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CulturingandMaintainingClostridiumdifficileinanAnaerobicEnvironment

JVisExp.2013(79):50787.
Publishedonline2013Sep14.doi:10.3791/50787

PMCID:PMC3871928
NIHMSID:NIHMS581253

CulturingandMaintainingClostridiumdifficileinanAnaerobicEnvironment
AdrianneN.Edwards, 1JoseM.Surez, 1andShonnaM.McBride1
1
DepartmentofMicrobiologyandImmunology,EmoryUniversitySchoolofMedicine
Correspondenceto:ShonnaM.McBrideatshonna.mcbride@emory.edu
Copyright2013,JournalofVisualizedExperiments

ThisarticlehasbeencitedbyotherarticlesinPMC.

Abstract

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ClostridiumdifficileisaGrampositive,anaerobic,sporogenicbacteriumthatisprimarilyresponsibleforantibioticassociateddiarrhea(AAD)andisa
significantnosocomialpathogen.C.difficileisnotoriouslydifficulttoisolateandcultivateandisextremelysensitivetoevenlowlevelsofoxygeninthe
environment.Here,methodsforisolatingC.difficilefromfecalsamplesandsubsequentlyculturingC.difficileforpreparationofglycerolstocksforlong
termstoragearepresented.Techniquesforpreparingandenumeratingsporestocksinthelaboratoryforavarietyofdownstreamapplicationsincluding
microscopyandanimalstudiesarealsodescribed.Thesetechniquesnecessitateananaerobicchamber,whichmaintainsaconsistentanaerobic
environmenttoensureproperconditionsforoptimalC.difficilegrowth.Weprovideprotocolsfortransferringmaterialsinandoutofthechamberwithout
causingsignificantoxygencontaminationalongwithsuggestionsforregularmaintenancerequiredtosustaintheappropriateanaerobicenvironmentfor
efficientandconsistentC.difficilecultivation.
Keywords:Immunology,Issue79,Genetics,Bacteria,Anaerobic,GramPositiveEndosporeFormingRods,Spores,Bacterial,GramPositiveBacterial
Infections,ClostridiumInfections,Bacteriology,Clostridiumdifficile,Grampositive,anaerobicchamber,spore,culturing,maintenance,cellculture

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Introduction

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ClostridiumdifficileisaGrampositive,sporeformingbacteriumthatisanobligateanaerobeandapotentiallyfatalgastrointestinalpathogenofhumans
andanimals.Initiallydescribedin1935asacommensalorganismfoundinfecalsamplesfromnewborns1,C.difficilewaslaterdemonstratedtobethe
causativeagentofpseudomembranouscolitisassociatedwithantibiotictreatment2.C.difficileinfections(CDI)aretypicallyprecededbyantibiotic
treatmentwhichresultsinthedisruptionofthenormalcolonicflora,creatinganicheforC.difficiletothrive2.C.difficileistransmittedasadormant
sporeviathefecaloralrouteandsubsequentlygerminateswithinthegastrointestinaltract,producingvegetativecellscapableofgeneratingseveraltoxins
andcausingseverediseaseandcolitis3.CDIareoftenrefractorytoconventionaltreatmentsandtheseinfectionsarefrequentlyrecurrent4.Asaresult,
CDIareresponsibleforupto$4.8billioninhealthcarecostsintheUnitedStates57.
C.difficileisverysensitivetoevenlowlevelsofoxygenintheenvironment.ForC.difficiletopersistintheenvironmentandbeefficientlytransmitted
fromhosttohost,theformationofametabolicallyinactivesporeiscritical8.BecausethelaboratorymaintenanceandmanipulationofC.difficilerequires
acontrolled,anaerobicenvironment,thesetechniquesnecessitatetheuseofananaerobicchamber.Useofanaerobicchambershasresultedinincreased
recoveryandisolationofobligateanaerobes911,andhasallowedanumberofmoleculartechniquestobeperformedinananaerobicatmosphere.
InadditiontoC.difficile,theanaerobicchamberuseandmaintenancedescribedhereareapplicabletootherobligateanaerobessuchasotherClostridial
species(e.g.C.perfringens),othergastrointestinalspecies(e.g.Bacteroidesspecies12)andperiodontalpathogens(e.g.Peptostreptococcusspecies13).

