Hintsandprecautionsforthecare,feedingandbreedingofNeurosporaSomeofthesenotesdescribewhatmaybeestablishedroutinesinolderlaboratories,butareeitherunpublishedorpublishedininaccessIBLeplacesorscatteredthroughtheoldliterature,andthusmaynotbeknowntothosejustbeginningtouseNeurospora.Othersmaybepracticesuniquetoourlab.TheattempthereistosupplementcompilationsofmethodssuchasDavisanddeSerres(1970MethodsinEnzymology17A:79-143)andtheolderStanfordNeurosporaMethods(1963NeurosporaNewsletter4:21-25).HintsandprecautionsregardingparticularmutantsorcategoriesofmutantswillalsobefoundinindividualentriesinPerkinsetal.1982Microbiol.Rev.46:426-570(referredtobelowas"Compendium").StatementsapplytoN.crassaunlessanotherspeciesisindicated.NeurosporaNewsletterisabbreviatedN.N.Alltemperaturesaredegrees°C.

ThisdocumentmaybebrowsedoronecanjumptoanyoftheheADIngsdescribedbelow:

CrossingTipsonhandlingascospoesGeneralLaboratoryPracticesStockkeepingMutagenesisandenrichmentTipsforencouragingcolonialgrowthSolutionsandmediaHandlingHeterokaryons

CROSSING

2.Mediaandsupplements.Thesyntheticcrossingmedium(SC)ofWestergaardandMitchellismostwidelyused.TheneedtoadjustpHofSCto6.5canbeavoidedbysubstituting0.7gK2HP04and0.5gKH2HP04perliterforthemonobasicsaltintheoriginalformula(1963N.N.4:21-25).TheformulainDavisanddeSerres,p.86,incorporatesthischange.ThetraceelementsusedforVogel"smediumNaresuitableforSCat0.1mlperliterfinalvolume;thereisnoneedforaseparateformula.

Russo,etal.(1985N.N.32:10-11)havemodifiedVogel"sMediumNforuseasacrossingmediumbyreducingNH4NO3tenfold.ThemodifiedVogel"scanbemadeupasa50Xstock,whereastheSyntheticCrossformulaofWestergaardandMitchellallowsonly2x.

Highammoniumoraminonitrogeninhibitscrossing.Keepaminoacid,purineorpyrimidinesupplementsataminimallevelincrossingmedium.Iftotalaminoacidsdonotexceed0.3mg/mlthereisusuallynoproblemwithfertility.Usuallytheparentwiththesimplestrequirementsispreferredasfemale.

3.Useofper-1toassurefemaleparentage.Wherethepedigreeofamitochondrialgenomeorplasmidiscritical,andassuranceisdesiredthatonlyoneparentisfunctioningasfemale,per-1(typeI)maybeused.Whenper-1ispresentinoneparent,allperitheciashouldbeblackandnowhiteperitheciashouldbepresentiftheprotoperithecialparentwasper-1+,andviceversaiftheprotoperithecialparentwasper-1-.

4.Falseperithecia.Insomesingle-mating-typestrains,protoperitheciamayenlargeandbecomepigmentedsoastoresemblesmallperithecia.These"falseperithecia"aredevoidofbeaks,asci,andascospores,andunfertilizedstrainsexhibitingthemremaincompletelysterile.Thestrainscandevelopnormalperitheciauponfertilizationwiththeoppositematingtype.Falseperitheciaarecharacteristicofsingle-mating-typeculturesofNeurosporatetraspermaandoftheKirbyville,TexaspopulationofN.discreta.FalseperitheciaarealsoencounteredsporadicallyinsomeN.crassagenotypes-theyaremostcommonlyofmatingtypea.Someisolatesoffla(fluffy)genotypehavetendedtomakefalseperithecia.

Thefalseperitheciacanbeanuisanceformating-typetesting,andacauseofalarmifcontaminationisincorrectlysUSPectedtoberesponsible.Ordinarily,continuedcompletesterilityandfailuretodevelopevenafterlongincubationdistinguishfalsefromtrueperithecia,andthereisnoseriousproblem.Falseperitheciacanbeofseriousconcern,however,incrosseswherelegitimatetrueperitheciaarebarrenasaresultofduplicationsorofgenesaffectingmeiosisandascusdevelopment.

Falseperitheciaarealsoanobjectofinterestforthoseinterestedinsexualdifferentiation.Forexample,inactivemutantsofmatingtypeashowdevelopmentofbarrenorfalseperitheciainabortivematingreactionswithAoratesters(Griffithsetal.1978Genetics88:239-254;1982Can.J.Genet.Cytol.24:167-176).

