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