Exercise12.7-ViABIlityCellCount

Materials

• SUSPensioncultureofcells
• SteriletransferPipettes
• Stock0.2%(w/v)Trypanblue
• Hemacytometerandmicroscope

Procedure

1. Gentlyswirlasuspensionculturetodistributethecellsevenly.Asepticallyremoveasmallsample(0.1ml)ofcellsfromthecultures.Placethesampleinaseparatetesttube(itneednotbesterile).

2. Dilute4partsofstockTrypanBluewith1partof5Xsalineandadd0.1mlofthediluteddyetoyoursample.Mixgently.

3. Setupahemocytometerandcoverslip.Immediatelyplaceadropofthestain/culturecombinationonthehemocytometer(remembertousebothsidesofthehemocytometer)andwaitoneminute.

4. Observethecellswithlowpowermicroscopy.Countthetotalnumberofcells,andthenumberofstainedcells.

5. Computetheconcentrationofviablecellsperml.ofculture.

Notes

TrypanBlueisastainthatisactivelyextrudedfromviablecells,butwhichreADIlyentersandstainsdeadcells.Therefore,thecellswhicharebluearedead.Thedifferencebetweenthetotalnumberofcellsandthenumberofdeadcellswouldbethenumberofviablecellsinagivenaliquotofyourculture.TrypanBlueactuallysignificantlyoverestimatesthenumberofviablecells,butissufficientforpurposesofthislab.

Approximately30%ofthecellsmeasuredasviablewithTrypanBluewillnotbeabletocontinuegrowthbeyonda24hourperiod.

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