ASCOSPORES.OBTAININGProgenY

6.Rehydrationofascospores.Ascosporesfromoldcrosstubesthataredesiccatedshouldberehydratedbeforeheatshocktoavoidpoorgermination(StricklandandPerkinsN.N.20:34-35).Thisismostlikelytobeaproblemwithcrossesinsmalltubes.Rehydrationcanbeaccomplishedconvenientlybyaddingwatertothecrosstubeorbyholdingisolatedascosporesovernightonmoistfreshmediumbeforeheatshock.Ifascosporesareisolatedtoslantspriortoheatshock,freshtubedmediumgivesbettergerminationthatmediumhaspartiallydrieddown.

7.AscosporeviABIlityandlongevity.Forascosporematuration,someauxotrophsrequiresupplementationofcrossingmediumeventhoughtheyareheterozygous,recessive,andusedasfertilizingparent.Thisistrueofpan-2(notpan-1),spe-1,andsomenic,cysandmetgenes(seeCompendiumentries).

N.crassaascosporesmaybestoredinsterilewatersealedinsmallvialswithoutappreciablelossofviabilityforatleastayearatroomtemperatureand18monthsat4°C(B.R.Smith1973N.N.20:34).MatureascosporesofN.crassaalsoremainviableinordinarycrosstubesandcanbegerminatedaftermanymonthsstorageat5°C.Incontrast,germinationofN.tetraspermaascosporesisreportedtodeclineafter19daysfollowingsimultaneousinoculationofA+aorafter11daysfollowinginoculationofconidiafroman(A+a)culture(Howeetal.1966Genetics54:293-302).

WhenN.crassaperitheciabecomedesiccatedbeforealltheasciwithinthemhavebeenshot,ascosporesremainviableintheunextrudedasciandshowhighgerminationmonthslaterwhenrehydratedandheatshocked.Intactlinearascimaybeextrudedsinglyafterperitheciathathavebeendriedinthiswayareplacedinliquid.Crossesmadein10X75mmculturetubesoftendrydownbeforetheperitheciahaveemptiedtheircontents(P.St.Lawrence),andareconvenientlystoredforfutureuse.

8.Heatshock.Forheatshock,a60°Cwaterbathispreferabletoahot-airoven.(Greaterlatentheat,betterheattransfer,morestable).Allowatleast30minutesat60°Ctoassurekillingofvegetativecellswithawaterbath,doublethisfordryair.Coverthebathtoassurekillingofconidiaontubewalls.

Neverremoveascosporesfrom60°Candthenreturnto60°C.Onceactivated,ascosporesarevulnerabletokillingbyheat.

9.Spontaneousgermination.Somegenotypesresultinspontaneousascosporegermination(forexample,theunpigmentedascosporesofper-1TypeI).Asignificantfractionofascosporescarryingfl(fluffy)germinatespontaneously,atleastinsomecrosses.Thisbecomesapparentwhenascosporesareagedafterisolatingtetrads.Off-ratiosmayresultifflascosporesbreakdormancybeforeheatshockandarethereforekilled.

10.Suspensionanddilutionofascospores.Whensuspendingascosporesfordilutionseries,add0.1%Difcoagartoincreaseviscosityandslowsedimentation;thisconcentrationremainsliquidatroomtemperature.Forover-layering,0.6%,0.8%or1%agarisused,andthissolidifies.

11.Germinationonsurfaceofplates.Itisconvenientforsomepurposestogerminateascosporesaftertheyhavebeenspreadonthesurfaceofprepoured3%or4%agarplatescontainingminimalmediumNorotherother,appropriatelysupplemented,medium.Percentgerminationcanbedeterminedeasilyandquickly.Mostauxotrophsandsomemorphologicalmutantscanbedistinguishedfromtheirwildcounterparts,andcountedorisolatedselectively.Ascosporesaretakenbyloopfromacrosstubetoadropofwaterinthemiddleofthe3%agarplate;thedesirednumber(about200perplate)canbeobtainedbyaddingorremovingsporeswhileexaminingthedropat40-60Xmagnificationunderthedissectingmicroscope.Sporesaredistributedevenlyovertheplate,usingasterileglassspreader.Donotspreadtotheextremeedge.Avoidscratchingthesurface.Ifthegerminatingascospresaretobescoredbutnotpicked,ascosporesmaybesuspendedin0.15%agarandspreadinnarrowparallelstreakswithaPasteurPipette(Maling1959Stanfordthesis).

