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Culturing Worms
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CulturingWorms

byMichaelKoelle

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.

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