SynchronizingWormCultures

Synchronizationisbasedonthefactnewlyhatchedlarvaewilllivebutnotdevelopifdeprivedofafoodsource.Anasynchronouspopulationofembryosisisolatedandthenallowedtohatchintheabsenceoffood.OncealltheembryoshavehatchedintoL1larvae,thenowsynchronouspopulationistransferredtomediumwithfoodandthepopulationoflarvaeresumetheirgrowthanddevelopment.

Materials:

M9:

22mMKH2PO442mMNa2HPO485.5mMNaCl1mMMgSO4

alkalinehypochloritesolution:

approx1%NaOCl(=140mM)250mMKOH

sterileculturedishor24-wellplate

Procedure:

Collectgravidadults
- Harvestgravidadultsbywashingwormsoffwellpopulatedplateswith~1mlM9
- Collectin15mlcentrifugetubeorinEppendorftubesdependingonscale
- Pelletwormsbycentrifugation20-30secondsat150-200xg(30secondsat1200rpminclinicalcentifuge)
- Removesupernatant
- WashoncewithequalvolumeofM9
- Removesupernatant

Isolateembryosfromadults

Add1-5mlsofFRESHhypochloritesolutiontoworms(1mlforeppendorfor5mlsfor15-mlconicaltubes)

Mixwellbyinversionandmonitorprogressunderthedissectingscope

wormswilldisolveinthebleachsolutionleavingtheembryoswhicharemoreresistanttobleach

Whenembryosarereleased(3-7minutes)collectbycentrifugation20-30secondsat150-200xg

Removesupernatant

Wash4XwithequalvolumeofM9

ResUSPendwormsinsmallvolumeM9

HatchoffL1s

Transferembryostoculturedishorwellofa24-wellcultureplatewithsterileglassPipette
Allowembryostohatchovernight(>12hours)at20degrees
TiterL1populationbycounting"swimming"L1sin10ulspotsof10-folddilutions
Transferdesiredamountofwormstomediumwithfoodsource
Allowdevelopmenttodesiredstage

Notes:

H2OcanbeusedinplaceofM9
sterilitydoesn"tmatteruntilafterthebleachingsteps
agitationoftheembryosduringthehatch-offisunnecessary-theymightnotbeswimmingthenextdaybutarejustfine
synchronizationisusuallyprettygoodthroughtheadultstage

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