Wildtypewormsadjustegg-layingbehaviorinresponsetofood.Theylayeggswhentheyarefedandstoplayingeggswhentheyarestarved.Ifstarvedwildtypewormsareputbackonfood,theyresumeegglayingalmostimmediately.Iusethefollowingassaytodeterminehowwellwormsregulateegglayinginresponsetofoodquantitatively. Measurethe#ofeggslaidby10wormsin30minunderthreeconditions: 1,starved;2,starvedthenre-fed;3,neverstarved. Note: 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. M9(withoutglucoseorMgSO4):MRCrecipe;isbasicallyM9forbacteriawithout20%glucose.usedasM9inHorvitzlab,e.g.foregg-layingassays,everythingelse.NotsameasM9bufferinwormbook. Na2HPO45.8g KH2PO43.0g NaCl0.5g NH4Cl1.0g dH20to1l M9buffer(Wormbookrecipe):alsoMRCrecipe;IsbasicallyM9bufferwithhighNaCltomakeuposmolarityfornoglucose.NotusedinHorvitzlab. KH2PO43.0g Na2HPO46.0g NaCl5.0g 1MMgSO41.0ml dH20to1l