APPROACHESTOTHEESTIMATIONOFMICROBIALVIABILITYUSING FLOWCYTOMETRY

1 2 1 1 HazelM.Davey ,ArsenyKaprelyants ,DieterWeichart andDouglasB.Kell

InstituteofBiologicalSciences,UniversityofWales,Aberystwyth,Ceredigion,SY233DDWales.U.K. Tel +441970623111 Fax+441970622307 EmailHLR@ABER.AC.UK

BakhInstituteofBiochemistry,RussianAcademyofSciences,Leninskypr.33, 117071Moscow,Russia

Keyterms:Celldivision,Death,Dormancy,Resuscitation,Thanatology,Viability

MiniAbstract Formicroorganismsinparticular,viabilityisatermthatisdifficulttodefineandastateconsequently difficulttomeasure.Thetraditional(andgoldstandard)usageequatesviabilityandculturability(i.e.the abilitytomultiply),butdeterminingculturabilityisoftentooslowforourneeds.Flowcytometryprovidesus withtheopportunitytomakerapidandquantitativemeasurementsofdyeuptakeinlargenumbersofcells, andwecanthereforeexploittheflowcytometricapproachtoevaluatesocalledviabilitystainsandto developprotocolsformoreroutineassessmentsofmicrobialviability.

Diskinformation:ThemanuscriptandfigureswerepreparedonanIBMcompatiblePCrunningWindowsNT4. ThemanuscriptwaswrittenusingMicrosoftWord6.0a.Thefilenameiscpcviab.doc. Figures1and3wereinitiallyproducedinGraFit3andpastedintoPowerpointforWindows95(v7.0b)forediting.Figure2was producedinthefreewareWinMDIprogramandtheresultingbitmapwaspastedintoPowerpoint.Figures1to3aretobefound infilefig1to3.ppt. Figure4wasproducedinMicrosoftExcelversion5andisstoredastp3.xls Figure5awasdrawninPowerpointforWindows95(v7.0b)andissuppliedassort_petri2.ppt. Figure5bisaphotographandwillbesentbyconventionalmail.

APPROACHESTOTHEESTIMATIONOFMICROBIALVIABILITYUSING FLOWCYTOMETRY

Introduction

Atpresentonemustacceptthatthedeathofamicrobecanonlybediscoveredretrospectively: a population is exposed to a recovery medium, incubated, and those individuals which do not dividetoformprogenyaretakentobedead........thereexistatpresentnoshortcutswhichwould permit assessment of the moment of death: vital staining, optical effects, leakage of indicator substancesandsoonarenotofgeneralvalidity"......Thetermviabilityappliestopopulations, notindividuals(exceptinanallornonesense:anindividualiseitherviableornonviable)(p.5) (Postgate,1976)

An organism is considered living or viable if itiscapableofcontinuedmultiplicationifitis notsocapableitiscalleddeadornonviable.(p.31) (Greenwoodetal.,1992)

Oneofthemostbasicquestionsthatamicrobiologistmightaskofamicroorganismiswhetheritisaliveor not,andinmicrobiology,itisoftennecessarytodeterminethenumberofliving(oftentermedviable)cells inasampleorcultureofinterest.However,perhapssurprisingly,thisisaquestionthatisnotalwayseasily answered(Kaprelyantsetal.,1993Kaprelyantsetal.,1999Kell etal.,1998),evenformacroorganisms (Watson,1987).Thegoldstandarddeterminationofthenumberofviablemicrobialcellsinasampleis usuallyachievedbyplatinga0.11mlsampleofcells(dilutedasrequired)ontoanagarplate(Hattori,1988 Postgate,1969),andscoringasviable(aposteriori)thosethatwereabletoformvisiblecolonies.Theculture viabilityisthentheratioofthesetothetotalcellcountintheoriginalsampledeterminedmicroscopically. However,thereareseveralproblemsassociatedwiththistechnique,notleastthelengthoftimerequiredto

obtaintheresults.Forsomeslowlygrowingorganisms(e.g.mycobacteria)itmaytakeseveralweeksto determinehowmanycellswere"viable"intheoriginalsample,andevenwhenthesamplecontainsfast growingorganismsandtheplatesareincubatedunderoptimalgrowthconditions,aminimumofovernight growthisusuallyrequiredbeforetheresultingcoloniescanbecounted.Inclinicalsituationsandforeconomic reasons,suchadelayisunacceptable,andmanysocalled"rapid"methodshavebeendevelopedtoallowa speedierassessmentoftheviablemicrobialloadinasample(e.g.AdamsandHope,1989Fung,1994 HarrisandKell,1985Jones,1987)

Thesealternativeandsocalledrapidmethodsofviabilitymeasurementincludeavarietyofstainbased methods.Suchsocalledvitalstainswhichhavebeenusedinattemptstoestimatemicrobialviabilityfall intothreebroadcategories:

Somedyes(e.g.propidiumiodide)areexcludedbytheintactmembranesofviablecellsandthereforethe presenceofthedyewithinthecellindicatesdisruptionofthecellmembraneandthusmaybeexpectedto becorrelatedwithcelldeath. Otherdyes(e.g.rhodamine123(Rh123))areactivelyaccumulatedbyviablecellsthusthenumberof brightlystainedcellswillreflecttheviabilityofthesample(althoughinsomecasesmoreactivecellscan actuallypumpsuchdyesout(JernaesandSteen,1994)!).Equivalently,somedyesarelesstightlyboundby energisedmembranesandsothemoreactivecellsarelessbrightlystained. Inthecaseof dyessuchasfluoresceindiacetateamembranepermeantnonfluorescentprecursoris convertedtoamembraneimpermeantfluorescentmoleculebytheactivityofintracellularenzymesand thusisanindicatorofmetabolicallyactivecells.Theseapproacheswillbediscussedinmoredetailbelow.

Afurtherproblemwiththeplatecountapproachisthatalthoughitisusuallyconsideredtobethegold standardmeasureofviability,aplatecountactuallyonlyindicateshowmanyofthecellscanreplicateunder theconditionsprovidedforgrowth.Inthecaseofenvironmentalsamplesthelaboratorymedia,temperature etcmaydiffersubstantiallyfromthoseintheoriginalsample(RoszakandColwell,1987)andthusthe

proportionofcellsthatcandivideandformcoloniesmaybeexpectedtobe(andindeedis(Amann etal., 1995))muchlowerthanthenumberofcellsthatwouldscoreasviableusingthedyebasedrapidmethods.

Althoughtherapidstainbasedmethodswouldthereforeseemtoofferthepossibilityofamajorimprovement overtheplatecountmethod,theplatecountmethodhasremainedthegoldstandard.Inpartthismaybedue tothefactthattraditionalmicroscopicanalysesofstainedcellsarestilltimeconsumingforthescientistand canleadtooperatorfatiguethusconclusionsarenormallydrawnfromtheanalysisofatbestafewhundred cells.Equally,microscopicexaminationislargelyaqualitativetechniqueandthusajudgementof"alive"or "dead"isallthatispossible,andtheinterpretationoftheextentofstainingofthecellsinthesamplemayvary betweenoperators.

