m et hod is t he use of an emeryboard or a fine p ie ce of s an dp ap er. Sh oul d theda ma ge be more serious, ot he r s teps m us t betaken to recondition the plate. O ne m et ho d isthe use of a diamond bar. has been found,however, that in a busy laboratory it is too timeconsuming and also causes the delicate Microsharp machines to break down more, requiringt he n ee d for r ep ai r mor e oft en than usual. An-other method is the use of coarse and fine sandpaper. The plate can be g ro un d dow n with analuminum oxide type of coarse sandpaperwrapped around a level, flat piece of wood youcan get from your maintenance department. f-ter you have ground down the plate enoughto get out your deep gouges, you can thenfinish the plate with a smooth finish with afine aluminum oxide sandpaper wrappedaround your wood. This should take you nolonger than a h al f ho ur . is advisable to weara face mask so that you w on t i nh al e t he copper dust, or you may finish u nd er ne at h ahood.To ensure that a proper bevel is maintained,t he knife shoul d always be sharpened on thesame plate. This can be accomplished by numbering the plate and recording both this numbera nd t he bevel s et ti ng o n t he knife box.

The third section deals with the use or misuseof diamond compounds. Numerous laboratorieshave reported inadequate results with recomm end ed s ha rp en in g times a nd m any laboratories are s ha rp en in g as long as 2 hours. Thisis not so much a p ro ced ur al e rr or a s i t i s a technical error. Some of the causes of this errorare as follows:

Contamination of plate or compound withd us t, grit, a nd t he l ike2 Improper lubrication3 Inadequate cleaning of plate4 Too much or too little diamond compound


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Employing the proper maintenance andcleaning procedures will hel p eli mi nate t heseerrors, with these recommended technics:1 Make sure s

03 Chap01 Instrumentation-e

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