Protocol

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Note:C.difficileisahumanandanimalpathogenthatcancausegastrointestinaldisease.ExperimentsinvolvingC.difficilemustbeperformedwith
appropriatebiosafetyprecautions(BSL2).
1.AnaerobicChamberUseandMaintenance
C.difficileisastrictanaerobeandisextremelysensitivetoevenlowconcentrationsofoxygenintheatmosphere.Therefore,acontrolled,anaerobic
environmentisneededforitssuccessfulmanipulation.Theuseofananaerobicchamber(Figure1A)providesthemoststableenvironmentandtheideal
conditionsforeffectivecultivationofC.difficileandotheranaerobicbacteria14.Here,anatmospherecontainingagasmixture(5%CO2,10%H2,85%
N2)canbestablymaintained.
Tointroduceanyitemsintothechamberwithoutsignificantoxygencontamination,anairlockmustbeused(Figure1B).Thisdevicefunctionsasagas
interchangeandworksautomatically,semimanuallyormanually.Theairlockhastwodoors:oneprovidingaccesstotheoutsideoftheairlockandthe
otherprovidingaccesstotheinterioroftheanaerobicchamber.Dependingonthemodel,theairlockmayhaveanadditionaldoor,whichcanofferaccess
toanadjoininganaerobicchamber,thussavingthecostofaseparateairlock.Unlessactivelymovingitemsinoroutofthechamber,bothdoorsshould
remainclosedatalltimestoavoidoxygencontamination.Theairlockpossessesanon/offswitchonthefrontandapanelcontainingfourbuttons.The
gaslinesandvacuumpumphoseareconnectedintherearoftheairlock,nearthepanelwheretheswitchesforfullmanualoperationoftheunitare
located.Itisrecommendedthattwopurgecycles,usingnitrogengas(N2),areusedbeforefillingtheinterchangewithgasmix(5%CO2,10%
H2,85%N2)toreducetheamountofoxygenintroducedintothechamber.Itisalsoimportanttoneverleaveeitherdooropenforlongerthanthe
amounttimerequiredtomoveitemsinandoutoftheinterchangeandtocarefullyplanexperimentstoreducethefrequencyofmovingitemsinandout
ofthechamber.Thedifferentoperatingproceduresforvinylanaerobicchambersareoutlinedbelow.Beforefollowingtheseprotocols,ensurethatthese
arecompatiblewiththemanufacturersinstructionsprovidedwiththechamber.
1.AutomaticModeThisisaprogrammableandcustomizablemodeandisperformedentirelybytheairlock.Toprogramtheairlock,refertothe
manufacturersinstructions.Arecommendedprogramfortypicalentryintothechamberinvolvestwopurgecyclesusingnitrogengasfollowedby
refillingtheairlockwithagasmixthatcloselymatchestheatmospheremaintainedwithinthechamber.Duringeachvacuumcycle,thestandard
factoryproceduressuggestpullingavacuumto20inHgandpurgingto1inHg.
1.Ensurethattheinnerairlockdoorisfullyclosed.
2.Opentheouterairlockdoor.
3.Placeitemsintoairlockandclosetheouterairlockdoor.Ifintroducingliquids,unscrewthecapshalfwaytoensureefficientgasexchange.
Sealedpackagesandcontainersshouldbeopenedpriortobeginningthecyclerefrainingfromdoingsomaywarpanddamageplasticware
andcontainers.Tomaintainsterility,openthepackagesenoughtoonlyallowefficientgasexchange.
4.PresstheStartbutton.
5.Waituntiltheairlockhascycledthroughtheprogramandthedisplayreads"Anaerobic."
6.Openinnerairlockdoor.
7.Moveitemsintothechamber.
8.Closeinnerdoor.
2.SemimanualModeThismodemaybeusedwhenmovingitemsinoroutofthechamberandisnecessarywhencyclingthegaswithinthe
chamberisrequired.
1.Ensurethattheinnerairlockdoorisfullyclosed.
2.Opentheouterairlockdoor.
3.Placeitemsintoairlockandclosetheouterairlockdoor.Ifintroducingliquids,unscrewthecapshalfwaytoensureefficientgasexchange.
Sealedpackages,suchasinoculatingloopsand96wellplates,mustbeopenedpriortobeginningthecycle.
4.PressMenu.