Flowcytometryoffersanalternativemethodofdeterminingtheamountoffluorescentdyetakenupbyeach cellinapopulation.Sincequantitativemeasurementscanbemadeveryrapidlyonalargenumberof individualcellsanaccuratepictureofthedistributionofdyeuptakebymanythousandsofcellsispossible withinafewminutesofobtainingasample.Theflowcytometricapproachwillbediscussedinmoredetail belowbutfirstwemusttrytodefinewhatismeantbythetermviability.

TheProblemofDeterminingViability

Amicrobialcellisgenerallyconsideredviableifitpossessesallthecomponentsandmechanismsnecessary forsustainedproliferation.Viabilityisevidentlybestdeterminedbyassessingcellularproliferationdirectly, andscoringonlythosecellsthathavevisiblymultiplied,buttheunderpinningassumptionofrapid microbiologyisthatwecanestimatesomethingwhichmightcorrelatewithculturabilityjustbyassessingthe presenceandfunctionalityof individualvitalfactorsandprocesses.

Thefirst(classical)methodofviabilitydeterminationinvolvestheprovisionofsuitablenutrientsand mediaandasuitablemethodofdetectinggrowth,whilethesecond(cytochemical)approachgivesan insightintothephysiologyofindividualcellsbyprovidingdataonparameterssuchasmembraneenergisation, enzymeactivityetc.Bothapproacheshavetheiradvantagesandproblems,andstudiesinwhichthesamecells arecomparedusingeachapproachareregrettablyrareindeed.Theclassicalapproachrequiresapriori knowledgeofthesuitablegrowthmediaandconditionsfortheorganismororganismspresentinthe sample,andasuitablemethodofgrowthdetectionfortheorganismsmustalsobeestablished.Inpractice, becauseoflimitationsoftime,materialsandpriorknowledge,alltoooftenthemostconvenientmethodis chosen.Thisusuallyinvolvestheaerobicincubationofthesampleatatemperaturethatishighrelativeto

o thosefromwhichthesamplewascollected(e.g. 30 Cmaybeusedeveninthecaseofenvironmental

samples).HighnutrientcomplexmediasuchasLuriabrothandTSB(trypticsoybroth)areoftenusedfor theseproceduresalthoughtheyareverydifferentfromconditionsfoundinnature.Growthdetectionis usuallymeasuredeitherascolonyformingunitsonasolidagarplateorasturbidityinliquidmedia.Thereare severalproblemswiththiskindofapproach:

Manymicroorganismshavegrowthrequirementsthatareverydifferentfromthestandard conditionsoftenappliedinfact,severalmedicallyimportantbacteria(suchasMycobacteriumleprae)and thevastmajorityofbacteriaintheenvironmenthavenotyetbeenculturedaxenicallybyanymethoddevised

sofar(Amann etal.,1995),andinmanycaseswherewehaveeventuallysucceeded(awellknownexample isLegionella(Meyer,1983)),organismshavedefiedoureffortstoculturethemuntilthecriticalcomponent hadbeenaddedtothemedium.

Microorganismswithknowngrowthrequirementsmayresideinaphysiologicalstateinwhich the(otherwiseappropriate)standardcultureconditionsdonotsupportgrowth,ordosoonlyforasmall fractionofthepopulation,oronlyafterlonglagphases.Physiologicalstatesthatcanbedifficultorimpossible todetectincludeinjury(stress),starvation(orstationaryphase),anddormancy(latencyor cryptobiosis)(Kell etal.,1998),and,insomecasesatleast,normallycopiotrophicbacteriacanbe recoveredonlyafterincubationincomparativelyoligotrophicconditions(MacDonellandHood,1982 Mukamolovaetal.,1998b).

Insomecases,growthofviablecellscanremainundetectedduetotheconstraintsofthe growthdeterminationmethodemployed.Organismsdisplayingslowgrowthratesorlonglagphasesmaynot becapableofproducingenoughbiomasstoformvisiblecoloniesordetectableturbidityduringtheperiodof incubationallowed.Insomecasescessationofgrowthmayoccurafteralimitednumberofdivisions(Kell et al.,1998),ortheorganismmaybeunabletoformcoloniesonsolidmedia.Thesefactors,aloneorin combination,mayleadtofalsenegativeresults.

Inaddition,itisofcourseentirelyplausible(andevenlikely (KaprelyantsandKell,1996 Kaprelyantsetal.,1999Mukamolovaetal.,1998a))thatdormantandunculturedmicroorganismsactually needautocrineorparacrinegrowthfactorsfortheircultivation invitro.

Thusthemaindrawbackoftheclassical,growthbasedviabilityassaysisthepossibilityoffalsenegative resultsfalsepositivescanbeexcludedbycorrectsteriletechnique.Butifweequateviabilitywith culturability,onlygrowthbasedviabilityassaysmakeanytruesense,andthusproliferationbasedviability assaysmaybeconsideredtobebothnecessaryandsufficientcriteriaforcellularviability.

Thissaid,thereareoccasionsinwhichitisthemetabolicactivityofthecellswhichmayconcernus,whether theyarecapableofmultiplicationornot.ClearlyacellwhoseDNAhadbeendamagedattheoriginof replicationcouldnotmultiply,buttherestofitsactivitieswouldprobablybeunaffected.Iftheseactivities includedtheproductionofatoxinweshouldbemuchmoreinterestedinamethodwhichdetectedmetabolic activitythanonewhichrequiredproliferation toscoreforcellularpresenceandactivity.

Cytochemicalassayscanhaveseveraladvantagesoverproliferationbasedassays.Theyaregenerallyless timeconsuming,insomecasesdeliveringinstantaneousresults,andfacilitate(atleastpotentially)amethod ofmeasuringsomethingwhichonemighthopetobeabletocorrelatewithothermeasuresofviability(such asculturability)inorganismsforwhichsuitablegrowthconditionshavenotbeenestablished.Fororganisms whichdisplayextremelyslowgrowthrates,longlagphasesorlowgrowthyields,proliferationbasedmethods areoftenimpossibleorimpractical,andthusthecytochemicalapproachoffersanattractivealternative.In somecases(e.g.flowcytometry),thecytochemicalapproachallowssimultaneousanalysisofthetaxonomic traitsbyspecificantibodiesorribosomalRNAprobes(Amann etal.,1995Vesey etal.,1995Wallneretal., 1993).Thusmultidimensionalsnapshotsofmixedpopulationscanbegenerated,providinginformationon speciescompositionandphysiologicalstatusofcellpopulations.