Platesareheatshocked30-40minabovethesurfaceofa60°Cwaterbathwithlidsoff,andwiththebottomofthedishjusttouchingthewater.Thebathshouldbecovered.Alternatively,lidsmaybekeptoniftheplatebeingheat-shockedisplacedonapartiallysubmergedwater-filledcontainersuchasapetridish,justabovethesurfaceofthe60°Cwaterbath.(Allow60minina60°Chot-airoven.)

Intheabsenceofsorbose,inspectionandisolationofgermlingsmustbetimedcarefullybecausefast-growinggerminantswillovergrowtheplate.Roughestimatesoftiming:18to23hoursincubationat20°C,15to16hoursat25°C,5to7hoursat34°C,11to12hoursat39°C.(39°Cisabovetheoptimum.)

Figure1.Cameralucidadrawingofgerminatingascosporesshowingthesegregationoftwogenesinacrossofcol-4xpyr-2.Sporeswereisolatedonminimalagarmediuminpetridishes,heatedat60°for30minutesforactivation,andincubatedat25°for18hours.Thisnutritionalmutantgrowssufficientlyonminimalmediumtopermitidentificationofpyr-2col-4doublemutants.Oneplateshowssegregationsintwoasciwithsporepairsisolatedinorder.Fromtoptobottomthetwoascishow,respectively,genotypespyr,pyr,col,col;andwild,pyr,col-pyr,col.Theotherplateshowsresultsfromplatingsporesfromthesamecrossatrandom.Geneticanalysescanbemadedirectlyusingeitherprocedure,orbytransferringgerminatedsporestoappropriatelysupplementedculturetubesforfurthertesting.Muchlargernumberscanbeobservedconvenientlyusingtherandommethod.Reproducedwithper-missionfromR.P.WagnerandH.K.Mitchell,1955.GeneticsandMetabolism,2ndedition.J.WileyandSons,Inc.N.Y.

12.MicroscopeandIllumination.Formostmanipulationsandobservations(ascosporeisolations;inspectionofspores,perithecia,etc.),anexcellentgeneral-purposemicroscopeistheStereoZoom7withbase,Bausch&LombCat.No.BVIO70with10Xoculars(1984price$1417).Thisprovides10X-70Xmagnification.Illuminationfromaboveisbestforisolatingascospores,withawhiteplateonthemicroscopestagetomaximizecontrastoftheviableblacksporesanddistinguishthemfromsporesthatareunripeordefective.Lightingfrombelowusingthefrostedreflectorisbestfordeterminingthepercentofdefective,hyalineascosporeswhenscoringchromosomeaberrations.Abalanceoflightfromaboveandbelowisusedforexaminingshotgroupsofeightascosporesandclassifyingthemaccordingtonumbersthatareblackornonblack(Perkins1974Genetics77:459-489).Fixed-focusilluminatorswithsnoutsadaptedforconvenientusewiththeStereoZoom7areavailablefromAmericanOptical(AO*363H,$94)andB&L(Nicholasilluminator,B&L31-33-05-28,$100).Lightingintensityofbothisjustadequateformostpurposes.Wheremoreintenselightisdesiredathighmagnification,afocusableilluminatormaybepreferred,suchasAO653H($182)orB&L31-33-60-40($220).

13.Toolsformanualisolationandtransfer.Isolatingandtransferneedlesarereadilyformedfrom70%platinum30%iridiumwire.020inchdiameter,obtainablefromEngelhardIndustries,700BlairRd.,CataretNJ07008.(Obtainquote.Quantitypurchaseismoreeconomicalbecausethereisaminimumtoolingchargeforeachorder).Platinum-iridiumcanbebeatenandcuttomakebladesorneedlesaresharp,flatandthin,asdesired.

14.Useofglassspreaderforascospores.Ifascosporesarebeingspreadonagarwithaglassspreader,useaseparatespreaderforeachcross.Alcoholflamingisinadequatetokillalladheringascospores(Newmeyeretal.1971N.N.18:13).

LABPRACTICE,HOUSEKEEPING

16.Decontaminationofworkspaceandglassware.Wipebenchsurfacesbeforeandafterusewith70%alcohol,ordilutephenol(Lysol),ordilutehypochlorite(householdbleach).Autoclaveallcontaminatedglasswareandmediabeforediscardingorsendingtodishwasher.Exceptions:Pipettesmaybediscardedintodilutehypochlorite.

