JoyceMooreHT/HTL(ASCP)TechnicalSpecialistJeffersonRegionalMedicalCenterHisto-PathLaboratories1515West42ndAvenue.PineBluff,AR71603(501)541-7544

ALittleAboutMicrowavesMasson"sTrichromeStainGrocott"sSilver-MethenamineMethodforFungusKinyonsCarbolFuschsinAcid-FastOrganismsGordonandSweet"sSilverImpregnationofReticulumWarthinStarryTechniquesforSpiRochetesAlcianBlueMethodSouthgate"sMuciarmineStainPeriodicAcidSchiffReaction

ALittleAboutMicrowaves

Microwavestraveldirectlytothesolution.Inamicrowaveoven,amagnetronconvertsordinaryelectricalenergyintomicrowaves,whichareelectromagneticwaves,justlikerADIoandtelevisionwaves,buthaveshorterwavelengthsandhigherfrequency.Themicrowavesentertheovencavitythroughawaveguide.Afan-likedevicecalledthefieldstirrerormicrowavestirrerhelpsdistributethemicrowavesevenlythroughouttheovenchamber.

Microwavesarewavesofenergy,notheat.Theamountofmicrowaveenergyabsorbedbyagivenspecimen(or"load")dependsonmanyfactors.Amongthesearethesizeoftheload,itsorientationwithrespecttothewaves,andthedielectricandthermalpropertiesofthematerial.

Dependinguponthematerial,microwavesmaybereflected,passedthrough,orabsorbed.Forexample,metalreflectsmicrowaves.Thisiswhythewallsofamicrowaveovenaregenerallymadeofmetal,confiningthemicrowavesinsidethecavity.Thesee-throughpanelinthemicrowaveovendoorcontainsametalscreen.ThemetalscreenreflectsthemicrowavesyetenablestheinteriorofthecavitytobeobservedbecausewavelengthsofvisIBLelightcanstillpassthrough.Themicrowavescannotpenetratethisscreen,becausetheholesinthescreenaremuchsmallerthanthemicrowaves.

So-called"microwavetransparent"materialssuchassomeglass,pottery,paperandmostplasticsallowthewavestopassthrough.Whenusedascontainers,thesematerialsdonottakeupmicrowaveenergy,butallowthesolutionsinsideofthemtoabsorbthemicrowaves.Theabsorbedmicrowaveenergycausesdipolarmolecules(suchaswater)torotateattherateof2.45billioncyclespersecond.Theinteractionbetweentherotatingdipolarmolecules,ionsandnon-movingmoleculesinducesfriction,whichinturnproducestheheatthatwarmsthesolution.Thisissomewhatlikethewayheatisgeneratedwhenyourubyourhandstogether.Microwavesheatthesolutionsfromtheoutsidein,justlikesourcesofordinaryheating,butmuchfaster.

EnergyAbsorption:Manycharacteristicsaffecttheheatingrateofamaterial,suchasvolume,size,compositionandstartingtemperature.

Volume:Thelargerthevolumeofsolutionthemoretimeisneededtoheatittoagiventemperature.

Size:Thefrequencyof2.45GHzusedinalldomesticmicrowaveovenscorrespondstoawavelength(inair)of12.2cm(about4.8").The"antennaproperty"ofamaterialisbestwhentimesthediameteriscloseto12.2cm.Therefore,acupofespressoisgoingtobeaverygoodantenna,whereasa30µldropletonaslidewillhavepoorreceivingproperties.Attheotherextreme,ifthesizeoftheloadisgreaterthan2timesthepenetrationdepthofthemicrowaves,theheatingwillnotbehomogeneousthroughoutthematerial.

Composition:Ittakesmicrowaveslongertoheatthecenterofasolidblockofmaterialifitcontainsdielectricmolecules.Thehigherthecontentofthesemolecules,themoreenergyisabsorbed,leavinglessenergyforthecenter.

