CulturingWorms 1.Bacterialstrainforfeedingworms:OP50,auracilauxotroph.StreakOP50outona9cmNGMagarplate,growovernightat37°.CanthenParafilmtheplateandkeepitat4°formonths.Tomakebacteriaforseedingplates,useaflamedwirelooptopickasingleOP50colonyintoa100mlbottleofBbroth.Setthebottle(noshaking)at37°overnight.Itshouldbecloudythenextday.Thisbottlecanbestoredat4°andusedfor~3months. 2.Seedingplates.NGMplatesshouldbeallowedtositoutatroomtemperatureinaclosedboxforacoupleofdaysafterpouringtoallowthemtodry,andsothatplateswithbacterialorfungalcontaminantsbecomeobvious.UsingasterilePipette,drop1-2dropsOP50sUSPensiononthemiddleofeach5cmNGMplate.Itisbestifthebacteriallawndoesnottouchthesidewall,aswormsthentendtocrawlupthewallanddie.(Don"tmovetheplatessoonafterseeding,orthepuddleofbacteriawilldefinitelysloshagainstthewall).Takecarenottodamagethesurfaceoftheagarwiththepipettetip;wormswillthenburrowunderthesurface.Lettheplatessitovernighttoformabacteriallawnbeforeusing.Platescanbeusedforabout7daysafterseeding,andarebest2-4daysafterseeding. Toseedlarge(9cm)NGMplates:drop5dropsOP50inthemiddleofaplate.UseasterilepipetteataveryshallowangletospreadtheOP50liquidaroundsothatathinpoolofliquidcoversthemiddle2/3oftheplate.Settheseededplatesat37°overnight,oratroomtemp.for2days.(ManypeopletrytoseedlargeplatesbydroppingahugeamountofOP50ontheplates,untilthethickpoolofliquidcoversalargeportionoftheplate;thisresultsinawetmessthattakesseveraldaystodryintoamealy-lookinglawn.) 3.Transferringworms.Useaplatinumwirepick.Toconstructthis,takeahammerandbangonapieceof~32gaugeplatinumwiretoflattenit.Othersuseplierstoflattentheendofthewire.Thencutthewiresothatonlyafewmillimetersofflatwireareleftattachedtoalongersectionofroundwire.Canusearazorbladetosliceoffthesharpcornersoftheflatend.Thenbendtheflatsectionsothatitisatashallowangletotherestofthewire.Finally,placethenon-flattenedendinsideofashortPasteurpipette,andsealintotheglassoveraBunsenburner.About2cmofwireshouldprotrudefromtheglass,butpersonalpreferencesvary.Eventuallytheendofthepickwearsout,andtheendcanbecutoffandanewendflattenedandbent. Topickupworms,flamethewiretosterilizeit,anddragthepickalongabacteriallawntocoattheundersideofthepickwithbacteria.Goopy,severaldayoldbacteriallawnsarethebestforthis;somekeepoldplatesnexttothemicroscopeespeciallyforcoatingtheirpicks.Gentlybrushagainstthewormtobepickedupsothatitstickstothebacteriaonthepick.Tosetthewormdown,gentlybrushagainstthenewplate,andallowthewormtocrawloff.Thewormshouldbeimmediatelyactiveonthenewplateifitisundamaged.ItispossIBLetopickupatleasttenwormsatatime,however,youdon"twantthewormstospendtoomuchtimeonthepick,astheywilldryout. 4.Wormsaretypicallyculturedina20°room,withtheplatesupsidedown,storedinoldscintillationvialboxes.Thedividersintheboxescanbereconfiguredtofitthe5cmwormplates.Theboxesanddividersarebakedat65°for>1hourbeforeusetosterilize.Wormscanbeculturedbetween15°and25°,andwillgrowslowerorfaster,respectively,atthesetemperatures.Tokeepastockavailableforalongtimewithoutmaintenance,parafilmtheplateandsetitat15°.Canthenrecoverthewormsfor~5monthsafterwards.Thelimitingfactoristhatthewormsdieeventuallyofdessicationwhentheplatedries. Ifyou"releavingtownforawhileagoodwaytokeepyourwormsistoputtheplatesina12.5°Cincubator.Thewormswillbealmostinstasis,andwillshowonlybarelyperceptabledevelopmentafter1week.Apparentlyprolongedincubationat12.5°killswormstocksbysterilizingthem.Oneweekiscertainlysafe,andafter2-3weeksyou"llprobablystillhavesomefertileworms. 5.Totransferastocktoanewplate,typicallyyou"chunkitout"insteadoftransferringwormswithapick.KeepsmallcappedbottleofEtOHbyyourmicroscope,andasmallspatula.DipthespatulainEtOH,thenplacethespatulainthebunsenburnerflamefor~15seconds:theEtOHwillburnoffandthenthespatulashouldgetalmostredhot.(IfyoujustquicklylettheEtOHburnoffthiswon"tsterilizethespatula,andyouwillalwaysendupcontaminatingyourworms).DipthehotspatulabackintheEtOHuntilthespatteringandfizzlingstops-thiscoolsitoffsotheheatwon"tkilltheworms.ThenquicklypassthespatulathroughtheflametoignitetheEtOH,whichwillburnoffwithoutheatingthespatulamuch.Usethenowsterilespatulatocuta~1cmsquarechunkoftheoldplateandplaceitupsidedownonthenewplate.Wormswillcrawloutontothefreshbacteriallawn. NGMagar:3gNaCl 17gagar 2.5gpeptone 1mlcholesterol(5mg/mlinEtOH) 975mlH20 Autoclave,andthensterillyaddthefollowing,mixingaftereachaddition: 1ml1MCaCl2 1ml1MMgSO4 25ml1MpotassiumphosphatepH6 Typicallypour5and9cmplates.Storeinplasticboxeswithcoversatroomtemperatureforacoupledaysbeforeusetoallowtheplatestodry. Bbroth:10gmbactotryptone 5gmNaCl 1literdH20 dissolveoverbunsenburner. Thendistributeto100mlbottlesandautoclave.