| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 484/520&630 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | MicroBIOLOGy FlowCytometry |
| Related | FluorescenceImaging |
| Spectrum | AdvancedSpectrumViewer |
Note1:Thawkitcomponentsatroomtemperaturebeforestartingyourexperiment.
Note2:TheKithasbeentestedatlogarithmicallygrowingculturesofthefollowingbacterialspecies:Micrococcusluteus,Staphylococcusaureus,S.warnerii,Bacilluscereus,Klebsiellapneumoniae,Escherichiacoli,andSalmonellacholeraesuis.
Note3:ManybacteriadonotshowaproportionalresponsetopartialmembranedepolarizationwithMycoStainIt™Green.Theresponseofeachbacterialsystemshouldbeinvestigatedandoptimized.OccasionallytheMycoStainIt™Greenconcentrationandstainingtimemustbeadjustedforoptimaldetectionofmembranepotential.Thefollowingistherecommendedprotocolforbacterialstaining.Theprotocolonlyprovidesaguideline,shouldbemodifiedaccordingtothespecificneeds.
Note4:Somecommonbuffercomponents,suchasTween20,sodiumazideandthimerosal,canaltermembranepotential,andshouldbeavoided.Besuretotestbufferadditivesfortheireffectonmembranepotentialduringoptimizationstudies.
1. Growbacteriainanyappropriatemedium.Bestresultsforhealthybacteriaareobtainedfromlog-phasecultures.Dilutethebacterialcultureto~106cellspermLinPBS(ComponentC)orequivalentsterilebuffer.Bacteriamaybediluteddirectlyfromtheculturemediumwithoutwashing.PreparesufficientsUSPensiontoprovide500µLpertest.
2. Aliquot500μlofthebacterialsuspensionintoaflowcytometrytubeforeachstainingexperimenttobeperformed.Preparetwoadditionaltubesforadepolarizedcontrolandanunstainedcontrol.
3. Add10μlof500μMCCCP(ComponentB)tothedepolarizedcontrolsampleandmix.
4. Add5μlof100XMycoStainIt™Green(ComponentA)toeachflowcytometrytubeandmix(donotaddstaintotheunstainedcontrolsample).Incubatesamplesatroomtemperaturefor30min.Stainedsamplescanbeanalyzedafter5min,butsignalintensitycontinuestoincreaseuntil~30min.
5. Stainedbacteriacanbeassayedinaflowcytometerequippedwithalaseremittingat488nm.Fluorescenceiscollectedinthegreen(fluoresceinfilter)andred(TexasRedfilter)channels. Theforwardscatter,sidescatter,andfluorescenceshouldbecollectedwithlogarithmicsignalamplification.
6. Instrumentadjustmentsareespeciallycriticalfordetectingrelativelysmallparticlessuchasbacteria.Usetheunstainedcontrolsampletolocatebacterialpopulationsintheforwardandsidescatterchannels.Usethesidescatterastheparameterforsettingtheacquisitiontrigger.
7. Applythedepolarizedcontrolsampleafteradjustingtheflowcytometerasdescribedabove.GateonbacteriausingforwardversussidescatterandadjustfluorescencephotomultipliertubevoltagessuchthatthegreenandredMFIvaluesareapproximatelyequal.Donotsetcompensation.
8. Whiletherelativeamountofredandgreenfluorescenceintensitywillvarywithcellsizeandaggregation,theratioofredtogreenfluorescenceintensitycanbeusedasasize-independentindicatorofmembranepotential.Thedatacanalsobeprocessedbygatingonbacteriausingforwardversussidescatter,andanalyzegatedpopulationswithadotplotofredversusgreenfluorescencereportingMFIvaluesaslinearvalues,notaschannels.
9. Onaratiometrichistogram,setMarkersaroundthepeaksofinterestandrecordthemeanratiovalues.Foradotplotofredversusgreenfluorescence,setregionsaroundthepopulationsofinterestandrecordredandgreenmeanfluorescenceintensity(MFI)valuesforeach.Toevaluatethedata,dividetheredpopulationMFIbythegreenpopulationMFI.
10. Intheflowcytometer,bacteriaareidentifiedsolelyonthebasisoftheirsizeandstainABIlity.Itisbesttoinspecteachsamplebyfluorescencemicroscopytoconfirmthattheparticlesdetectedareindeedbacteria.
| References&Citations |  PrinterFriendlyVersion |
1. MyintzuHlaing,M.;Wood,B.;McNaughton,D.;Ying,D.;Augustin,M.A.,RamanspectroscopicanalysisofLactobacillusrhamnosusGGinresponsetodehydrationrevealsDNAconformationchanges.JBiophotonics2017,10(4),589-597.
2. Shi,C.;Jia,Z.;Chen,Y.;Yang,M.;Liu,X.;Sun,Y.;Zheng,Z.;Zhang,X.;Song,K.;Cui,L.;Baloch,A.B.;Xia,X.,InactivationofCronobactersakazakiiinreconstitutedinfantformulabycombinationofthymoquinoneandmildheat.JApplMicrobiol2015,119(6),1700-6.
3. Booyens,J.;Thantsha,M.S.,Fouriertransforminfra-redspectroscopyandflowcytometricassessmentoftheantibacterialmechanismofactionofaqueousextractofgarlic(Alliumsativum)againstselectedprobioticBifidobacteriumstrains.BMCComplementAlternMed2014,14,289.
4. Zeidan-Chulia,F.;Keskin,M.;Kononen,E.;Uitto,V.J.;Soderling,E.;Moreira,J.C.;Gursoy,U.K.,AntibacterialandantigelatinolyticeffectsofSaturejahortensisL.essentialoilonepithelialcellsexposedtoFusobacteriumnucleatum.JMedFood2015,18(4),503-6.
5. DeLamo-Castellvi,S.;Toledo,R.;Frank,J.F.,ObservationofinjuredE.colipopulationresultingfromtheapplicationofhigh-pressurethrottlingtreatments.JFoodSci2013,78(4),M582-6.
6. Cai,P.;Huang,Q.;Walker,S.L.,DepositionandsurvivalofEscherichiacoliO157:H7onclaymineralsinaparallelplateflowsystem.EnvironSciTechnol2013,47(4),1896-903.
7. Nocker,A.;Fernandez,P.S.;Montijn,R.;Schuren,F.,Effectofairdryingonbacterialviability:Amultiparameterviabilityassessment.JMicrobiolMethods2012,90(2),86-95.
8. Lindback,T.;Rottenberg,M.E.;Roche,S.M.;Rorvik,L.M.,Theabilitytoenterintoanavirulentviablebutnon-culturable(VBNC)formiswidespreadamongListeriamonocytogenesisolatesfromsalmon,patientsandenvironment.VetRes2010,41(1),8.
9. Sawaya,K.;Kaneko,N.;Fukushi,K.;Yaguchi,J.,Behaviorsofphysiologicallyactivebacteriainwaterenvironmentandchlorinedisinfection.WaterSciTechnol2008,58(7),1343-8.
10. Alleron,L.;Merlet,N.;Lacombe,C.;Frere,J.,Long-termsurvivalofLegionellapneumophilaintheviablebutnonculturablestateaftermonochloraminetreatment.CurrMicrobiol2008,57(5),497-502.
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