Startingtemperature:Thetemperatureatwhichsolutionsandtissueareplacedinthemicrowaveaffectsthelengthofheatingtime.Moretimeisneededtoreachaspecifictemperatureforrefrigeratedmaterialsthanforroomtemperaturematerials,eventhoughenergyabsorptioninacoldersolutionisgreaterthaninawarmersolution.

Containers:Metalcontainersshouldneverbeusedbecausetheyreflectthemicrowavesandshieldthesolutionsfromheating.Anyconductivematerialplacedinthemicrowavecavitycanradicallychangetheelectromagneticfieldswithintheovenandmaycausesparking(alsoknownasarcing).Formostapplications,microwave-transparentcontainersarepreferred.Containersshouldalsobeselectedbasedontheirsizeandshape,tooptimizepenetrationofthemicrowaves(seenoteonantennaproperties,above).

Howtochecktoseeifcontainersaremicrowaveproof.Fillaglasscontainerwithapproximately50mlofwater.Placeinthemicrowavenexttothecontaineryouaretesting.Setmicrowaveonmaximumpowerandheatforoneminute.Ifthenewcontainerishot,itisabsorbingmicrowaveenergy;ifitremainsatroomtemperature,itismicrowave-proof.Alwaysusethermalmittsorpotholderswhenhandlinganycontainerwithamicrowaveoven.Eventhoughthecontaineritselfmaybemicrowave-transparent,theheatedsolutioninsidecanstillconductheattothehands.

Agitation:Agitationpromotesevenheatingthroughoutavolumeofliquid.AgoodexampleisthepinkringcommonlyseenatthetopofthefluidinmicrowavePASstainingprocedures.ThislayerrisestothetopduetovaporizationofSO2.AgitationwouldovercomeanylargeSO2differenceinthefluid.

Rotators:Themicrowavestirrermoves("stirs")theelectromagneticwaveswithrespecttotheload;aturntablemovestheloadwithrespecttothestandingelectromagneticfieldswithintheovencavity.Thetheorybehindthisisthatthedistributionofmicrowaveenergywithinthecavityisalwaysimperfect,andtherotatorwillcausethematerialtopassthroughhotterandcoolerspots,averagingouttheexposuretomicrowaves.

Covering:Allcontainersusedinamicrowaveovenshouldbeopen,vented,orlooselycoveredtopreventpressurization.Ventedcontainersareadvisedwhenworkingwithvolatilefluids.Theyreduceevaporationandallowformoreefficientheating.Papertowelingcanbeusedasalightcoveringtopreventsplatterandtoabsorbmoisture.Waxedpaperhelpstoretainheatandmoisture.

StandingTime:Heatcontinuestobeconductedfromtheoutsidetothecenter.

HighAltitudeAdjustments:Solutionsmayrequireaslightlylongerheatingtime,aswaterboilsatalowertemperature.

Cost:Thecostcontainmentfactorisabigconcerninanylaboratory.Usingamicrowaveoveninthelabisalaborsavingdevice.Mostsolutionsusedmustbediscarded.Measuretechtimevs.costofsolution.

Safety:Aremicrowaveovensdangerous?MostresearchersagreethathighdosesofexposurecanleadtoBIOLOGicaldamageduetoheat.Thereisstillnoclearconsensusregardingthedangerfromlowlevelsofmicrowaveexposure.Microwaves,likeradiowaves,havemuchlessenergythantheamountneededtobreakeventheweakestmolecularbond.Thepotentialhazardsfrommicrowaveenergyshould,therefore,notbeconfusedwiththoseofionizingradiationfrom,say,gammarays.Microwaveovenscanbesafelyusedinalaboratory,providedthatcertainprecautionsarescrupulouslyobserved.Hereisalistofsimplesafetydosanddon"ts:

• Usemicrowave-transparentcontainerswheneverpossible.
• Alwayshandlecontainerswithpotholdersorthermalmitts.
• Nevercovercontainerstightly.
• Neveroperatethemicrowavewithoutaminimumvolumeofmicrowave-absorbingmaterialinsidethecavity.
• Neverputaconductivematerial(suchasametalcontainer)insidethemicrowave.
• Periodicallyinspectandcleandoorsealsandhinges.
• Periodicallyuseamicrowaveleakagedetectortocheckformicrowaveleakagefromthedoorseals.
• Neverheatfoodinamicrowaveovenusedforlaboratoryprocedures.
• Neverusetoxicchemicalsinamicrowaveovenunlessyouhavealaboratorymicrowavewithahigh-volumeexhaust(100cubicfeetperminuteshouldbethestandard)ventilatingthecavityintoafumehood.
• Neveruseflammablesolventsinakitchenmicrowaveoven;thesechemicals(e.g.alcoholconcentrationsof50%orhigher,xylene)canonlybesafelyheatedinalaboratorymicrowavedesignedforthispurpose,withatemperaturefeedbacksystemwhichwillpreventoverheating.

References

1. Login,G.R.,andDvorak,A.M.(1994).TheMicrowaveToolBook.APracticalGuideforMicroscopists.Boston:BethIsraelHospital.

Masson"sTrichromeStain

Fixation:Bouin-fixedtissueispreferable.MaymordantinBouin"sfluidforonehourat56Corovernightatroomtemperature.

Technique:Cutparaffinsectionsat6microns.

Solutions:

Picricacid,saturatedaqueoussolution

Weigert"sIronHematoxylin

SolutionA

Hematoxylin1.0g

Absolutealcohol100.0ml

BiebrichScarlet-AcidFushin

1%aqueousBiebrichscarlet90.0ml

1%acidfuchsinsolution10.0ml

Glacialaceticacid1.0ml

PhosphotungsticAcid

Phosphotungsticacid5.0g

Distilledwater100.0ml

AnilineBlueSolution

AnilineBlue2.5gm

Aceticacid2.0mlDistilledwater100.0ml

AceticWater

Glacialaceticacid1.0ml

Distilledwater100.0ml

StainingProcedure:

1. AttachtoautomaticstainerXylene.
2. Absolutealcohol.
3. 95%alcohol.
4. Rinseindistilledwater.
5. Place50mlofsaturatedpicricacidinthemicrowavefor45seconds.Placeslidesonstainingrack.DrophotBouin"sonslides.Letset3minutes.
6. Coolandwashinrunningwaterfor5minutes.
7. Rinseindistilledwater.
8. Weigert"sironhematoxylinsolutionfor7minutes.(Note:thisstepcanalsobeperformedinalaboratorymicrowaveat80%powerfor15seconds)
9. Rinseindistilledwater.
10. Biebrichscarlet-acidfuchsinsolutionfor30seconds.
11. Rinseindistilledwater.
12. Phosphotungsticacidsolutionfor1-3/4minutesandrinse.
13. Anilinebluesolutionfor3minutes.
14. Rinseindistilledwater.
15. 1%aceticwaterforoneminute.
16. 95%alcohol.
17. Absolutealcohol-2changes.
18. Xylene-2changes
19. Mountwithxylenesolublemedia.

Results:

Nuclei-blackCytoplasm,keratin,musclefibers,intercellularfibers-redCollagen,mucin-blue

AdaptedfortheJeffersonRegionalMedicalCenterHisto-PathLaboratory

References

P.J.:Jour.Technicalmethods,12:75-90,1929(AFIPModification)

Grocott"sSilver-MethenamineMethodforFungus

Fixation:10%Neutralbufferedformalin

Technique:Paraffinsectionscutat4microns

Control:Tissuecontainingfungus

Solutions:5%aqueoussilvernitrate,with3%methenamine

10%ChromicAcidSolution

Chromiumtrioxide5.0g

Distilledwater50.0ml

1%SodiumBisulfiteSolution

Sodiumbisulfite0.5g

Distilledwater50.0ml

2%SodiumThiosulfate

Sodiumthiosulfate2.5g

Distilledwater50.0ml

Methenamine-SilverNitrateSolution(Stock)

5%Silvernitrate5.0ml

3%Methenamine100.0ml

Awhiteprecipitateformsbutimmediatelydissolvesonshaking.Clearsolutionsremainusableformonths.Storeinrefrigerator.