5.PressStart.Theairlockwilldisplaythefunctionofeachbuttonandwillnotresponduntilthisiscompleted:
Uparrow:activatesthevacuumpump.
Downarrow:initiatesthenitrogen(purge)gasflow.
Start:initiatesthegasmixflow.
Menu:returntothedefaultdisplay.
6.Press"Up,"removinggasfromtheairlock,untilthedisplayreads20inHg.
7.Press"Down,"fillingtheairlockwithnitrogen,untilthedisplayreadslessthan1inHg.Donotovershoot,asthiswillcreatepositive
pressurewithintheairlock,whichmayaffectitsintegrity.
8.Repeatsteps6and7toperformanadditionalpurgecycle.
9.Press"Up"untilthedisplayreads20inHg.
10.Press"Start,"fillingtheinterchangewithgasmix,untilthedisplayreadslessthan1inHg.
11.Openinnerairlockdoor.
12.Moveitemsintothechamber.
13.Closeinnerdoor.
3.ManualModeThismodemaybeusedwhenevertheautomaticorsemimanualmodesarenotworkingorthereisnopowertotheairlock.This
modedoesnotfunctionwhentheairlockisontherefore,itisdifficulttodeterminethepressurewithintheairlock.Becausethevacuumpulland
gaspurgetimesisdependentuponvacuumandgasflowrates,respectively,theuseofapressuregaugewithintheinterchangeisrequiredto
monitorthepressureandreducetheriskofpersonalinjuryanddamagetotheairlock.
1.Ensurethattheinnerairlockdoorisfullyclosed.
2.Opentheouterairlockdoor.
3.Placeitems,includingthepressuregauge,intoairlockandclosetheouterairlockdoor.Ifintroducingliquids,unscrewthecapshalfwayto
ensureefficientgasexchange.Sealedpackages,suchasinoculatingloopsand96wellplates,mustbeopenedpriortobeginningthecycle.
4.Locatethethreeswitchesonthebackoftheairlocklabeled"ManualControlSwitches."Theseareindividuallylabeledas:
ToVacuum
Nitrogen
GasMix
5.FliptheToVacuumtoggleswitchuntilthepressuregaugereadsapproximately20inHg.
6.FliptheNitrogentoggleswitchuntilthepressuregaugereadsapproximately1inHg.
7.FliptheToVacuumtoggleswitchuntilthepressuregaugereadsapproximately20inHg.
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8.FliptheNitrogentoggleswitchuntilthepressuregaugereadsapproximately1inHg.
9.FliptheToVacuumtoggleswitchuntilthepressuregaugereadsapproximately20inHg.
10.FliptheGasMixtoggleswitchuntilthepressuregaugereadsapproximately1inHg.
11.Openinnerairlockdoor.
12.Moveitemsintothechamber.
13.Closeinnerdoor.
2.Culturing,EnumeratingandStoringC.difficilefromStoolSamples
ThisprocedureisdesignedtorecoverC.difficilefromfecalsamplescontainingsporesandsubsequentlymaintainisolatedcoloniesasvegetativecellsor
sporesinlongtermstorageasglycerolstocks.Alternatively,thisproceduremaybeusedtoenumeratethenumberofC.difficilepresentinstoolsamples
(e.g.fromanimalstudies).ToselectivelyanddifferentiallyenrichforC.difficile,taurocholatecefoxitincycloserinefructoseagar(TCCFA)isusedto
inhibitgrowthofnormalfecalflora1516.CycloserineisbacteriostaticforGramnegativebacteria,whilecefoxitinmorebroadlyinhibitsgrowthofboth
Gramnegativeandpositivebacteria,withtheexceptionofC.difficileandmostentercoccistrains.ApHindicator,neutralred,canbeincludedinthe
medium,asthefermentationoffructosewillresultinadecreaseinpHandasubsequentcolorchangefromred/orangetoyellow.Toefficientlyrecover
sporesofC.difficileasvegetativebacteria,thebilesaltsodiumtaurocholateisusedtoinducegermination1718.BecauseC.difficileformsspores,alcohol
orheattreatmentofsamplesmaybeusedtoreduceoreliminatevegetativecells,limitingthegrowthofcontaminatingflora,whichmayincreasethe
efficiencyofC.difficilerecovery19.Asmentionedabove,itiscriticaltoprereduceallplatesforatleast12hrintheanaerobicchamberpriortouseto
ensuretheremovalofresidualoxygen.Airdryingtheplatespriortouseinthechambercanreducecondensation.Liquidmediummayneedupto24hr
