Anactivelygrowingcultureofcellsisrequired,ie2-3dEScellculture.Thetotalnumberofcellsneedstobebetween106-107cells.

NBReadnotesatendofmethod.

1)Harvestcellsintheusualmanner,intoaconicalbottomcentrifugetube.

2)Centrifugetoremovemedium.

3)ResUSPendcellbuttonbyflickingthebottomofthetube.

4)Add8mlofwarmedKC1solutiontoeachtubeandmixgentlywithaPipette.

5)Returnto37oCwaterbathfor10-14mins,(thisneedstobedeterminedforeachcelltype)

6)Add2mloffixativeandmixbyinversion.

7)Centrifugeculturesat1500rpmfor5mins.

8)Aspiratemostofthesupernatant.

9)Usingapasteurpipettegraduallyresuspendthecellsin~2mlsoffixative.Thisneedstobedoneverygently.Thenaddfixativeto8mls,andmixgentlybyinvertingthetube.

10)Centrifugecellsagain.

11)Repeat8,9,103-4times.

12)Gentlyresuspendcellsatappropriateconcentration.

13)Makespreadsby"huffing"onslides,holdingat45oCanddroppingonedropofcellsontothetopoftheslide.

StainslideswithLeishmanns.

STAINING

1)Stainslideswith1mlLeishmann"sstainand5mlpH6.8bufferfor7-9mins(enoughfor2slides).

2)Rinsebrieflyinrunningwaterandairdry.

3)ClearinX2changesofxylene.

4)CoverslipusingDepexmountant.

SOLUTIONS.

HYPOTONICSOLUTION(0.075MPOTASSIUMCHLORIDE)

Add0.56gmKC1to100mlsH20.

LEISHMANN"SSTAIN

BDHPROD35022

0.2%w/vsolutioninmethanol.

GURRSBUFFER.TABLETSpH6.8

BDHPROD33193

FIXATIVE

3partsmethanolto1partglacialaceticacid.

NOTES

ThetimeinKC1iscrucial.Ifthetimeistooshort,thechromosomeswillbetootightlypacked.Toolong,theywillnotbecontainedintheirappropriategroup.

Whenstainingspreads,theLeishmannsmustbemadeupfreshfor2-4slidesatatime,asitprecipitatesout.

Slidesneedtobesoakedinabsoluteethanolovernightandwipedwithalint-freetissue.Ifitistoowarmto"huff"ontheslides,theycanbeplacedinthefridge/freezer.

Fixativemustbemadeupfresheachtime.Disposeoffixativewasteproperly,ieintosolventwaste.

Avoidbreathingvapour.

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