Methenamine-SilverNitrateSolution(working)

Methenamine-silvernitratesolution(stock)25.0ml

Distilledwater25.0ml

5%Borax2.0ml

Makethissolutionfresheachtime.

0.1%GoldChlorideSolution

1%GoldChloridesolution5.0ml

Distilledwater45.0ml

Thissolutionmaybeuseduntilbrownprecipitateappearsandthesolutioniscloudy.

1%LightGreenSolution(stock)

LightgreenSFyellowish1.0g

Distilledwater100.0ml

Glacialaceticacid0.2ml

Thymol2.0grains

1%LightGreenSolution(working)

Lightgreen,stock10.0ml

Distilledwater40.0ml

StainingProcedure:

1. AttachtoautomaticstainerDeparaffinizesectionsandhydratetodistilledwater.Slidespreviouslystainedwithmostotherstainsmaybeusedbyremovingcoverslipsandrunningthroughxyleneandalcoholstowater.Subsequentchromicacidtreatmentwillremoveanyremainingstain.
2. Oxidizeinfresh10%chromicacidsolutionfor10minutesatroomtemperature.(Note:thisstepmayalsobeperformedinaventedlaboratorymicrowavesetat60Cfor90seconds).
3. Rinseindistilledwaterforafewseconds.
4. Rinseuntilclearin1%sodiumbisulfitetoremoveanyresidualchromicacid.
5. Rinseindistilledwater5times.
6. PlaceslidesinSilverMethenamineSolutionandmicrowavefor45seconds.Letslidessetfor2minutes.Examinemicroscopically.Fungishouldbedarkbrown.Ifstainingisnotcompletemicrowaveagaininthehotworkingsolutionfor10seconds.Re-examineslides.Repeatthisstepuntilthedesiredstainingintensityisreached.
7. Rinsein5changesofdistilledwater.
8. Tonein0.1%goldchloridesolutionuntilsectionsaregray-lavender,1minute.
9. Rinseindistilledwater
10. Removeunreducedsilverwith2%thiosulfate(hypo)for1minute.
11. Washthoroughlyintapwater.
12. Counterstainwithworkinglightgreensolutionfor15seconds.
13. Dehydrate,clearandmountwithxylenesolublemedia.

Results:

Fungi-sharplydelineatedblack

Mucin-taupetodarkgray

Innerpartsofmycelia&hyphae-oldrose

Background-palegreen

AdaptedfortheJeffersonRegionalMedicalCenterHisto-PathLaboratory

References

Grocott,R.G.:AmericanJournalofClinicalPathology,25:pp975-979,1955.

KinyonsCarbolFuschsinAcid-FastOrganisms

forParaffinSection

Fixation:10%NeutralBufferFromalin

Technique:Paraffinsectionscutat4microns

Control:Tissuecontainingacid-fastBacillusorganisms

Solutions:

Kinyoun"sCarbolFuschinSolution

Basicfuchsin4.0g

Phenolcrystals,melted8.0ml

95%Alcohol20.0ml

Distilledwater100.0ml

Mixinordergivenandfilterbeforeusing.

