Stockkeeping

1.MechanicsMoststockscanbesuccessfullyculturedbyperiodicmasstransferofadultstofreshfood.Bottlesorvialsaretappedonthepoundingpadtoshakefliesawayfromtheplug,theplugisrapidlyremovedandtheoldcultureinvertedoverafreshbottleorvial.Fliesaretappedintothenewvessel,orsomeshakenbackintotheoldone,asnecessary,andthetwoarerapidlyseparatedandreplugged.Goodtossingtechniquecombinedwithplugsthatareeasilyremovedandreplacedresultinveryfewescapees.Youwilllearnfromexperiencewhichstocksrequireamediumorlargeinoculationofadultsandwhichdobetterwithonlyafew.

Thefrequencywithwhichnewsubculturesneedtobeestablisheddependsonthehealthandfecundityofthegenotype,thetemperatureatwhichitisraised,andthedensityofthecultures.TemperaturehasalargeeffectontherateofDrosophiladevelopment.Generationtime(fromeggtoadult)isapproximately:7daysat29^oC,9daysat25^oC,11daysat22^oC,19daysat18^oC.Formostpurposesstocksaremaintainedbyliveculture,transferringadultstofreshmediumeveryfewgenerations.Stockskeptatroomtemperatureshouldbetransferredtofreshfoodevery20to30days.Mitesandmoldaremorelikelytobeaprobleminolderstocks,soitisgoodpolicytoset30daysasanabsoluteupperlimitforroomtemperaturestocks.Thisperiodcanbeextendedbykeepingstocksatlowertemperature.18^oCbuysmoretimethan22^oC,forexample,butasignificantnumberofgenotypesfailtothriveat18^oC,andmoldcanbeaseriousproblem.Itiswisetokeeparoomtemperaturebackupofstockstobemaintainedatlowtemperatureforthefirsttwoorthreetransfersincasethestocksdopoorly.Ifthequalityofyourflyfoodisunreliableitiswisetohaveatleasttwoculturesforeachstock,staggeredtoassuretheuseofdifferentbatchesofmedium(atleastuntilyoufindanewcook).

Cryopreservationofovaries(seeAshburner,1989)orembryos(Coleetal.,1993;SteponkusandCaldwell,1993)areviablealternativestocontinuouscultureforsomepurposes.Genotypesthatareunstableduetoreversion,breakdown,oraccumulationofmodifiers,especiallythosewithnon-visIBLephenotypesthataretimeconsumingtoselect,aregoodcandidatesforfreezing.Also,ifyouaregeneratinghundredsofstocksthatwillnotbeinusebutmustbekeptformanyyearsitmightbecost-effectivetomaintaintheseasfrozenstocks.Formostroutinestockkeepingpurposes,however,livecultureremainsthepreferredroute.

Identifystockswithtagsshowingthecompletegenotypeofthestock,sansshorthand.Writingthegenotypeonthevialorbottleateachtransferinvitestranscriptionerrorsandtakeslongerthanmovingatag.Don"tuseastockcenterstocknumberorotherpotentiallycrypticsymbolastheonlyidentifierofastock.Stocksareoftenkeptformanyyearsandwhatisobvioustoyounowmaybemeaninglesseventoyouinafewyears,andiseasilymisinterpretedbysomeoneinheritingyourstocks.Unlessyouarecarefultomaintaincompletestockdataelsewhere,recordallrelevantinformationonthetag.

2.BalancedstocksandbalancersMutationsthatarehomozygousviableandfertilearemosteasilykeptashomozygousstocks.Lethalorsterilemutationsmustbemaintainedinaheterozygousstate.Abalancedstockisonethatregeneratesthesamesetofheterozygoteseachgenerationsothestockcanbemaintainedbymasstransferofadultsinsteadofbymatingspecificgenotypeseachgeneration.Asimplebalancedlethalstockcarriesdifferentrecessivelethalsoneachofthetwohomologues,allowingonlyheterozygotestosurvive.Dominantmaleorfemalesteriles(MsorFs)canbemaintainedinstockwithoutselectionbydoublebalancing-oneMs,oneFsandonerecessivelethal.Fs/lethalmaleandMs/lethalfemaleswillbetheonlyfertilegenotypespresentinthestockeachgeneration.

Inmostcases,balancedlethalschemesworkonlyifoneofthelethalchromosomesisitselfabalancerchromosome.Recombinationbetweenlethal(orsterile)mutationsondifferenthomologuescanproduceonehomologuewithbothmutationsandonewild-typehomologue.Thewild-typechromosomewillrapidlypredominateorbecomefixedinthestock.Balancersarestructurallyrearrangedchromosomesthatpreventrecombinationbetweenhomologuesinfemales(meioticrecombinationisabsentinD.melanogastermalesandinthetiny4thchromosomeinfemales).Thisisaccomplishedinpartbyreducingrecombinationdirectlyandinpartbypreventingtransmissionofrecombinantchromosomes.Themostcommonlyusedbalancerscarryoverlappingsetsofinversionsandpreventrecombinationthroughoutmostofthelengthofthechromosome.Somespecialpurposebalancersworkwellonlyforspecificregionsofachromosome.Suppressionofrecombinationislesseffectivewhenbalancersfortwoormoreheterologuesarepresentinastock.