17.Mitecontrol.Infestationofculturesbymitescanbeaseriousproblem.Mitescanpassbyorthroughplugsandclosures,carryingconidiaandothercontaminants.Don"tleaveculturesunnecessarilyforlongperiodsatroomtemperature,soastoriskmiteinfestation.Forthesamereason,don"tbringsoil,plants,vegetables,fruitorotherpossiblesourcesofmitesintotheworkareaorintorefrigeratorswhereculturesarestored.Largeslants,whichretainmoistureforweeksormonthsbeforedryingdown,arethemostpronetobecomeinfested.Myimpressionisthatmitesdonotproliferateeffectivelyinculturesthatarecompletelydried.Thisisbasedonverylimitedexperience.

Ifmorethanonedifferenttypeofcontaminantisfoundinatubeorplate,inspectformites(aboutthesizeandshapeofprotoperithecia;use20-40Xmagnification).Ifmitesarefound,autoclaveorfreezeculturesandaccessories.Botheggsandadultsarekilledafter24hoursat-18°C(SuBDenandThrelkeld1966N.N.10:14).Cleaninfestedareascrupulouslyandtreatequipment,benches,shelves,incubators,refrigeratorsandsurroundingswithamiticide.SubdenandThrelkeldreccommendKelthane.Becauseresistancemaydevelop,Threlkeld(personalcommunication)suggestsalternatingKelthaneandMalathion.Wehave,inthepast,cautiouslyusedLindaneappliedtoincubatorsandbenchesinalcoholicsolution(whichleavesaresidue),andhavefoundithighlyeffective.Lindanehassincebeenlistedasacarcinogen(seeMerckIndex),butcarcinogenicitydataaresaidtobeequivocal("AnAssessmentoftheHealthRisksofSevenPesticides...".N.R.C.BoardonToxicology,Nat.Acad.Press,1982).

Freezingkillsascosporesbutnotconidia.AmethodforusingcarbondioxidetoridculturesofmiteswithoutkillingascosporeshasbeendescribedbyMetzenberg(1985N.N.32:22).

18.Avoidsaturatedatmosphereinclosedcontainers.Neverencloseinoculatedplatesortubesinasmallairtightcontainer.Condensateinasaturatedatmospherecreatesafilmofmoistureallowingmyceliatogrowthroughplugsandoutofpetridishes.

STOCKKEEPING

20.Long-termpreservation.Minimizevegetativetransferof"stem"stocks,toavoidacquisitionofmutationsandrearrangements.Subculturefromtheprimarystocktomakeaworkingstockforfertilizingcrosses,etc.Theprimarystockshouldpromptlybeputintosuspendedanimation,suchassilicagel(Perkins1977N.N.24:16-17;seealsoN.N.26:24-27),-20°Cdeep-freeze(Perkins1973N.N.20:33),cryopreservation(Jong,etal.1979N.N.26:26),orlyophil(Barrattetal.1959Science112:122-123).TheFGSChasadescriptionofthesetechniques.

21.Silicagel.Makecertainthatsilicageltubesaresecurelysealedtoexcludemoisture.Longevityrequiressustaineddesiccation.Double-thicknessParafilmiseffectiveforcotton-pluggedtubesifthefilmisstretchedtorenderitadherentandtosealittightlytotheglass.

Wedecidedtouseplain13X100mmculturetubeswithcottonplugsandparafilmsealsforsilicagelstocks,inpreferencetoscrewcaptubes.Withthelatter,wecouldnotbeconfidentthatthesealwastight,andtheconstrictedneckseemedtomakepipettingmoredifficult.

22.Heterokaryonsforpreservingdifficultstocks.Aconidiatestocks,whichshowlowsurvivalafterfreezing,canbehomogenizedandsilicagelled,thoughthisisconsiderablymorelaboriousthanpreparingconidialsuspensions.IfaconidiatestrainsarehetcompatiblewithOakRidgeandhaveaphenotypeappropriateforforcing,theycanbepreservedmosteasilyasphenotypicallywild-typeconidiatingheterokaryonsincombinationwitham1ad-3Bcyh-1asanonmating"helper"component(Perkins1984N.N.31:41-42).Suchheterokaryonscanalsobeusedtoadvantageforshelteringandpreservingdifficultgenotypes.Becauseosmoticstocksarereadilylost,osstocksshouldbereplicatedandmonitoredorcarriedinheterokaryons.

Auxotrophssuchasinlandothersaffectingmembranecomponentshaveshorthalflivesinabsenceofsupplementbutshowgoodsurvivalifsupplemented.Idoubtthattheirlong-termsurvivalonsilicagelhasbeendetermined.