MethyleneBlue(stock)

Methyleneblue0.7g

95%alcohol50.0ml

MethyleneBlue(working)

Methyleneblue(stock)5.0ml

Tapwater45.0ml

1%ACIDALCOHOL

HCLconcentrated1.0ml

70%Alcohol99.0ml

StainingProcedure

1. Deparaffinizeslidesectionstodistilledwater.
2. StainslidesinCarbolFuchsinfor10secondsinmicrowave(Note:inalaboratorymicrowave,stainat60Cfor20seconds).
3. Washwellinrunningtapwater
4. Differentiateinacidalcoholuntiltissueislightpink.
5. Washwellinrunningtapwater.
6. StaininMethyleneBlue1minute.
7. Rinseindistilledwater.
8. Dehydrate,clearandmountinpermount.

Results:

Acidfastorganisms-red

Background-blue

AdaptedfortheJeffersonRegionalMedicalCenterHisto-PathLaboratory

References

Kinyoun,J.J.:Am.J.Pub.Health,5,867,1915.

GordonandSweet"sSilverImpregnationofReticulum

Demonstrates:Reticulum

Fixation:10%NeutralBufferedFormalin

Techniques:Paraffinsectionscutat4microns

Control:Lymphnode

Solutions:

PermanganateSolution

Potassiumpermanganate0.5%0.5g

Distilledwater50.0ml

Wilder"sSilverSolution

To5.0mlof10%silvernitrate,add58%ammoniumhydroxidedropbydropuntiltheprecipitatethatformsisjustredissolved.Add5.0mlof3%sodiumhydroxide.Ablackprecipitateforms.Add58%ammoniumhydroxideagaindropbydropuntiltheprecipitateisagainjustdissolved.Bringupto40.0mlwithdistilledwater.

Thissolutionwillkeepseveralweeksifstoredinadarkbottleintherefrigerator.Severalhundredmilliliterscanbemadeatonetimeandonlyusedin50.0mlquantitiesanddiscarded.Disposeofsolutionwhenppt.appears.pH11-12.

1%NeutralRed

NeutralReddiluent0.5g

Distilledwater50.0ml

2%FerricAmmoniumSulfate(IronAlum)

Ferricammoniumsulfate1.0g

Distilledwater50.0ml

1%OxalicAcidSolution

Oxalicacid1.0g

Distilledwater100.0ml

0.2%GoldChlorideSolution

Stockgoldchloridesolution(1%)10.0ml

Distilledwater40.0ml

5%SodiumThiosulfateSolution

Sodiumthiosulfate2.5g

Distilledwater50.0ml

StainingProcedure:

1. Deparaffinizesectionsandhydratetodistilledwater.
2. Oxidizeinfreshpermanganatesolutionfor2secondsinmicrowave.
3. Rinseindistilledwater.
4. Bleachuntilwhitein1%oxalicacidfor30seconds.
5. Rinseindistilledwater,2changes.
6. Mordantinfresh2%ironalumfor5secondsinmicrowave.
7. Rinseindistilledwater,2changes.
8. Impregnatefor5-7secondsinWilder"sSilverSolution.
9. Rinse1dipindistilledwater.
10. Reducein37-40%formaldehydefor15seconds.
11. Rinseindistilledwater,2changes.
12. Tonein0.2%goldchloride1minute-sectionsturngray-lavender.
13. Rinseindistilledwater,2changes.
14. Removeunreducedsilverin5%sodiumthiosulfatefor1minute.
15. Rinseindistilledwater,2changes.
16. Counterstainin1%neutralredfor1minute.
17. Rinse10dipsinrunningtapwater.
18. Dehydrate,clearandmountwithxylenesolublemountingmedia.

Results:

Reticulumfibers-blackNuclei-redCollagen-yelloworbrown

AdaptedfortheJeffersonRegionalMedicalCenterHisto-PathLaboratory

Reference:

AmericanJournalofPathology,12,549,1936.

