Ahemacytometer(alsospelledhemocytometer)isanetchedglasschamberwithraisedsidesthatwillholdaquartzcoverslipexactly0.1mmabovethechamberfloor.Thecountingchamberisetchedinatotalsurfaceareaof9mm2. Calculationofconcentrationisbasedonthevolumeunderneaththecoverslip.Onelargesquare(seeWinFigure2)hasavolumeof0.0001ml(lengthxwidthxheight;i.e.,0.1cmx0.1cmx0.01cm). Inbothmethods,thehemacytometerisfilledbycapillaryaction-placethePipettefilledwithawell-sUSPendedmixofcellsatthenotchattheedgeofthehemacytometerandthenslowlyexpelsomecontentssothatthefluidisdrawnintothechamberbycapillaryaction. Stainingofcellsoftenfacilitatesvisualizationandcounting.Eithermixcellswithanequalvolumeoftrypanblue[0.4%(w/v)tyrypanblueinPBS]todeterminelive/deadcount(deadcellsareblue)orkillcellswith10%formalinandthenstainwithtrypanblueorotherstain(toimprovevisualizationofallcells. Herearetwosimplemethodsforcountingcellsbasedonthesurfaceareaofthehemacytometerusedtodeterminecellcount.Othercountingschemesareaccetablealso.Thechoiceofmethodsdependsuponthecellconcentration-theaccuracyoftheproceduredependsuponthenumberofcellscounted.Whencellconcentrationislow,oneshouldcountmoregrids. MethodACountthenumberofcellsinthe4outersquares. Thecellconcentrationiscalculatedasfollows: Cellconcentrationpermilliliter=Totalcellcountin4squaresx2500xdilutionfactor Example:Ifonecounted450cellsafterdilutinganaliquotofthecellsuspension1:10,theoriginalcellconcentration=450x2500x10=11,250,000/ml MethodBEstimatecellconcentrationbycounting5squaresinthelargemiddlesquare. Thecellconcentrationiscalculatedasfollows: Cellconcentrationpermilliliter=Totalcellcountin5squaresx50,000xdilutionfactor Example:Ifonecounted45cellsafterdilutinganaliquotofthecellsuspension1:10,theoriginalcellconcentration=45x50,000x10=22,500,000/ml