Theserapidassays,however,oftenhavetheirowndrawbacks.Theycanbedifficulttointerpret,andtheir utilitymaybehamperedbythefollowingproblems:

Sofar,noviabilityassayhasbeendevelopedthatselectivelyandreliablydetectsviablecells, without,underanycircumstances,givingasignalwithdeadcells(operationallydefined(Bareretal.,1998 Kell etal.,1998)ascellsunabletoformacolonyonaplateunderanyconditiontested(Kaprelyantsetal., 1993)).Thisisduetothefactthatassaysarenormallybasedonsingleparameterssuchasmembrane energisation(oftenreferredto,despitetheabsenceofevidenceforitinbacteria(Kell,1988Kell,1992),as membranepotential),enzymeactivity,oruptakeofsubstrate.Someofthesecriteriamightbeconsidered

necessarytodefineviabilityinmostcases,butnoneofthemissufficienttoexcludenonviablecells.Thus theseassayscangiverisetofalsepositiveresults.Forexample,acellcoulddisplaysomeenzymeactivitybut mayhavelostitsabilitytodividebylethallesionsinthechromosome.Thusitisoftheutmostimportanceto ascertainthereliabilityofanyviabilityassaybynegativecontrolexperiments,preferablyinvolvingsamples ofcellskilledbyarangeoftreatments(heat,ethanol,chlorineetc.).

Commercialviabilityassays(seelater)canalsoproducefalsenegativeresults,evenifthe suppliersofassaykitsincludeseeminglyconvincingdatasupportingtheirreliability(albeitunderrather restrictedconditions).Ingeneral,thetestpopulationsemployedtodemonstratedetectionofviablemicrobes areeithergrowingcellsorcellssubjectedtorathershortperiodsofstress(heat,coldetc).Innatural environmentsstarvationand/orstressmaybelongterm,andtheactivityofcellsmaybereducedtoextremely lowlevels(especiallyinthecaseofdormantcells),such thatpositiveresultsmightbebelowthelimitsof detectionoftheassay.Similarly,injuredcellsmayhavedamagedmembranesandwouldscoreasnonviable inthesekits,butrepairofthedamageduringcultivationonarichmediumwouldallowthemsubsequentlyto grow(suchthattheyshouldhavebeenscoredasviable).Theapparentparadoxisavoidedbytheuseof operationaldefinitions(Bareretal.,1998Kell etal.,1998)inwhichviabilityisnotinfactan innate propertyof acellbutisscoredasaresultofourexperimentalmeasurements.Usingthesedefinitions however,weshouldnotbesuprisedifdifferentexperimentsleadtodifferentresults!

Takentogether,theviabilityassayswhicharenotbasedonproliferationmightbelesstimeconsumingand moreconvenientinalotofcases,buttheydonotnecessarilyprovidereliabledata,astheprinciplesonwhich theyarebasedarenotsufficient(orsometimesevennecessary)criteriaforviability.Thus,beforeuse,each methodforviabilityassessmentmustbevalidatedforeachorganismandforeachtypeofsampleinorderto avoidfalsepositiveorfalsenegativeresults.

FluorescentStainsforMicrobialViabilityDeterminationsbyFlowCytometry
Flowcytometryoffersarapidmethodforthequantitativedeterminationofstainuptakewithinasampleof cells(DaveyandKell,1996Kell etal.,1991Lloyd,1993Shapiro,1995).Thereareseveralclassesof moleculeswhoseuptakeand/orfluorescencemaybeexpectedtoreflecttheviabilityofthecells(seeTable 11.7.1)andthesearediscussedbelow.

i)DyeExclusion Fluorescentstainsnormallyexcludedbylivingcellshavebeenusedtoassessviabilityonthegroundsthat deadcellswillhaveleakymembranesthatarepermeabletothestains.Nucleicacidstainssuchaspropidium iodideorethidiumbromideareindeedgenerallyexcludedbyintactplasmamembranesandtheiruptakeis oftenusedtoindicatecelldeath (Aeschbacheretal.,1986Bhmer,1985Green etal.,1994Groganand Collins,1990Jones,1987Lapinsky etal.,1991LpezAmorsetal.,1995Schmidetal.,1992).

Propidiumiodideisoftenthedyeofchoiceforviabilitydeterminationsinanimalcells,whethertheassayis doneusingflowcytometryorfluorescencemicroscopy (seee.g.Garneretal.,1997MaxwellandJohnson, 1997Ronotetal.,1996).Thereishowever,aninherentdangerinblindlytransferringprotocolsdeveloped foronecelltypetoanotherparticularlywhen,ofthetwocelltypesinquestion,oneisaeukaryoticcelland theotherisaprokaryote.Thus,theapplicabilityofsuchdyesformicrobialviabilitydeterminationsneedsto becarefullyconsideredforeachtypeoforganism,notleastbecauseefficienteffluxpumpscapableof removingethidiumbromidefrom EscherichiacolihavebeendemonstratedbyJernaesandSteen (1994),and manyothersuchpumpsareknown (Lewis,1994).

ii)Dyeuptake Itiswelldocumentedthatthemitochondriaofeukaryoticcellshavetheabilitytoaccumulate"lipophilic" cationssuchasRh123concentratively (Chen,1988Chen etal.,1982GroganandCollins,1990Johnson et

al.,1981Johnson etal.,1980),inanuncouplersensitivefashion,andthestainingofmitochondriawith Rh123,inconjunctionwithflowcytometry,hasbeenusedtostudytheiractivity (Darzynkiewiczetal.,1981 Iwagaki etal.,1990Lizardetal.,1990).ViablebacteriaalsoaccumulateRh123,butnonviableonesdo not(Diaperetal.,1992),andundercertainconditionstheextenttowhichindividualbacteriatakeupRh123 quantitativelyreflectstheextentoftheirviability (KaprelyantsandKell,1992),i.e.whethertheyare immediatelyculturable,nonculturableordormant.

Onaverage,largercellsmaybeexpectedtoaccumulatemoremoleculesofRh123thandosmallercells,but sinceflowcytometryallowscollectionofbothfluorescence(Rh123uptake)andforwardlightscattering(cell size)fromeachcell,thedatacanbeplottedasadualparameterhistogram,enablingonetotakesize differencesbetweencellsintoaccountwheninterpretingthedata.

Incontrasttosomeoftheotherviabilitystains(e.g.acridineorange(BackandKroll,1991)),theuptakeof Rh123notonlydoesnotrequiretheuseoffixativestopermeabilisethecell,buttheconcentrativeuptakeis dependentonanintactandenergisedcytoplasmicmembranethuslivingcellscanbeusedforstaining purposes.Thishasthegreatadvantagethat,followingstainingofthecells,furtherphysiologicalstudiesmay beconductedifrequired(Davey etal.,1993).

Therearehoweverexperimentalproblemswiththeuseoflipophiliccationsformicrobialviability determinations.Theseincludethefactthattheymaybepumpedoutofviablecellsbymicrobialdrugefflux pumps,andthusbothviableandnonviablecellsmayappeartobenonfluorescent.Inaddition,whilethe stainisreadilyconcentratedbyGrampositivebacteriasuchasMicrococcusluteus,thepermeabilityofthe staininGramnegativeorganismsislowunlessthecellsarepretreatedwithEDTA (KaprelyantsandKell, 1992).Howeveroneshouldbearinmindthatsuchpretreatmentispracticallyimpossibletostandardise,and thustheextentoflipophiliccationaccumulationmayvaryfromexperimenttoexperiment.Inaddition,ina protocolforviabilitydetermination,itisgenerallydesirablethatthenumberofpreprocessingstepsbekept toaminimuminordertoavoidthepossibilityofaffectingtheviabilityofthesample.