WarthinStarryTechniquesforSpirochetes

Fixation:Formalin

Technique:Sectionscutat4microns

Control:TissuecontainingSpirochetes

Solutions:

AcidulatedWater

Acidulate1literdistilledwaterwith0.1gcitricaciduntilpHof3.8-4.4isreached.ApHof4.0isidealforstainingspirochetes.FordemonstratingDonovanBodiesofgranulomainguinale,apHof3.6isrecommended.

2%SilverNitrateSolution(developer)

SilvernitrateC.P.crystals0.5g

Acidulatedwater25.0ml

1%SilverNitrateSolution(impregnation)

SilvernitrateC.P.crystals0.5g

Acidulatedwater50.0ml

0.5%HydroquinoneSolution

Hydroquinonecrystals,photographicquality0.35g

Acidulatedwater25.0ml

5%GelatinSolution

Gelatin1.5g

Acidulatedwater25.0ml

StainingProcedure

1. Attachslidestoautomaticstainer,deparaffinize,hydratetowater.
2. Rinseindistilledwater,2changes.
3. Placeslidesin1%silvernitratesolutionfor45secondsinmicrowave.Letstandfor1minuteatroomtemperature.(Note:alternatively,inalaboratorymicrowave,heatat80%power,60Cfor5minutes,nostandingtimerequired).
4. Preheatfor45secondsinmicrowaveinseparateflasks:2%silvernitrate,5%gelatin,0.15%hydroquinone.Preheatemptyflaskinmicrowavewiththese.
5. Mixdevelopersolutioninordergiven:Usewarmemptyflask:

   2%silvernitrate1.5ml

   5%gelatin3.75ml

   0.15%hydroqinone2.0ml

6. Whenstep5iscompleted,removeslidesfromsilversolution.Donotrinse.Placeslideshorizontallyonasliderackandcoverwithdeveloper.Allowsectionstodevelopuntiltheyarelightyellowtogoldenbrown,approximately1minuteorless.(Note:developingstepcanalsobecarriedoutinalaboratorymicrowaveat80%powersetfor60Cfor20seconds).
7. Rinsethoroughlyin50mltapwaterwhichispreheatedtoapproximately56Cinthemicrowaveat450Wfor45seconds.
8. Rinseindistilledwater.
9. Attachtoautomaticstainer,dehydrateandclear.Mountinaxylenesolublemountingmedium.

Results:

Spirochetes-black

Background-paleyellowtolightbrown

AdaptedfortheJeffersonRegionalMedicalCenterHisto-PathLaboratory

AlcianBlueMethod

Fixation:10%neutralbufferedformalin

Technique:Paraffinsectionscutat4microns;eyesectionsat8microns.Avoidoverheating.

Stains:Acidmucopolysaccharides

Control:Appendix

Solutions:

Eosinsolution

3%AceticAcidSolution

GlacialAceticAcid3.0ml

Distilledwater97.0ml

AlcianBlueSolutionpH2.5

Alcianblue8GS1.0g

Aceticacid3%97.0ml

AdjustthepHto2.5.Filterandaddafewcrystalsofthymol.Note:duetotemperature,theaceticacid"buffer"solutioncandriftinpH;therefore,itishelpfultosetthepHat60C.

StainingProcedure

1. Deparaffinizesectionsandhydratetorunningtapwaterusingtheautomaticstainer.
2. Staininalcianbluesolutionfor20secondsinthemicrowave.(Note:alternatively,inalaboratorymicrowave,heatat80%power,60Cfor45seconds).
3. Rinsewellindistilledwater.
4. Placein2ndEosinandattachtoautomaticstainer.
5. Dehydrate,clearandmountinxylenesolublemedia.

Results:

Acidmucopolysaccharides-blue

Nuclei-pink

AdaptedfortheJeffersonRegionalMedicalCenterHisto-PathLaboratory

Reference:

Lev,R.andSpicer,S.S.J.Histochem.Cytochem.12:309,1964.Williams&WilkinsCo.