toreducedependingonthevolumeandthesurfacetoairratioofthecontainerused.
Beforebeginning,thefollowingitemsshouldbeplacedintotheanaerobicchamber:
Stoolsample
Sterileswabs
Sterileinoculatingloops
1xPBS(seeMaterials)
TCCFAplates(seeMaterials)
BHIS(BrainHeartInfusionmediumwithyeastextract)platesandbroth(seeMaterials)
50%glycerol(sterilized)
10%Lcysteine(sterilized)
Cryogenicstoragevials
*Alternatively,isolatedcoloniescanbespreadontoBHISagarplates,andsubsequentlyscrapedoffandresuspendedinBHISliquidmediumwith15%
glycerolforlongtermstorageat80C.**ItisimportanttonotethatGramstainingisnotaneffectivestrategytoidentifyC.difficiledirectlyfromstool
samples23C.difficilemustfirstbeisolatedfromtheotherflorapresentinthestool.
1.Resuspendthestoolsamplein1xPBS(thismaybeperformedinaerobicconditions),andensurethatthestoolsampleisfullyresuspendedin1x
PBSbyvortexing.IfenumeratingC.difficilefromthestool,weighthestoolsamplepriortotheadditionof1xPBS.
2.Makeserialdilutionsoftheresuspendedstoolsamplein1xPBSforappropriateisolationorenumerationofcolonyformingunits(CFU)pergram
ofstool.
3.Usingaseptictechnique,apply100lofeachserialdilutiontoTCCFAplates.
4.Usingaseriesofsterileinoculatingloops,streaktheappliedcultureforisolatedcoloniesorevenlyspreadtheappliedcultureonthesurfaceofthe
TCCFAplateforenumerationofcolonies.
5.Incubatetheplateanaerobicallyfor48hrat37C.ItispossibletodetectandidentifyC.difficilecolonieswithin24hrbasedontheflat,irregular,
groundglassappearanceofthecolonies15,althoughmoreaccurateidentificationandenumerationisachievedafter48hr.
6.Usingsterileinoculatingloops,subcultureanycoloniesthatappeartobeC.difficileontoprereducedBHISagarplates,supplementedwith0.03%
Lcysteine20.Pickanindividualcolony,andstreakontotheplateinquadrants,usinganewsterileinoculatingloopforeachquadrant,toobtain
isolatedcolonies.Alternatively,counttheC.difficilecoloniesofeachdilution(thismaybeperformedinaerobicconditions),andcalculatethe
numberofcolonyformingunitspergramofstoolsample.
7.ConfirmC.difficileidentificationviaGramstaining.AfterGramstaining,C.difficilewillappearaspurplerodsandsomecellsmaycontain
terminalendospores.Polymerasechainreaction(PCR)identificationofthegenesthatencodetoxinBmayprovidefurtherconfirmationof
toxigenicstrainsofC.difficile21whilemultilocussequencetyping(MLST)issuccessfulforidentificationandaccuratetypingofunknownand
nontoxigenicstrainsofC.difficile22.
8.TomaintainastockofC.difficileat80C,pickanindividual,isolatedcolonyfromtheBHISagarplateusingasterileinoculatingloopand
resuspendthecolonyin10mlofprereducedBHISliquidmediumsupplementedwith0.03%Lcysteine.*
9.Incubateovernightanaerobicallyat37C,oruntiltheculturebecomesturbid.
10.Add333lof50%glyceroland666loftheC.difficileculturetoa1.8mlcryogenictubetocreatea15%glycerolstockoftheisolatedstrain.
11.Tightlycapthecryogenictube,mixwellandimmediatelyremovethestockfromtheanaerobicchamberandplaceina80Cfreezerforlong
termstorage.
3.CulturingC.difficilefromFrozenGlycerolStocks
ThisprocedureprovidesforrecoveryofC.difficilefromglycerolstocksstoredat80C.Becauserepeatedfreezethawcyclesmaykillvegetativecells,
itisimportanttokeeptheglycerolstocksfrozenatalltimes.Wedonotrecommendtheuseofdryicefortransferringstrainsinandoutofthechamberas
theevaporationofdryicecanchangetheenvironmentwithinthechamber.Instead,werecommendusingfrozencoolingrackstokeepglycerolstocks
frozenduringtransport.Forvariousselectiveanddifferentialpurposes,threemediaarecommonlyusedforculturingC.difficile.TCCFA,asdiscussed