Culturecontaminants

Drosophilaisrelativelypestilence-free,butmites,fungiandbacteriacanbeproblemsinlaboratorycultures.Itisgoodpracticetocleanyourbenchtopandflypushingequipmentregularly.Thisisparticularlyimportantifaproblemisevident.Cleanthebenchtopandallequipmentthatcomesintocontactwithpotentiallycontaminatedstockswith10%bleach,70%ethanolorsoapandwaterafteruse.Sharingpoundingpads,CO₂pads,flypushersandsortingplatescanaidthespreadofcontaminants.Ifsharingisunavoidable,theneedforcleanlinessshouldbeunderstoodbyallandenforced.

1.Mites"Ihavemites"isnotanadmissionyouwanttohavetomaketoyourflycolleagues(butyoumustmakeit,iftrue).Themostdangerousspeciesareeggpredators,buteventhosethatsimplyfeedonthemediumcanout-competeweakgenotypesandcompromiseexperimentalobservations.Frequentstocktransfer,tightplugs,andzeromite-tolerancebyalltheflyworkersinabuildingarethebestdefenses.Ingeneral,culturesgrownat24-25^oCshouldneverbekeptformorethan30days.Ifmitesareknowntobeaprobleminyourlaborbuilding,culturesshouldbecheckedanddiscardedafter18to20days.Liningstocktrayswithbenzylbenzoate-treatedcheesecloth(soakclothin10%benzylbenzoatein95%ethanol,airdry;replacetheclothsevery6months)mayhelppreventinfestation.Somekindsofplasticaredissolvedbybenzylbenzoate,sotestfirstifyouuseplasticvialsortrays(theclothwillfusewiththeplasticwithin24hours)andprotectpaperitemssuchasstocktagsfromdirectcontactwiththecloths.

Topreventtheimportationofmitesfromoutsidesourcesallstocksnewtoyourlabshouldbequarantinedforatleasttwogenerations.Neveropenaforeignbottleorvialatyourflybench(oryourneighbor"s)withoutfirstinspectingthecultureformites.Usingamicroscope,examinethesurfaceofthemediumandthewallsofthecontainer,especiallyaroundpupaeorpupalcases.Ifnomitesareevident,replacefoamorpaperstopperswithtightcottonplugsandisolateculturesinaquarantinetray.Asanaddedprecaution,culturescanbewrappedinthemiteclothsdescribedabove.Keeptheoriginalbottleorvialforabout20days,eventhoughyouhaveestablishedfreshcultures,recheckingformitesevery5to10days.Wecheckthenewculturestoo,justtobesafe,butwehaveneverfoundmitesinasubculturewhentheparentalvialwasmitefree.

Anyculturefoundtocontainmitesshouldbefrozenorautoclavedimmediatelyifitcanbereplacedfromamite-freesource.Ifreplacementisnotpossible,useoneofthemethodsdescribedinAshburner(1989)todisinfecttheculture,suchasdailytransferofadultsforaboutaweek,usingonlythefinaltransfertoestablishanewandmite-free,itishoped,culture.Keepinfectedcultureswrappedinmiteclothsuntiltheyhavebeenmitefreeforthreegenerations.

2.FungiandBacteriaIfmoldisaprobleminisolatedculturesitcanusuallybeeliminatedbydailytransferofadultsfor7-10days.Visuallyinspectculturesfromthelatertransfersforhyphae(lookaroundthepupalcases)anduseonethatappearstobefreeoffungalgrowthforfurthersubculture.Inextremecasesthisprocessmayneedtoberepeatedforanadditionalgeneration.Iffungalcontaminationisawidespreadproblembesurethatfungalinhibitor(p-hydroxy-benzoicacidmethylester)isbeingaddedtothemediumafteritiscooked(boilingdestroystheinhibitor),addasmallamountoflivebaker"syeasttoeveryculture(theyeasttendstoinhibitthegrowthofunwantedfungi)andcheckforsourcesofinfectioninthelab,suchasincubatorfanhousings.CleananycontaminatedorsUSPiciousareaswithdisinfectant.

Avarietyofbacterialcontaminantscanoccurinflycultures.Themostcommonproblemsarecausedbymucusproducingbacteria.Althoughnotdirectlytoxic,larvae,andtosomeextentadults,becometrappedintheheavylayerofmucusthatcoatsthesurfaceofthefood.Largenumbersoflarvaeovercometheeffectsofthebacteriainahealthystock,butweakstocksorpairmatingscanbeseriouslycompromised.AwidespreadbacterialproblemmayindicatethatthepHofyourmediumistoohigh;tryloweringthepHtoabout5.Individualstockscanbetreatedwithantibioticsforonegeneration.Aquickapproachthatoftenworks:add100祃ofpenicillin-streptomycinsolution(10,000u/mland10,000礸/ml,respectively)tothesurfaceofthemediuminavialandallowitbeabsorbed.Addasmallamountofyeastandtransferfliestothetreatedmedium.DiscardadultsbeforeProgenyeclose;subcultureprogenyonuntreatedmedium.Otherantibioticsmaybetriedifthecontaminantprovestoberesistanttopenicillinandstreptomycin.

