Wildtypewormsadjustegg-layingbehaviorinresponsetofood.Theylayeggswhentheyarefedandstoplayingeggswhentheyarestarved.Ifstarvedwildtypewormsareputbackonfood,theyresumeegglayingalmostimmediately.Iusethefollowingassaytodeterminehowwellwormsregulateegglayinginresponsetofoodquantitatively.

Measurethe#ofeggslaidby10wormsin30minunderthreeconditions:

1,starved;2,starvedthenre-fed;3,neverstarved.

Note:

• TransferofwormsinliquidshouldbedonewithaglasspasteurPipette,notplasticpipettetowhichwormsstick.
• Alwaysusedryandnon-crackedNGMplates.Theplatesneedtoquicklyabsorbafewdropsofliquidplacedonthem.

1.Picklargedark-lookinglateL4larvae,~60L4s/platex2plates.L4sarerecognizedbyawhitecrescentinthepresumptivevulvalregion,whichacquiresablackcentraldotinlateL4.WormsneedtobestagedaspreciselyaspossIBLe,becausethisseemstoreducefluctuation.

2.Incubatewormsat20ºCfor30hrs.

3.Washwormsoffplateswith2.5mlM9bufferatatimex2times,combinethewashesina15mlfalcontube.

4.Spindownwormsinacliniccentrifugefor10secatmaximalspeed.

5.Removesupernatantbyaspiration,leave~0.5mlliquid.(soyoudon"tlosewormsduringwashing)

6.Rinsewormswith~7mlM9,spin10sec,removesupernatantbyaspirationandleaveabout0.5mlliquid.

7.Thencarefullyremoveasmuchsupaspossiblewithapasteurpipette,usuallyabout100ulliquidisleftinthetube.

8.Takea5cmNGMplateseededwithalawnofO.P.50bacteria,dropontheedgeoftheagar2-3dropsof4Mfructosefromapasteurpipetteandrotatetheplatesothatthesolutionflowsalongtheveryedgeandmakesafructosecircle.Takeanunseededplateanddothesametocircletheedgewithfructose.Youcandostep7whileyouarewaitingforthecentrifugetostop.The4Mfrunctosecreatesanosmoticbarrierthatpreventswormsfromcrawlingofftheplateanddying.

9.Underadissectingscope,useapasteurpipettetotransfer20-30wormstotheseededplatefromstep7.Icountthe#ofwormstransferredbecauseIdon"twanttowastetoomanywormsonthisplate.Putwormsdownnearthebacteriallawnandlettheliquidbeabsorbedintotheplate.Icomebacktothisplateafewminuteslaterandmoveanywormsthatareofffoodbackonfoodusingawormpickingcoatedwithbacteria.Transfertheremaining~90wormstotheunseededplatefromstep7.

10.Incubatethewormsat20ºCfor2hrs.Neartheendofthe2hrs,prepare6fructosecircledplates(4seeded+2unseeded)inthesamewayasstep7.

11.Transfer20starvedwormsto2unseededplatesfromstep9withabareplatinumwormpick(nobacteria!),10worms/plate.(Hereismywaydoingit:Useashortflat-endedwormpick,pressagainsttheagarbesideawormsothatsomeliquidgetssqueezedouttolubricate.Shovethewormpickunderneaththewormandpickitup.Onanewplateputdownthewormpick[withawormontopofit]againsttheagarandsqueezeoutsomeliquid.Thewormwillfloatupandswimoff.Don"tteartheagarsurfacewhenyouputdownaworm,justmakeadent.)

12.Transfer20starvedwormsto2seededplateswithawormpickcoatedwithbacteria,10worms/plate.

13.Transfer20nonstarvedwormsto2seededplateswithawormpickcoatedwithbacteria,10worms/plate.

14.Waitfor30minatroomtemperate(shouldbecloseto20ºC).Thencountthe#ofeggslaidoneachplate.Youcanjustremovethewormsandcounteggslaterifyoudon"thaveenoughtime.Maximumof3strainscanbeassayedatatime.

15.Repeattheprocedure4times(8repetitionstotal)andaveragetheresults.Forunknownreasons,thereissomefluctuationinthisassaythatrequiresthisnumberofrepetitionstoaverageout.