Analternativeapproachistheuseoflipophilicanions,which,incontrasttocationsbindpreferentiallytonon viablecells.Thelipophilicanionbis(1,3dibutylbarbituricacid)trimethineoxonol(DiBAC4(3))hasbeen showntoentereukaryoticmembranesonlyiftheirmembraneshadbecomedeenergised(WilsonandChused, 1985).Thesamestainhasbeenusedfortherapidassessmentofmicrobialresponsestoantibiotics(Jepraset al.,1997Mason etal.,1994Mason etal.,1995bSulleretal.,1997),allowingtheanalysisofheterogeneity withinamicrobialpopulationintermsofsusceptibilitytoanantibiotic.

iii)MetabolicActivity Athirdclassofviabilitystainsareusedinmammaliancellbiology,oftenasapositiveviabilitymarkerina dualstainingprotocolwithethidiumbromideorpropidiumiodide(Aeschbacheretal.,1986).Oneexampleis fluoresceindiacetatewhichitselfisnonfluorescentbutmembranepermeant.Itiscleavedbyintracellular esterasestoproduceaproduct(inthiscasefluorescein)thatisfluorescentbutunderidealconditionsnon membranepermeant.Deadcellswithleakymembranesdonotstainbecausetheylackenzymeactivityand/or fluoresceindiffusesfreelythroughthemembrane.Flowcytometricanalysesofmammaliancellswiththisclass ofdyesiswellestablished(e.g.Aeschbacheretal.,1986Frey,1997).DiaperandEdwards(1994a1994b) usedflowcytometrytodetectavarietyofviablebacteriaafterstainingwithfluoresceindiacetateandits derivativesandwithChemChromeB.Importantly,noneof thedyestestedwasfoundtobeuniversalforthe detectionofviablebacteria.However,ChemChromeBwasfoundtostainthewidestnumberofGram positiveandGramnegativespecies,whereastheFDAderivativespreferentiallystainedGrampositive bacteria.BreeuwerandcolleaguesshowedthatFDAandcarboxyFDApenetratedyeastrapidly,andthat esteraseactivitywasprobablymostlimiting(Breeuweretal.,1995)anenergydependenteffluxfromviable cellsofcarboxyfluoresceinwasalsoobserved(Breeuweretal.,1994Ueckertetal.,1995).Itisprobable thatfluoresceincan bepumpedoutoforleakrapidlyfromviablebacteria,thusgivingtheappearanceoflack ofmetabolicactivityincellsthatarenonethelessviable.

iv)Commercialkitsformicrobialviabilityassessment Avarietyofkitshavebeenproducedspecificallyforthemeasurementofviabilityofspecifictypesof organisms.Forexample,theLIVE/DEADBacLightBacterialViabilityKitfromMolecularProbes(Oregon, USA)givesatwocolour(SYTO9(green=live)/propidiumiodide(red=dead))viabilityassessmentofboth GramnegativeandGrampositivebacteria.However,asfreelyadmittedbythecompany,thekitequatesthe presenceofintactplasmamembraneswithviability.Thus,bacteriarenderednonviablebyexposuretoagents thatdonotnecessarilycompromisetheintegrityoftheplasmamembrane,suchasformaldehyde,usually appearviablebythiscriterion(Haugland,1996).Despitethislimitationthekitisbecomingwidelyusedin microbialflowcytometry (Braux etal.,1997BuchmeierandLibby,1997Decampetal.,1997Duffyand Sheridan,1998Jacobsen etal.,1997 Joux etal.,1997Korberetal.,1997LangsrudandSundheim,1996 Rigsbeeetal.,1997Swartsetal.,1998Taghikilani etal.,1996Terzievaetal.,1996Virtaetal.,1998 Weiretal.,1996).Thegrowinguseofsuchkitsreflects,atleastinpart,theireaseofuse.Inthecaseofthe BacLightkitthereagentsaresimultaneouslyaddedtothebacterialsuspension,whichisthenincubatedfor5 10minutes.Thesampleisthenanalysedwithoutwashingsoliveanddeadbacteriacanbedistinguished andquantitatedinafewminutes.Thereisagreatdangerthatbecauseofthenametheuninitiatedmayuse thesetestsblindly,despitethemanufacturerswarnings,withoutcheckingthereliabilityofthedyeswiththe organismsandconditionsusedintheirexperiments.

Despitetheproblemsassociatedwithfluorescentstainingprotocolsforviabilitymeasurements,providingone carefullyselectsandtestsanappropriateprotocolfortheproblemunderinvestigation,usefulinformationcan stillbeobtained.Toillustratethis,wepresentacasestudyoftheuseofflowcytometricviabilitytestinginthe Grampositivebacterium Micrococcusluteus.

EstimationsofViabilityofMicrococcusluteus
Innatureandunderconditionsofstress,bacterialculturesdisplaysignificantheterogeneityintermsofthe percentageofviable(culturable)cells,andwithrespecttocellularmetabolicactivities(Kaprelyantsetal., 1993).Animportanttaskisthereforetofindreliableandrapidmethodsforestimatingthenumberofcells withdifferentcharacteristicsinthewholebacterialpopulation.Tothisend,theapplicationofflowcytometric methodsseemsverypromising,sincetheyallowonetodistinguishthepropertiesof individualcellsinthe population.Sincefluorescenceandlightscatteringaremeasuredonaquasicontinuousscaleitispossibleto quantifytheheterogeneityofasamplefullyratherthanasimpleclassificationintooneoftwoclasses(liveor dead).Thefollowingexperimentswith Micrococcusluteusillustratetheapplicationofflowcytometryfor discriminationbetweencellsindifferentphysiologicalstates.

AvarietyofflowcytometricapproacheshavebeeninvestigatedforthedeterminationofviabilityintheGram positive,nonsporeformingbacterium MicrococcusluteusNCIMB13267(Kell etal.,1995).Thebacteria aregrownaerobicallyat30CinshakeflasksinalactateminimalmediumcontainingLlactate(seee.g. KaprelyantsandKell,1992forfulldetails).Inordertoobtainsamplesoflowviability(asjudgedbyplate counts)thecellsmaythenbesubjectedtoastarvationstressbyallowingthemtoreachstationaryphase

o beforeholdingthemat30 Caerobicallyforupto1month,followedbyafurtherincubationofupto3months

atroomtemperaturewithoutagitation.Asaresultoftheseprocedurescellpopulationsoflowviabilitycanbe obtained(lessthan0.01%ofthecellsmaygrowonagarplates(solidifiedNutrientBrothE)thatwould normallysupportgrowth).Howeverthetotalcellcount,estimatedmicroscopicallyusingacountingchamber, remainscloseto100%oftheinitialprestarvationvalue(KaprelyantsandKell,1993a).Infact,asillustrated below,starvedpopulationsconsistofdifferentsubpopulationswhichcanbevisualisedflowcytometrically.

i)Estimationofnumbersofactiveversusinactivecells Theproportionofactivecellsinapopulationof M.luteuscanbeestimatedusingthemembraneenergisation sensitiveprobeRh123.Figure11.7.1showsthetypicaldistributionofthefluorescenceofnonstarvedM.

luteuscells,stainedwithRh123andanalysedbyflowcytometry.Theregions(A,BandC)weredemarcated followingtheanalysisofafreshlyharvestedsampleofviablecellsstainedwithRh123,whichintheabsence ofuncoupler,exhibitedaleveloffluorescencebetweenchannels80and136(Figure11.7.1a),whichwasfully uncouplersensitive(Figure11.7.1b).