Alternatively,cleanculturescanbeestablishedfromembryosdechorionatedwith5.25%sodiumhypochlorite(liquidhouseholdbleach,fullstrength).Aconvenientmethodistotransfereggstoableachsoakedwedgeoffilterpaper,wickawaybleachafterchorionshavedissolved(3-5minutes),washeggsseveraltimeswithwaterandtransfertoafreshpieceoffilterpaper(smallenoughtositonthesurfaceofthemediuminavial)moistenedwithwater.Placethefilterpaperwitheggsintoafreshvialoffoodandplacealargerstripoffilterpaperalongthewallofthevial.Wetthisstripofpapertomaintainhighhumidityinthevialuntiltheeggshatch.

Experimentalpopulations

1.MatingsWhilemasstransferofadultsworkswellformoststockkeepingpurposes,itoftenresultsinovercrowdedcultures.Overcrowdingcaneffecttheoutcomeofcrossesandexperimentalprocedures.Developmenttimeisslowed,differentgenotypesmaybedisproportionatelyaffectedbycompetitionforfoodandpupationsites,andmanypupaeandadultswilldrowninthesoupoflarvaeandliquifiedmedium.Thebestyieldofhealthyadultsisobtainedfromculturesestablishedwithanoptimumnumberofanimals.Expect50-100adultsfromavigorous8dramvialculture,300-600fromacomparablehalf-pintbottleculture.Formostgenotypestheoptimumnumberoffemaleswillrangefrom1to3pervialand5to20perbottle.Setupafewtestbottlesorvialstodeterminethisnumberempiricallyforthegenotypesinvolved;controltheageofthefood,theageofthefemales,andthenumberofdaysthefemalesareleftinthevials.Oneortwomalesareusuallysufficienttorapidlyinseminateseveralfemales,butsomegenotypeswillrequireanequalorexcessnumberofmalestoinsurerapidmating.Ifnecessary,theeffectsofovercrowdingcanbereducedmid-culturebyaddingbaker"syeastandatissue(suchasaKimwipe®)toprovideadditionalnutritionandpupationsites,respectively,fortheexcesslarvae.Extremelycrowdedculturesarebestdealtwithbydistributinglarvae(scoopthemoutwithaspatula)toseveralfreshbottlesorvials.

Ifyoucannotdistinguishparentfromprogenybyphenotype,parentsshouldbediscardedbeforetheprogenybegintohatch.Experimentalcrossesmaintainedat25^oCshouldbediscardedafter18daystopreventrecoveryofsecondgenerationprogeny.Aneffectivescheduleistoestablishcrossesonday0(startonaFridayifyouwanttobeginvirgincollectiononaMonday),discardadultsandaddyeastandpapers(optional)onday7,collectvirginsorscoreprogenyondays10through18.

Somemutantphenotypesareaffectedbytemperatureorgeneticbackground.Beforesettingupalargescaleeffortsuchasascreen,makeatestcrossoftherelevantgenotypesundertheconditionstobeusedandconfirmthatallphenotypesarescorable.Itisalsoprudenttoassuretheabsenceof"background"lethalsinastocktobeusedformutagenesisbyisogenizingachromosomeforuseinascreen.Toisogenizeariechromosome,forexample,crosstoanappropriatebalancerstock,recover10-20progenyheterozygousforrieandthebalancer,backcrossthemindividuallytothebalancerstrain,crosssiblingrie/balancerprogeny,andthenrecoverriehomozygotesfromoneofthelinesandestablishastock.Onlylinescarryinglethal-freechromosomeswillproducehomozygotesamongtheprogenyofthesibmatings.

2.VirginsMostexperimentalschemesrequirevirginfemales.D.melanogasteradultsdonotmateforabout10hoursaftereclosion,allowingvirginstobecollectedwithin8-12hoursaftertheculturehasbeenclearedofadults.Thetimingofthisvirginwindowappearstobegenotypedependent.Collectfemaleswithin8hourstobesafe,ordetermineempiricallyforagivenstrainhowlongyoucanwaitandstillrecovervirgins.Ashburner(1989)describesavarietyofenvironmentalandgenetictrickstofacilitatethecollectionoflargenumbersofvirginfemales.

Formanymatingschemesvirginityisdesirableforefficiency"ssake,butnotessentialbecausetheprogenyofnon-virginfemalescanbedistinguishedphenotypicallyfromtheprogenyofinterest.Ifyourschemerequiresvirginity(e.g.,malefertilitytesting,ortheprogenyofnon-virginfemalesareindistinguishablefromthoseoftheintendedmating),holdfemalesfor3-4daysandcheckforlarvaeintheholdingvialbeforeusingthefemalesinmatings.Don"tovercrowdfemalesinholdingvials-50orsoinan8dramvial,fewerifyouaren"tsureoftheirvirginity(you"llhavetodiscardallofthefemalesintheholdingvialifanyhavemated).Thepeakoffemalefertilityisgenotype-dependent,butonaveragefemalesarebestusedbetween4and10daysold.