Accuracyofmanualcountswithanhemacytometerdependon:

• accuratemixingofthesample(nobubbles!)
• numberofchamberscounted
• numberofcellscounted(practical:200-500per0.1mm3)

Thechamberofvariouscomposition,allhaveacommonunitof1x1mmsquares,whichmaydividedinninesmallersquares.Theheightofthechamber,delimitedbythecoverslipis0.1mm.

Suppliers:

HYCOR:KovasystemforstandardizedurinalisysN^o87144(expect10%variABIlityormore)HycorBiomediacalInc.GardengroveCA92841HemacytometerFisherscientificN^o026715

DiluteyourcellsampleinTrypanBluedyeexclusionmedium.Deadcellswillbeblue,livecellswillbebirefringent.Suggesteddilution1:20(5µlcellsUSPensionin95µlTrypanBlue).Carefullyandcontinuouslyfillevenlyatoncethehemacytometerchamber.Leaveundisturbedfor1-2min.(Ifyouneedtoleaveforlonger,useahumidchamber).Thiswillallowdepositionofthecellsonthecountingplane.Countunderthemicroscopetwo1x1x0.1mmareas(delimitedbyadoubleline;ninesmallsquares),fromtwochambers.Ideallyyoushouldcountmorethan200cellsandlessthan500perchamber.

Thenumberofcellsper1x1x0.1mmareas=cellsx1*10^-4cm³(1*10^-4ml).Witha1:20dilutiontheeasycountis:numberofcellsintwo1mmsquaresdividedby10=cellsx10⁶/ml.(2squares/2x20x10,000/1,000=10⁶cells/ml)(2squaresx10x10,000/1,000=10⁶cells/ml)(2squaresx100=10⁶cells/ml)(2squares/10=millions/ml)

Ifyourcellsuspensioniswayabove500/square,countasmanysmallestsquaresyoucan,calculatethemean:thisnumbermultipliedby0.9willapproximatethenumberofmillions/ml.

Usefulrefence:

UseoftheHemacytometerfortheDeterminationofCellNumbershttp://www.imgen.bcm.tmc.edu/molgen/labs/bradley/hemacyto.txt