AgoodcorrelationisobservedbetweenthepercentageofviableM.luteuscellsinthepopulationandthe percentageofcellswithCCCPsensitiveaccumulationofRh123,judgedflowcytometrically (Kaprelyantsand Kell,1992).Similarresultswereobtainedbytheflowcytometricstudyof M.luteuscellsstainedwithCTC (5cyano2,3ditolyltetrazoliumchloride),thereducedformofwhich(afluorescentformazan)allowsoneto monitortherespiratoryactivityofindividualcells(KaprelyantsandKell,1993bRodriguezetal.,1992).

ii)Estimationofdormantanddeadcells Ithasbeenshownthat1050%of M.luteuscellsin3montholdpopulationscanberesuscitatedtonormal, colonyformingbacteriaunderconditionswhichexcludeanysignificantregrowthofinitiallyviablecells (Kaprelyantsetal.,1993KaprelyantsandKell,1992KaprelyantsandKell,1993aKaprelyantsandKell, 1996Kaprelyantsetal.,1996Kaprelyantsetal.,1994Kaprelyantsetal.,1999Mukamolovaetal.,1998a Mukamolovaetal.,1998bVotyakovaetal.,1994Votyakovaetal.,1998).Thisindicatesthepersistenceof asignificantpercentageofcellsinthedormantstate.ThishypothesiswasconfirmedusingtheMostProbable Number(MPN)methodbytheresuscitationofcellsfromsampleswhich,statistically,containedno"initially viable"cells(Kaprelyantsetal.,1994).Itwasfoundthatwhenthemediumalsocontainedspentgrowth mediumfromacultureinlatelogphase,asubstantialincrease(1000to100,000fold)inthenumberof viablebacteriawasobservedwhencomparedwiththoseestimatedwiththeagarplatemethod(Table11.7.2). Theseexperimentswerethefirstthatservedconclusivelytoexcluderegrowthasacontributortothe observedresuscitation anenormousproblemthatisrarelytackledsatisfactorilyinthiscontext(Kell etal., 1998)(andonethatisalsohighlysignificantfortheisolationofslowlygrowingstrainsfromnatural ecosystems(Button etal.,1993Schutetal.,1993)).Itwasalsoconcludedthatviablecellsof M.luteuscan secreteapheromonelikesubstance,whichisapparentlynecessary(thoughnotonitsownsufficientinall

cases)fortheresuscitationofstarved,dormantcellsofthesameorganism (Kaprelyantsetal.,1994).This substance(ResuscitationPromotingFactor(RPFfactor))isasecretedsmallproteinwithaMWofca1718 kDa(Kaprelyantsetal.,1999Mukamolovaetal.,1998a).

AsshowninFigure11.7.1,theproportionofdormantcellscanalsobedeterminedusingflowcytometry. Figure11.7.1cshowsatypicaldistributionofthefluorescenceof M.luteuscellsthathadbeenstarvedfor5 months,stainedwithRh123andanalysedbyflowcytometry.Abimodalfluorescencedistributionisevident. RegionA(channel0tochannel80)representscellswhichbindRh123nonspecifically:98%offreshlate logarithmicphaseM.luteuscellsstainedwiththesameconcentrationofRh123followedbytreatmentwitha suitableconcentrationoftheuncouplerCCCPexhibitedafluorescenceinthisregion(Figure11.7.1b). AlthoughstarvedcellsalsooccurredinregionB(betweenchannels80and136)theirsensitivitytoCCCP wasverylow(only25%ofthecellsinregionBexhibitingadecreaseinfluorescenceafterCCCPtreatment). Thisphenomenonwasnotduetoanyinabilityoftheuncouplertoactperse,sinceoctanoltreatmentalso failedtodecreasetheextentofstainingofsuchcells(notshown).Ithasbeensuggestedthatcells accumulatedinregionAandBrepresentbacteriaindifferentphysiologicalstates(Kaprelyantsetal.,1996). Todeterminewhetherthisisindeedthecaseacellsortingapproachwasused(seethesectionontheuseof cellsorting,below).

iii)Estimationofinjuredcells Thepresenceofinjuredbacteriainstarvedpopulationsorinpopulationssubjectedtostress(freezing,drying etc)isverylikely.Commonly,suchcellshaveanimpairedmembranepermeabilitybarrierwhichhasbeen testedbythefollowingflowcytometricapproaches:

a)Membraneimpermeantprobes Theuseofdyeexclusionformonitoringviabilitybyflowcytometrywasdiscussedabove,butinthecaseof bacteriaadamagedorleakymembranemaynotbeasufficientcriterionfordefiningacellasnonviablebutit canbeusedasanindicationofstressinducedinjury.Thepermeabilitybarrierofthecellsthatwerestarved

for5monthswasmonitoredbystainingwithPI(Mukamolovaetal.,1998b).ItwasshownthatPIdoesnot penetratethecytoplasmicmembraneofintactM.luteus,whiletheadministrationof0.5%v/voctanoltothe cellsuspensionresultedin100%ofthecellsbeingstainedwithPI(seeFigure11.7.2).Observationof differentstarvedculturesof M.luteusrevealedthatinsomecultureswherethepercentageofPIpositivecells iscloseto100%theresuscitationofcellswasnotsuccessful(eveninthepresenceofRPFfactor).This indicatesacorrelationbetweenthestateofthepermeabilitybarrierandtheabilityofstarvedcellstorecover andthismayallowtheuseofPIstainingfordiscriminationbetweendormantanddeadcellsinapopulation.

