EfficientsporulationofBY4743requiresgrowthasapatchonfreshlypreparedGNApresporulationplatesat30°Covernightpriortotransfertoliquidsporulationmedium.(Note:ifGNAisinoculatedwithanoldculture,transfertoanotherGNAplateforanadditionalday.)ResUSPendonecolonyequivalentofcellsinto2mlsofliquidsporulationmediumina17x150mmglasstesttube.Sporulationculturesshouldbeincubatedonarollerwheelfor3-5daysat25°C.Theculturesshouldbereadytodissectandcanbestoredat4°Cforacoupleofweeks.However,dissectionwithintwoweeksisthebest.Sporulationefficienciesshouldbearound20-30%.IfyoupresporeonYPDinsteadofGNAthesporulationefficiencyismuchlower. GNAPresporulationPlates Autoclavefor15minutes.Platesshouldalwaysbefreshlymadeandlastonlyfor1-2weeksat4°Cforoptimumsporulation. LiquidSporulationMedium 2mls ResuspendthestocksindH2Oandfiltersterilize.Thestocksaremixedinsterilewaterjustbeforesporulation.FinalConcentrations recipefor2plates recipefor10plates 5%D-glucose 3%Difconutrientbroth 1%Difcoyeastextract 2%Bactoagar FinalConcentrations Stocksolutions 1%PotassiumAcetate 10%PotassiumAcetate(20g/200ml) 0.005%ZincAcetate 0.5%Zincacetate(1g/200ml)
