FreezingWorms byMichaelKoelle 4/6/94 I.Thepreferredmethod 1.Washwormsoffof1largeplateor3smallplatesin3mlsSBasalintoasterile15mldisposablecentrifugetube.WormsshouldbeharvestedoffofjuststarvedplateswhicharepredominantlyL1andL2.Thisshouldbe1dayafterthebacteriahavebeenexhausted.Thewormbooksaysdauersdon"tfreeze,butE.Jorgensonsayshefreezesfromreallyoldplatesanditworksfine.Rumorhasitthatiftheplatesaren"tcompletelystarved,thefreezingwon"twork-i.e.foodinthegutsomehowpreventsthewormsfromsurviving.Towashthewormsoffplates,add~2mlsSBasaltoeachsmallplate(w/sterile5mlglassPipetteandapasteurpipettebulb),swirlbrieflytodislodgeworms,andsuckoffthesUSPensionwiththesterileglasspipetteandplaceintoa15mlsterileplasticcentrifugetube. 2.Spindowninaclinicalcentrifugefor~30sec.Removeallbut1.5mlofthesupernatant. 3.Add1.5mlfreezingsolution,mixwell,andaliquot1mleachinto3sterilefreezingvials(NuncCryoTubes#363401). 4.Freezeslowlyto-80deg..Thisisaccomplishedbyplacingthevialsinastyrofoamrack(thekindthat15mldisposablesterilecentrifugetubescomein),placinganotherinvertedsuchrackontopofthefirst,fasteningthetworackstogetherwithrubberbands,andplacingina-80deg.freezer. 5.Thenextday,movetwovialstoapermanentlocation.Recordthestrainnumber,genotype,andcommentsinacomputerdatabase. 6.Thethirdvialshouldbeusedforatestthaw.Takethevialoutofthefreezer,thawquicklybyholdinginyourhandorina37deg.waterbath(leaveinthebathonlyuntiltheiceisgonesoasnottoheatitup).Candumpthewholevialoutinaseededlargeplate,oruseasterile200ulpipettemantiptowithdrawthebottom200ul(containingthesettledworms)andplaceitonasmallplate.Thenextday,picklivewormstoanewplate. II.Theagarmethod.ThisgivesmuchlowerviABIlitythantheabovemethodinmostpeople"shands,buthastheadvantagethatonedoesn"tneedtothawanentirevialatatime.Onbalance,Idon"tthinkitisworthriskinglosingstrains(especiallyriskywithheterozygousmixtures)togetthisminorsavings.However,somepeopleseemtohavebetterluckwiththismethodthanme. 1.Washwormsoffof1largeplateor3smallplatesin3mlsSBasalintoasterile15mldisposablecentrifugetube.WormsshouldbeharvestedoffofjuststarvedplateswhicharepredominantlyL1andL2.Thisshouldbe1dayafterthebacteriahavebeenexhausted.Thewormbooksaysdauersdon"tfreeze,butE.Jorgensonsayshefreezesfromreallyoldplatesanditworksfine.Towashthewormsoffplates,add~2mlsSBasaltoeachsmallplate,swirlbrieflytodislodgeworms,andsuckoffsuspensionwithasterileglasspipetteandplaceintoa15mlsterileplasticcentrifugetube. 2.Chillonice15"orlonger. 3.Meltfreezingsolution+agarinmicrowave(carefulltofullymeltbutnotboilover). 4.PutFS+agarbottlein50deg.waterbathfor~15". 5.Spinthewormsdown(30sec.inaclinicalcentrifuge),andremoveallbut3mlsofsupernatantwithasterilepipette. 6.Pipette3mlFS+agarintoatubeofwormsandimmediatelymixbyinversionoftube8-10X. 7.Pipette1.8mlofthemixtureinto3preparedfreezervials. 8.Repeatsteps6and7foreachstrain. 9.Freezeslowlyto-80deg..Thisisaccomplishedbyplacingthevialsinastyrofoamrack(thekindthat15mldisposablesterilecentrifugetubescomein),placinganotherinvertedsuchrackontopofthefirst,tapingthemtightlytogether,andplacingina-80deg.freezer. 10.Thenextday,movetwovialstoapermanentlocation.(Or,toaboxinthe-80deg.,andwhentheboxisfull,moveallthevialstoaliquidN2freezer).Recordthestrainnumber,genotype,andcommentsinacomputerdatabase. 11.Thethirdvialshouldbeusedforatestthaw.Caneitherthawthewholevialanddumponalargeplate.Or,usingaflamedspatula,scrape~0.1mloffrozenwormsontoasmallplate,andputtheremainderofthevialbackinthefreezer. FreezingSolution 200ml1MNaCl 100ml1MKPO4,pH6.0 600mlglycerol Bringto2literw/dH20 Distributeto200mlbottles;autoclave Add0.06mlsterile1MMgSO4per200mlbottle Freezingsolutionwithagar freezingsolutionwith0.4gagaraddedper100mlandreautoclavedfor20min. Sbasal 5.8gNaCl 50ml1MKPO4 950mldH20 1mlcholesterolin95%EtOH(5-10mg/ml) swirltodisperse,andautoclave