b)NADHinducedrespiration Theabilitytomonitortherespiratoryactivityofindividualcellsallowsthedesignofexperimentsforthe quantitativedeterminationofinjuredcellsinapopulationfollowingstresssuchasfreezing.Itiswellknown thatsomebacteriainstressedpopulationsbecomeinjured,asreflectedforexampleintheirelevatedsensitivity tosurfaceactiveagents(RayandSpeck,1973).Thiseffecthasbeenusedforenumeratinginjuredbacteriaby platingthemonselectivemediacontainingdetergents(RayandSpeck,1973).Howeverthisapproachcan reflectonlyinjuriesconnectedwithdamagetotheouterportionofthecellenvelopeofGramnegative bacteria(Ignatov etal.,1981RayandSpeck,1973),whilstitisdamagetothecytoplasmicmembranethatis moreimportantindeterminingtheviabilityofbacteriaafterfreezing(Ignatov etal.,1981).Thus,ithasbeen shownthatanincreaseinthepermeabilityofthecytoplasmicmembranetoNADHafterfreezing(whichin contrasttonormalcellsresultedinthestimulationofendogenousrespirationbyNADH),waswellcorrelated withadecreaseintheviabilityof E.coli(Ignatov etal.,1981).Theflowcytometricbehaviourof frozen/thawedM.luteuscellsaftertheadditionofCTCandinthepresenceorabsenceofexogenousNADH revealedthatafterthefirst5minutesofincubationinthepresenceofCTCapproximately25%ofthe populationgaveasignificantfluorescencewhenNADHwasalsopresentbutonly1%whenitwasnot.After 17minofincubationwithCTCthepercentageofcellsfluorescingaboveachannelnumberof20had increasedinbothcases(NADH),whilsttheirdifferencehadnot.Furtherincubationofthecellsresultedina decreasingdifferenceinthedistributionpatternforthetwotypesofsample.ThekineticsofCTCreductionin thetwosamplesaresummarisedinFigure11.7.3.

ThereductionofCTCwithinthefirstfewminutesofincubationinthepresenceof NADHindicatesthe existenceofcellswithaninjuredpermeabilitybarrierbutwithanintactrespiratorychain (Ignatov etal., 1982Ignatov etal.,1981).ThesecellsveryrapidlyreduceCTCtoformazan,toaconcentrationcomparable withthatinintactcells(asjudgedbythechannelnumberofthefluorescence),whilstsomeendogenous substratesleftinthecellsafterfreezingandthawingpermitaslowerreductionofCTCinthesampleswithout NADH.Wecanconcludethatatleast2530%ofthecellsinafrozenpopulationof M.luteusareinjured (althoughthefinalviabilityofthissample,asjudgedbyplatingonarichmediumwhichpermittedrepair processestotakeplace(Ignatov etal.,1982RayandSpeck,1972),was9095%).

DesigningaProtocolforMicrobialViabilityAssessment

Whenselectingastainforaparticularapplicationthereareseveralfactorsthatneedconsideration.Someof thesesuchastheextinctioncoefficient,quantumyield,photostabilityetcareofgeneralapplicabilitytoflow cytometricfluorescencemeasurementsandarediscussedindetailelsewhere(DaveyandKell,1996Shapiro, 1995).Thewavelengthsavailableforexcitationmustalsobeconsideredandalistofviabilitystainsthatare compatiblewithcommonflowcytometriclightsourcesisshowninTable11.7.1.

Factorsthatareofparticularrelevanceinthemeasurementofviabilityincludethetoxicityofthestain.When oneismakingsuchmeasurementsitisdesirablethattheptotocolsuseddonotperturbtheviabilityofthecells understudy.Thisbecomesessentialwhenonewishestoperformfurtherphysiologicalstudiesonthecells forexamplebyexploitingflowcytometriccellsortingtoisolatesubpopulationswithdifferentfluorescence properties.Inthiscasethetoxicityofthestain(andindeedofanyotherchemicalsused)mustbeassessedat theconcentrationsusedintheprotocoltoensurethattheydonothaveanyunwantedeffects.

Evenwherefurtherphysiologicalstudyisnotrequired,itisgenerallydesirabletousecellsthathavenotbeen fixedtoavoidanypossibleperturbationofwhatoneistryingtomeasure.Thisapproachdoeshowever,have thedisadvantagethatlevelsofcellularautofluorescencearegenerallyhigherforunfixedcellsthanfore.g. ethanolfixedcells.Forthemajorityofmicroorganisms(chlorophyllcontainingorganismsarethemost notableexception)cellularautofluorescencetendstodecreasesubstantiallytowardstheredendoftheoptical spectrum,andthisisdrivingthedevelopmentofredexcitedfluorophores(Fabian etal.,1992Patonayand Antoine,1991Shealy etal.,1995aShealy etal.,1995b).Suchdyescanbeexploitedinflowcytometry usinga633nmHeliumNeonlaserora635nmlaserdiode.

Laserdiodescanbeusedtoconstructsmaller,cheaperandmorerobustflowcytometerse.g.theMicrocyte (seeInternetResourcessectionforfurtherinformation).TheMicrocytewasdevelopedbyGjelsnesand

Tangen (1994)primarilyfortheanalysisofmicroorganisms.Usingthisinstrumentitispossibletoobtainboth atotalcountandaviablecountinabsolutetermsveryrapidly (DaveyandKell,1997).Flowcytometric analysesofsamplesof M.luteusstainedwiththeexclusiondyeTOPRO3areshowninFigure11.7.4,see thelegendfordetails.

Whentheappropriatestainandexcitationsourcehavebeenselecteditisimportanttoperformaseriesof experimentstodeterminetheoptimumconcentrationofthestainandtheoptimumlengthoftimebetween additionofthestainandthesubsequentanalysis.Theoptimumconcentrationwillinevitablybeacompromise betweenchoosingahighconcentrationtogivemaximumsignal,andalowconcentrationtogivespecificity.It maybenecessarytomeasureandadjustthecellconcentrationtoensurethatstainuptakeisnotlimiting.In thiscasetheuseofaflowcytometerthatallowsdeterminationofabsolutecellnumbersisanidealapproach.

Insomesituationsitmaybedesirabletoexploitthemultiparametricnatureofflowcytometrytousetwo differentviabilitystainthatrelyonmeasuringdifferentcellularparameters(e.g.YurkowandMcKenzie, 1993).Alternatively,onemay wishtocombinetheviabilityassaywithmeasurementsofothercellular properties.Ineithercase,carefulselectionofallofthestainsinvolvedisrequiredtoensurethatthereis minimaloverlapintheemissionspectra.

TheUseofCellSortinginViabilityStudies

Whileflowcytometricanalysisallowstheinvestigatortoperformarapidandquantitativeversionofthe experimentsthatcouldotherwisebeperformedbyfluorescentmicroscopy,flowcytometriccellsorting allowstheprocesstobetaken oneveryimportantstepfurther.Withflowcytometricanalysisonecansimply saythatthedistributionofdyeuptakeiscorrelatedwithaplatecountofthesamesample.However, providingthestainingprotocoldoesnotaffecttheviabilityofthecells(whichmaybedeterminedbyplate countsofstainedandunstainedsamples)onecanexploitthesortingcapabilityofappropriateinstrumentsto separatecellsfromthepurportedviableandnonviablefractionsofthehistogram.Thisallowsdetermination of theculturabilityofexactlythosecellswhosecytologicalpropertieshadalreadybeendetermineddirectly.

TothisendwesortedcultureswhosefluorescencewasofthetypedisplayedinFigure11.7.1cintotwo populations:(i)cellsofwhichtherhodaminestainingwassensitiveorpartiallysensitivetoCCCP(regions B+C)and(ii)cellswhoserhodaminedependentfluorescencewasnotsensitivetoCCCP(regionA).After sorting,cellswereplatedonnutrientagarandexaminedinanMPNassayforviablecountdeterminations, whilethetotalcountofsortedsampleswasalsoexamined.Theseexperimentsrevealedthattheresuscitation ofcellsasjudgedbytheMPNassaywassuccessfulforcellsinregionsB+CbutnotforcellsinregionA.This constitutesdirectevidencethatdormantcellsareconcentratedinregionsB+C(Kaprelyantsetal.,1996).

