UseoftheHemacytometerfortheDeterminationofCellNumbersCountingcellsbytheuseofahemacytometerisaconvenientandpracticalmethodofdeterminingcellnumbersinthecasethattheCoultercounterisout-of-ordertemporarily.(Itisnotthatbad.)Thehemacytometerconsistsoftwochambers,eachofwhichisdividedintonine1.0mmsquares.Acoverglassissupported0.1mmoverthesesquaressothatthetotalvolumeovereachsquareis1.0mmx0.1mmor0.1mm3,or10-4cm3.Since1cm3isapproximatelyequivalentto1ml,thecellconcentrationpermlwillbetheaveragecountpersquarex104.Hemacytometercountsaresubjecttothefollowingsourcesoferror:1.Unequalcelldistributioninthesample2.Improperfillingofchambers(toomuchortoolittle)3.Failuretoadoptaconventionforcountingcellsincontactwiththeboundarieslinesorwitheachother(beconsistent)4.StatisticalerrorWithcarefulattentiontodetail,theoverallerrorcanbereducedtoabout15%.Itisassumedthatthetotalvolumeinthechamberrepresentsarandomsample.ThiswillnotbeavalidassumptionunlessthesUSPensionconsistsofindividualwell-separatedcells.Celldistributioninthehemacytometerchamberdependsontheparticlenumber,notparticlemass.Thus,cellclumpswilldistributeinthesamewayassinglecellsandcandistorttheresult.Unless90%ormoreofthecellsarefreefromcontactwithothercells,thecountshouldberepeatedwithanewsample.Asamplewillnotberepresentativeifthecellsareallowedtosettlebeforeasampleistaken.Alwaysmixthecellsuspensionthoroughlybeforesampling.Thecellsuspensionshouldbedilutedsothateachsuchsquarehasbetween20-50cells(2-5x105cells/ml).Atotalof300-400cellsshouldbecounted,sincethecountingerrorisapproximatedbythesquarerootofthetotalcount.Acommonconventionistocountcellsthattouchthemiddlelines(ofthetriplelines)totheleftandtopofthesquare,butdonotcountcellssimilarlylocatedtotherightandbottom.Hemacytometercountsdonotdistinguishbetweenlivinganddeadcells.Anumberofstainsareusefultomakethisdistinction.Trypanblueamongothers(ErythrosinB,Nigrosin)canbeused:thenucleiofdamagedordeadcellstakeupthestain.Ifmorethan20%ofthenucleiarestained,theresultisprobablysignificant.Althoughthetrypanstaindistinctionhasbeenquestioned,itissimpleandgivesagoodapproximation.Materials1.Cleanhemacytometerandcoverglass,orcoverslips2.PasteurPipetsorTransferPipets3.BalancedSaltSolution(BBS)orPBS4.Trypanblue,0.4%inBBS(orPBS)5.Microscope5.Tubes6.Handcounter(Colonycountercanbeused)7.CellsuspensionProcedure1.Dilute0.2mlofTrypanbluewith0.8mlofBBS.2.Placecoverglassoverhemacytometerchamber.3.Transfer0.5mlofagitatedcellsuspensiontoa15mltubeandadd0.5mlofdilutedtrypanblue.4.WithaPasteurortransferpipet,fillbothchambersofthehemacytometer(withoutoverflow)bycapillaryaction.Cellswillsettleinthetubeandinthepipetbygravitywithinafewseconds.Workquickly.5.Usingthemicroscopewitha10Xocular(anda10Xobjective),countthecellsineachof10squares(1mm2each).Ifover10%ofthecellsrepresentclumps,repeatentiresequence.Iffewerthan200ormorethan500cellsarepresentinthe10squares,repeatwithamoresuitabledilutionfactor.6.Calculatethenumberofcellsperml,andthetotalnumberofcells,intheoriginalcultureasfollows:Cells/ml=averagecountpersquarex104Totalcells=cellspermlXanydilutionfactorXtotalvolumeofcellpreparationfromwhichthesamplewastaken.7.Repeatcounttocheckreproducibility(+/-15%).References1.Berkson,J.,T.B.MagathandM.Hurn(1939).Am.J.Physiol.128,309.2.Sanford.K.K.,W.R.Earle,V.J.Evans,H.K.WaltzandJ.E.Shannon(1951).3.Absher,M.inTissueCultureMethodsandApplications,Eds.Kruse,P.F.andPatterson,M.K.,Jr.AcademicPress,N.Y.,1973,p.395.FromtheLaboratoryofDr.AllanBradleyBaylorCollegeofMedicine,Houston,Texas

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