Southgate"sMuciarmineStain

Demonstrates:Acidmucosubstances

Fixation:10%Neutralbufferedformalin

Technique:Paraffinsectionscutat4microns

Control:Appendix

Solutions:

Muci-10plusproductofAmericanHistologyCo.,TartrazineSolution

WorkingMuciarmineSolution

Theworkingsolutionsare1:4dilutionsofthestockandthediluentisdistilledwater.Strongerorweakerdilutionsmaybeneeded,dependingonthestrengthofthestocksolution.

StainingProcedure:

1. Attachtoautomaticstainer.Deparaffinizesections,hydrateandrunslidesthroughHarrisHematoxylin,acidalcohol,blueingsolutionandwater.Removefromautomaticstainer.
2. Stainwithtartrazine,1minute.
3. Stainindilutedmucicarminefor40secondsinmicrowaveandletstandfor3minutesatroomtemperature.(Note:alternatively,inalaboratorymicrowave,drop2mlmucicarmineonthesection,andmicrowavetheslideat100%powerfor1minute;placeslideinstainingjarcontaining50mlofdilutedmucicarmineandmicrowaveat80%powerat60Cfor10minutes;standingtimenotrequired).Checkundermicroscope.
4. Stainiscomplete,drainslides.
5. Rinsequicklyindistilledwater.
6. Attachtoautomaticstaineranddehydrateandclear.Mountinxylenesolublemedia.

Results:

Mucin-rosetored

Nuclei-bluetoblack

CapsuleofCryptococcus-deeprosetored

Othertissueelements-yellow

AdaptedfortheJeffersonRegionalMedicalCenterHisto-PathLaboratory

PeriodicAcidSchiffReaction

Fixation:Formalin

Technique:Cutparaffinsectionsat4microns

Control:TissuecontainingPASpositivematerial

Solutions:

0.5%PeriodicAcidSolution

Periodicacidcrystals0.5g

Distilledwater100.0ml

Schiff"sReagent

Pourapproximately40mlofSchiffsolutionincoplinjar.Storeinrefrigerator.Discardwhensolutionbecomespink.

0.5%Diastasesolutionlightgreen

Ifstainingforglycogen,useliverwithglycogeninitforacontrol.

StainingProcedure:

1. Deparaffinizeandhydratesectionstodistilledwaterwiththeaidoftheautomaticstainer.
2. Rinseindistilledwater.
3. IfPASreactionwithdigestionisdesired,placesectionsincoplinjarin0.5%diastasefor45secondsinmicrowave.Parallelslidesshouldbeplacedindistilledwaterincoplinjarandmicrowavedfor45seconds.Rinseslidesinrunningtapwater.Rinseindistilledwater.
4. Periodicacidsolutionfor10secondsinmicrowave.
5. Rinseindistilledwater,2changes.
6. PlaceinSchiff"sreagentforandmicrowavefor30seconds(Note:inalaboratorymicrowave,use100%powerat30Cfor30seconds).Inalaboratorymicrowave,stirsolutionwhileheatingwithanairbubbleagitator.Otherwise,removefrommicrowaveandagitatethesolutionbyhandtoequilibratethesolutiontemperature.Thiswillallowformoreevenstaining.Letstandinstirredsolutionfor30secondsto5minutesafterremovingfrommicrowave,untilsectionshaveassumedapinkishcolor.Checkwithmicroscopeforcorrectstaining.
7. Washinrunningwaterfor5minutes.
8. Attachtoautomaticstainer.StaininHematoxylinsolutionfor15seconds.
9. Rinseinwater.
10. Differentiateinacidalcohol.
11. Rinseinwater.
12. Blueinrunningtapwater.
13. Rinseinwater.
14. Dehydrate,clear.Mountwithxylenesolublemedia.

Results:

Glycogen-rosetopurplishredMucopolysaccharides,fungi-rosetopurplishredBackground-blue

AdaptedfortheJeffersonRegionalMedicalCenterHisto-PathLaboratory

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