SomeflowsorterssuchastheCoulterEpicsElitehaveamotorisedstage(theAutoclonemodule)forthe collectionofsinglesortedcells.Whilethisisprimarilydesignedforthecollectionofcellsintothewellsof microtitreplates,itisalsopossibletomodifythestageandcollectionprotocol(seeFigure11.7.5)toallow microbialcellstobecollecteddirectlyontoagarplates.Thusaneventfromatightlydefinedregionona histogramcanbecorrelateddirectlytogrowth(orfailureofgrowth)ofacolonyonanagarplate.This approachhasbeenpioneeredbyNebevonCaronandcolleagues(seee.g.NebevonCaronandAnderson, 1996NebevonCaronandBadley,1996seealso

http://www.cyto.purdue.edu/flowcyt/research/micrflow/gerhard/genmic10.htm)andhasgreatpotentialfor manymicrobiologicalapplications.

Whilethesortingapproachoffersmanyadvantages,onemustbeawareofpotentialpitfallsofthemethod. Microbialcellsoftengrowinclumpsorformaggregatesduringsamplepreparation.Ifonecellinaclumphas aleakymembraneitwilltakeupanexclusiondyesuchaspropidiumiodideandthustheclumpwillbe fluorescentandwillscoreasdeadintheviabilityassay.However,ifthisclumpalsocontainsoneormore livecells,acolonywillresultwhentheclumpissortedontoanagarplate.Thisproblemcanbeovercometo someextentbyusingtheforwardscattersignalasanindicatorofsize.However,sincethesizeofmicrobial cells,evenwithinasinglespecies,canvarygreatlywithgrowthconditions,amorerobustapproachmaybeto usetwoormoreviabilitystainstogiveabroaderpictureofthephysiologicalstatusofthecells.Anadditional problemmayarisewithdamagedcells.Theprocessofflowcytometricanalysis,followedbysortingontoan agarplatemayinitsownrightbeconsideredasanadditionalstresswhichmayconvertaninjuredcellintoa nonviableone.Suchstressescanbequantifiedtosomeextentbyplatinginjuredcellsbeforeandaftersample preparation.Theeffect,ifany,ofthesheathfluidon injuredcellsshouldalsobedetermined.

Conclusions
Therapidcytologicalestimationoftruemicrobialviabilityisextremelydifficult(ifnotimpossiblein principle),notleastbecauseoftheproblemsofdefiningviabilityinmicrobialcells.Despitethedifficulties mentionedabove,theviewtowhichwesubscribe(Kell etal.,1998)isthatonlyculturabilitycanprovidea goldstandardforpositiveviability.Althoughtheflowcytometricapproachhasmuchtoofferforthe determinationofmicrobialviabilityitmustbeemphasisedthatnosinglestainnorevencocktailislikelytobe auniversalindicatorofviability,especiallyifwerequirethatitsinterpretationreflectsourabilitytoinducethe cellstodivide(seeTable11.7.3).

Acellthatiskilledbyexposuretoenvironmentalextremessuchasheat,pHetcislikelytobeverydifferent fromacellthatiskilledbyexposuretoanantibioticorotherchemical,anddifferentagainfromacellthat diesduetoalackofnutrientsinitsenvironment.Thustheflowcytometricpropertiesofacellandthe distributionofdyeuptakewithinapopulationwilldependonhowthecellsdie,andmoregenerallyontheir entirephysiologicalstateanditshistory.

Whilemostinstrumentsdesignedspecificallyfortheanalysisofmicroorganisms(e.g.theBioradBryteorthe AberInstrumentsMicrocyte)donotpermitcellstobesorted,microbesmaybesortedusingthestandard commercialinstruments.Theexploitationofthesortingcapabilityofflowcytometerspermitsthedesignof experimentsthatcarefullyevaluatetheapplicabilityofsocalledviabilitystains,andwestronglyrecommend authorstoadoptthisapproach.

Weconcludethatalthoughtherearecurrentlynoperfectstains,carefulprotocoldevelopmentcurrently allowsvaluableinformationtobeobtainedregardingspecificproblems.Inthecaseoforganismsthathavenot beenexposedtoexcessivestress(e.g.laboratoryculturesundernormalconditionsandinsomecasesclinical samples)substantialprogressisbeingmadetowardstherapidandroutineflowcytometricassessmentof microbialviabilityorvitality.

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KeyReferences
Amann,R.I.,W.Ludwig,&K.H.Schleifer(1995).Phylogeneticidentificationandinsitudetectionof individualmicrobialcellswithoutcultivation.MicrobiologicalReviews59(1):143169.

Davey,H.M.andD.B.Kell(1996).Flowcytometryandcellsortingofheterogeneousmicrobial populationstheimportanceofsinglecellanalyses.Microbiologicalreviews60(4):641696.

Porter,J.,D.Deere,M.Hardman,C.Edwards&R.Pickup(1997).Gowiththeflowuseofflow cytometryinenvironmentalmicrobiology.FEMSMicrobiologyEcology 24(2):93101.

Shapiro,H.M.(1995).Practicalflowcytometry(3rdedition).NewYork,AlanR.LissInc.

Troussellier,M.,C.Courties&A.Vaquer(1993).Recentapplicationsofflowcytometryinaquatic microbialecology.Biol.Cell 78:111121.

InternetResources
http://pcfcij.dbs.aber.ac.uk/home.htm TheAberystwythflowcytometrysitehasinformationonmicrobialflowcytometryincludingviability determinations. http://www.cyto.purdue.edu/flowcyt/research/micrflow/index.htm Microbialflowcytometrysectionwithcontributionsfromseveralauthorsonviabilitymeasurements. http://www.aberinstruments.co.uk/microcyt.htm Aflowcytometerdesignedfortheanalysisofmicrobialsamplesemployinga635nmlaserdiodeas theexcitationsource. http://www.probes.com/handbook/ch162.html Sourceforvariousviabilitystainsincludingviabilitykits.

FigureLegends
Figure11.7.1:Distributionofthefluorescenceof M.luteusfollowingstainingwith0.3 mMRh123(Sigma). FlowcytometrywasperformedusingaSkatronArgus100instrumentwhichwassetupasdescribedinthe manufacturersmanual.ThePMTvoltageforthefluorescencechannelwas700V,andthefullscaleofthe abscissarepresents3.5decadesinfluorescenceintensity.a)Thecellsweregrowninlactateminimalmedium untillatelogarithmicphase,harvested,washedandresuspendedinlactateminimalmediumlackinglactate (Kaprelyantsetal.,1996)priorto20folddilutionandstaining.b)Thecellswerepreparedasin a)but 15 mMCCCPwasalsoaddedpriortoanalysis.c)Cellsweregrowninlactateminimalmediumandthen starvedfor5monthsbeforedilutionandstaining.