MUTAGENESIS,ENRICHMENTANDREPLICATION

24.Chemicals.Chemicalmutagensarepotentiallyhazardous.ProtocolsforinactivatingmutagensandfordecontaminatingglasswaremaybefoundinchaptersbyL.EhrenbergandC.A.Wachtmeister1977,pp.401-418in"HandbookofMutagenicityTestProcedures"(Kilbeyetal.,eds.).Elsevier/N.HollandPress.

25.Filtration-enrichment.Forfiltration-enrichment,themethodofApplegateetal.,employingsorboseliquidmedium,isrecommended(1978N.N.25:17;Yoder1979N.N.26:2324).Avoidlossofsurvivingconidiainconcludingstepsbypelletingwithdeadconidiaascarrier(D.E.A.Catcheside;describedinYoder1979).

26.Replication.Protocolsforreplication,applicabletostrainswithwildtypeorcotmorphology,aregivenbyNilhedenetal.1975Hereditas79:239-250(seepp.246-248);Littlewoodetal.,1972J.Bact.110:1017-1021.ForexamplesofreplicationusingconidiatingcolonialstrainsseeSchroeder1970MGG107:291-304,DeLangeetal.1981Genetics97:247-259,Stadleretal.1979MGG171:59-68,1984Mutat.Res.127:39-47.

Platesforreplicationshouldbeallowedtodryenoughbeforeusesothatthereisnocondensateorwater-filmonthesurface.

COLONIALGROWTH

28.Useofcot-1.cot-1,whichgrowscoloniallyabove30°C,canbeavaluableadjuncttosorbose(orcanbeusedwithoutsorbose)forplatings.(seee.g.D.G.Catcheside1966Aust.J.Biol.Sci.19:1039-1046;D.E.A.Catcheside1970ibid23:855-865;Schroederetal.1980pp.55-62indeSerresetal.(eds.),"ConferenceonDNARepairandMutagenesisinEukaryotes").

29.Spot-testingonplates.Spot-testingtoscorerequirementsorresistanceisaccomplishedroutinely25spotsperplatein5X5arrays,holdingplatesinvertedtoavoidscatter,andcoolingtheneedleinmediumorinsterilewater(R.H.Davis).Basicspot-testingmedium:0.4%sucrose,0.8%sorbose;incubationat250(DavisanddeSerres,p.88).

SOLUTIONS,MEDIA

31.Inhibitionbysupplements.Indesigningmediaandsupplementation,bealerttoinhibitionsduetocompetitionfortransport.SomeofthesearedocumentedinCompendiumentriesforhis,hom,ser-3,gua,arg,lysandleu.Forexample,ifhis-orhom-ascosporesaregerminatedonorganiccompletemediumcontainingcaseinhydrolysate,growthmaynotgobeyondthegerminationtube.

32.Color-coding.Color-codingofdiagnosticmediaisconvenientandlesserror-pronethanmarkingplugs,tubes,orplatelids.Grocery-store"U.S.CertifiedFoodColor"(e.g.,McCormick,SchillingorCo-op)iseffectiveandisnotinhibitoryatconcentrationsgivingpalecolors.

33.Effectofmediumonconidiation.Conidiationofwildtypevariesdependingonsubstrate.Glycerolcompletemedium(no.2inTatumetal.1950Am.J.Bot.37:38-46)wasdevelopedtomaximizeconidiation.DifcoNeurosporaMedium(arichcomplete)tendstoinhibitconidiation.FactorsaffectingconidiationareconsideredbyTurian1964Nature202:1240andbyDavisanddeSerres,p.138.

HETEROKARYONS

Strainsofoppositematingtypesarehet-incompatiblewitheachotherinN.crassa.Thevegetative-incompatibilityofoppositematingtypes(butnotofotherhetgenes)maybecircumventedbyintroducingtherecessivesuppressortolintobothpartners(Newmeyer1970Can.J.Genet.Cytol.12:914-926),orbyusingamutantmating-typeallele.am33haslostthehet-incompatibilityfunctionbutnotabilitytomate;am1haslostbothvegetativeincompatibilityandmatingfunctions;thesemutantmatingtypeswereinducedinORbackground(Griffithsetal.1978Genetics88:239-254).am1hasprovedusefulasahelpercomponentinheterokaryons(Perkins1984N.N.31:41-42).

35.Countingnuclei.Nuclearcountsinconidia(orhyphae)arereadilymadewithafluorescentmicroscopeafterstainingwithDAPIandHoechst33258(Raju1982N.N.29:24-26).Alternatively,conventionalstainingmethodsmaybeused(seee.g.SernaandStadler1978JBact.136:341-351),buttheyaremorecomplicatedandtime-consuming.

================  蚂蚁淘在线  ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。