LIVE-and-DEAD-kits

LIVE/DEAD®BacLight™BacterialViabilityKitsMP07007Revised:15-July-2004ProductInformationLIVE/DEAD®BacLight™BacterialViabilityKitsL7007LIVE/DEAD®BacLight™BacterialViabilityKit*formicroscopy*L7012LIVE/DEAD®BacLight™BacterialViabilityKit*formicroscopyandquantitativeassays*L13152LIVE/DEAD®BacLight™BacterialViabilityKit*10applicatorsets*IntroductionMolecularProbes’LIVE/DEAD®BacLightTMBacterialViabilityKitsprovideanoveltwo-colorfluorescenceassayofbacterialviabilitythathasprovenusefulforadiversearrayofbacterialgenera.Conventionaldirect-countassaysofbacterialviabilityarebasedonmetaboliccharacteristicsormembraneintegrity.However,methodsrelyingonmetaboliccharacteristicsoftenonlyworkforalimitedsubsetofbacterialgroups,1andmethodsforassessingbacterialmembraneintegritycommonlyhavehighlevelsofbackgroundfluorescence.2Bothtypesofdeterminationssufferfrombeingverysensitivetogrowthandstainingconditions.3,4Becauseofthemarkeddifferencesinmorphology,cytologyandphysiologyamongthemanybacterialgenera,auniversallyapplicabledirect-countviabilityassayhasbeenverydifficulttoachieve.OurLIVE/DEADBacLightBacterialViabilityKitsnowallowresearcherstoeasily,reliablyandquantitativelydistinguishliveanddeadbacteriainminutes,eveninamixedpopulationcontainingarangeofbacterialtypes.TheLIVE/DEADBacLightBacterialViabilityKitsutilizemixturesofourSYTO®9green-fluorescentnucleicacidstainandthered-fluorescentnucleicacidstain,propidiumiodide.Thesestainsdifferbothintheirspectralcharacteristicsandintheirabilitytopenetratehealthybacterialcells.Whenusedalone,theSYTO9staingenerallylabelsallbacteriainapopulation—thosewithintactmembranesandthosewithdamagedmembranes.Incontrast,propidiumiodidepenetratesonlybacteriawithdamagedmembranes,causingareductionintheSYTO9stainfluorescencewhenbothdyesarepresent.Thus,withanappropriatemixtureoftheSYTO9andpropidiumiodidestains,bacteriawithintactcellmembranesstainfluorescentgreen,whereasbacteriawithdamagedmembranesstainfluorescentred.Theexcitation/emissionmaximaforthesedyesareabout480/500nmforSYTO9stainand490/635nmforpropidiumiodide.Thebackgroundremainsvirtuallynonfluorescent.Furthermore,althoughthedyeratiossuggestedfortheLIVE/DEADBacLightBacterialViabilityKitshavebeenfoundtoworkwellwithabroadspectrumofbacterialtypes,thesekitsalsoaccommodatefine-tuningofthedyecombinationssothatoptimalstainingofbacteriacanbeachievedunderavarietyofenvironmentalconditions.Acommoncriterionforbacterialviabilityistheabilityofabacteriumtoreproduceinsuitablenutrientmedium.Exponen-tiallygrowingculturesofbacteriatypicallyyieldresultswiththeLIVE/DEADBacLightbacterialviabilityassaythatcorrelatewellwithgrowthassaysinliquidorsolidmedia.Undercertainconditions,however,bacteriahavingcompromisedmembranesmaybeabletorecoverandreproduce—suchbacteriamaybescoredas“dead”inthisassay.Conversely,somebacteriawithintactmembranesmaybeunabletoreproduceinnutrientmedium,andyetthesemaybescoredas“alive.”5TheLIVE/DEADBacLightBacterialViabilityKitshavebeenthoroughlytestedwithavarietyoforganismsandunderseveraldifferentconditions(seeBacteriaThatHaveBeenTested,below).Thekitsarewellsuitedforuseinfluorescencemicroscopyorforuseinquantitativeanalysiswithafluorometer,fluorescencemicroplatereader,flowcytometer6orotherinstru-mentation.InouroriginalLIVE/DEADBacLightKit(L7007),thedyesareprovidedmixedatdifferentproportionsintwosolutions.KitL7007isstillavailableforcustomerswhohavealreadydevelopedprotocolsusingthatformulation.KitL7012,however,ismoreflexiblebecauseitprovidesseparatesolutionsoftheSYTO9andpropidiumiodidestains.Havingseparatestainingcomponentsfacilitatesthecalibrationofbacterialfluorescenceforquantitativeprocedures.Foraddedconvenience,ourLIVE/DEADBacLightkit(L13152)containstheseparatedyespremeasuredintopairsofpolyethylenetransferpipets.Besideshavingtheconvenienceofbeingpackagedinhandyapplicatorpipets,kitL13152hasaformulationthatdoesnotrequiredimethylsulfoxide(DMSO),nordoesitrequirerefrigeratedstorage.TheLIVE/DEADBacLightBacterialViabilityKitsareintendedasresearchtoolsandourTechnicalAssistanceDepartmentwelcomesanyfeedbackontheperformanceofthesekitswithbacterialstrainsandenvironmentalconditionsnotdescribedinthisenclosure.Storageuponreceipt:KitsL7007andL7012•≤–20°C•ProtectfromlightKitL13152•Roomtemperature•ProtectfromlightNote:DonotuseComponentCasimmersionoil.LIVE/DEAD®BacLight™BacterialViabilityKits2MaterialsKitContentsforViabilityKit,L7007$SYTO9dye,1.67mM/Propidiumiodide,1.67mM(ComponentA),300μLsolutioninDMSO$SYTO9dye,1.67mM/Propidiumiodide,18.3mM(ComponentB),300μLsolutioninDMSO$BacLightmountingoil(ComponentC),10mL,forbacteriaimmobilizedonmembranes.Therefractiveindexat25°Cis1.517±0.003.DONOTUSEFORIMMERSIONOIL.KitContentsforViabilityKit,L7012$SYTO9dye,3.34mM(ComponentA),300μLsolutioninDMSO$Propidiumiodide,20mM(ComponentB),300μLsolutioninDMSO$BacLightmountingoil(ComponentC),10mL,forbacteriaimmobilizedonmembranes.Therefractiveindexat25°Cis1.517±0.003.DONOTUSEFORIMMERSIONOIL.Notethata1:1mixtureofComponentsAandBofkitL7012isexactlyequivalenttoa1:1mixtureofComponentsAandBofkitL7007.KitContentsforV