Figure11.7.2:Distributionofthefluorescenceofcellsof M.luteuscellsthathavebeenstarvedfor5 months,stainedwithpropidiumiodideandassessedbyflowcytometry.Octanolwasaddedtothecellsas indicatedtogiveafinalconcentrationof0.5%.

Figure11.7.3:Flowcytometricfluorescencebehaviourofasampleof M.luteusthathadbeenfrozenand thawed.Cellswerediluted10foldin50mMphosphatebufferandincubatedwith4mMCTCforthetimes indicated,eitherwithorwithout1mMNADH.Theordinategivesthedifference,betweenthesampleswith orwithoutaddedNADH,inthepercentageofcellswhosefluorescencewasinachannelnumbergreaterthan 20.

Figure11.7.4:Flowcytometricanalysisof M.luteus.CellsweregrownovernightinNutrientBrothEina
o shakingwaterbathat30 C.Sampleswereremovedfromthecultureandstainedwith0.1 mMTOPRO3

(MolecularProbes).ThesampleswerethenanalysedonaMicrocyteflowcytometer.Undertheseconditions unstainedsamplesdidnotgiveanydetectablefluorescence.Thethicklinerepresentscellsfreshlyharvested fromtheculture.Thethinlinerepresentscellsthathadbeenfreezethawtreatedpriortostaining.Thesample

o o wasfrozen(20 C)for30minutes,rapidlydefrostedbyplungingthetubeintoa50 Cwaterbath,thenre

frozenandthenrethawedinthesamemanner.Thedottedlinerepresentscellsthathadbeenpermeabilisedby fixingin70%ethanol.Thefixativewasremovedbycentrifugationpriortostaining.Inallcasesthetotalcell count(basedonforwardlightscatterdatanotshown)hasbeennormalisedto1millioncells.

Figure11.7.5:a)FlowcytometriccellsortingusingtheAutoclonemoduleoftheCoulterElitecanbeused toplaceindividualcellsontoapproximately6065discreetlocationsonastandard90mmagarplate.b)An agarplatewith M.luteuscoloniesfollowingsorting.

Table11.7.1:Fluorescentdyeswhoseusehasbeenconsideredfortheflowcytometricmeasurementof viability. Stain BacLightKit(Molecular Probes) ModeofAction ExclusionofPIand stainingwith SYTO9 ExcitationSource Argon(488nm) Reference(s) Joux etal.,1997 LangsrudandSundheim, 1996Swartsetal.,1998 Virtaetal.,1998 BeckandHuber,1997 Deereetal.,1995Jepras etal.,1995Lpez Amorsetal.,1997 Mason etal.,1994Mason etal.,1997Sulleretal., 1997 Berglundetal.,1987 Mason etal.,1995a PresentedbyBergersen et al.attheNORDFOOD conference,Turku(bo) Finland,1995.Seealso DaveyandKell,1996 ClarkeandPinder,1998 Deereetal.,1998Diaper andEdwards,1994b Joux etal.,1997 KaprelyantsandKell, 1993b Aeschbacheretal.,1986) (Aeschbacheretal.,1986 Berglundetal.,1987 DiaperandEdwards, 1994bNorden etal.,1995 Plovinsetal.,1994 Millardetal.,1997 Prudencioetal.,1998 Wenisch etal.,1997 Augeretal.,1993 Berglundetal.,1987 Deereetal.,1998Gantet al.,1993Nivenand Mulholland,1998) (Augeretal.,1993Comas andVivesRego,1998 Davey etal.,1993Diaper etal.,1992Kaprelyants andKell,1992Porroet al.,1994 LangsrudandSundheim, 1996Roth etal.,1997 DaveyandKell,1997

bis(1,3dibutylbarbituric acid)trimethineoxonol (DiBAC4(3))

Uptakebydeadcells Argon(488nm)

CalcofluorWhite Carboxynaphtho fluoresceindiacetate

Uptakebydeadcells HeliumCadmium (325nm) Enzymicactivity HeliumNeon(633nm)/ Laserdiode(635nm)

ChemchromeB/Y

Proprietary information Respiratoryactivity

Argon(488nm)

5cyano2,3 ditolyltetrazoliumchloride (CTC) Ethidiumbromide Fluoresceindiacetate

Argon(488nm)

Exclusion Enzymicactivity

Argon(488nm) Argon(488nm)

FluoresceindibD galactopyranoside FUN1kit(Molecular probes) PropidiumIodide

Enzymicactivity Metabolicactivity

Argon(488nm)

Excluion

Argon(488nm)or HeliumNeon(544nm)

Rhodamine123

Uptakebylivecells

Argon(488nm)

SytoxGreen TOPRO3

Exclusion Exclusion

Argon(488nm) HeliumNeon(633nm)/ Laserdiode(635nm)

Table11.7.2:ResuscitationofdormantM.luteuscellsinliquidmedium. Culture Timeofstarvation

1 Totalcount(cells.ml )

Viablecountbycfu
1 (cells.ml )

ViablecountbyMPN
1 (cells.ml )* 9 3.510 9 9.210 9 9.210 9 5.410

1 2 3 4

2months 4.5months 6months 9months

9 5.310 10 10 10 1.210 9 6.210

6 510 6 1.310 4 3.610 5 5.210

*estimationwasperformedinthepresenceofRPFfactor.Forthedetailsoftheseexperimentssee Mukamolovaetal.,(1998a).

Table11.7.3:Summaryofadvantagesanddisadvantagesofviabilitydeterminationmethods Advantages Providessufficientproofthatcellwas alive Generallystraightforwardtointerpret Disadvantages Slow Requiresknowledgeofgrowth requirements Underestimatesviablecellnumbers Cytologicalassay Rapid Canbeusedwithoutknowledgeof growthrequirements Totalcountcanbedetermined simultaneously Canbedifficulttointerpret Viabilityisnotmeasureddirectly Falsepositivesandfalsenegativesmay occur

Multiplicationassay

Daveyetal1998Figure11.7.1a

60 50 Counts 40 30 20 10 0 0 50 100 150 200 Fluorescencechannel

Daveyetal1998Figure11.7.1b

250

60 50

Counts

40 30 20 10 0 0 50 100 150 200 250

Fluorescencechannel

Daveyetal1998Figure11.7.1c

60 50

Counts

40 30 20 10 0 0 50 100 150 200 250

Fluorescencechannel
Daveyetal1998Figure11.7.2

Daveyetal1998Figure11.7.3

PercentagedifferenceNADH

35 30 25 20 15 10 5 0 0 20 40 Time(min)
Daveyetal1998Figure11.7.4

60

10000 9000 8000 NormalisedCounts 7000 6000 5000 4000 3000 2000 1000 0 0 50 100 150 200 250 FluorescenceChannelNumber

Daveyetal1998Figure11.7.5a

Flow Cell

Deflection Plates Petri Dish

Autoclonearm

Topviewofpetridish showingsortgrid
Daveyetal1998Figure11.7.5b