above,isselectiveforC.difficileandcontainssodiumtaurocholate,agerminant.Brainheartinfusionsupplementedwithyeastextract(BHIS)isa
commonlyused,enriched,nonselectivemediumwhichallowsforthegrowthofawidevarietyoforganisms(Figure2A)24.Frequently,Lcysteineis
addedtoBHISasareducingagent20.Finally,theadditionofbloodtothemedium(Figure2B)allowsformoreefficientsporulationthanonTCCFA
andprovidesfordetectionoftheuniquegreenishorchartreusefluorescenceexhibitedbyC.difficileunderlongwaveultraviolet(UV)light15(Figure
2C).WhenculturingC.difficilefromsporestocks,itiscriticaltorememberthatsodiumtaurocholatemustbeaddedtothemediumtoensure
germination.
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Beforebeginning,thefollowingitemsshouldbeplacedintotheanaerobicchamber:
Frozenglycerolstock(incoolingrack)
Sterileinoculatingloops
TCCFAorBHISplates(seeMaterials)
BHISbroth(seeMaterials)
50%glycerol(sterilized)
Cryogenicstoragevials
1.PlacethefrozenglycerolstockofC.difficileinacoolingrackthathasbeenstoredat80Cpriortousetopreventthawing.
2.Bringthefrozenglycerolstockondryiceintotheanaerobicchamber.
3.Usingaseptictechniqueandasterileinoculatingloop,placeasmallamountofthestockontotheplateandstreakacrossonequadrantoftheplate.
4.Rotatetheplate90and,usinganewsterileinoculatingloop,continuetostreakacrossthesecondquadrant.
5.Repeatforthethirdandfourthquadrantstoensuretheisolationofindividualcolonies.
6.Immediatelyremovethefrozenglycerolstockfromtheanaerobicchamberandreturntothe80C.
7.Incubatetheplateanaerobicallyovernightat37C.Individualisolatedcoloniesshouldbeobservedafterovernightgrowth.
4.PurifyingSporesfromC.difficile
Assporulationisrequiredforsurvivalinoxygenrichenvironmentsandforefficienttransmissionofdisease8,thepreparationofsporestocksisoften
necessaryfordownstreamapplications,notlimitedtomicroscopyandanimalsstudies.Itisimportanttonotethatenumeratingsporesrequiresrepetition
toensurereproducibilityofcounts.Pipettingupanddownseveraltimesbetweendilutionsalsoreduceslosssincesporesadheretoplasticwell.
SporulationofC.difficileisnotasrapidorhomogeneousasothersporogenicspecies.Tooptimizesporeproductionandrecovery,eithersporulation
medium(SMC)17,25or70:30medium26isrecommended.OthermediacommonlyusedareBHIS,whichrequires45daysofgrowthbeforeefficient
sporulationisseen27,andClospore,aliquidmediumthatproduceshightitersofspores(107108sporespermilliliter)after72hrofgrowth.Other
protocolsuseicecoldwaterratherthan1xPBS20however,theuseofanisotonicsolutioncanreducesporesfromstickingtoeachotherandplastic
surfaces.Alternatively,someinvestigatorsfurtherpurifytheirsporesusingasucrosegradienttofullyremovevegetativecellsanddebris29.
Beforebeginning,thefollowingitemsshouldbeplacedintotheanaerobicchamber:
Sterileinoculatingloops
BHIS,SMCand/or70:30plates(seeMaterials)
BHISbroth(seeMaterials)
1xPBS,filtersterilized(seeMaterials)
1.CulturestrainsfromfrozenglycerolstockontoprereducedBHISplatesandincubateanaerobicallyovernightat37C.
2.RestreakontoseveralprereducedSMCor70:30platesandincubateanaerobicallyat37Cfor2448hours.Sporeformationcanbefollowedvia
phasecontrastmicroscopy.Sporeswillappearphasebrightwhilevegetativeandmothercellswillappearphasedark.*Asanalternative,spores
canbepurifiedfrom70:30liquidmediumafter2448hours,whichyields105106sporespermilliliter,dependingonthestrainused.
3.Usingasterileinoculatingloop,scrapeplatesandresuspendthecellsin10mlsterile1xPBS.
4.Discardplatesandremovesporesuspensionfromtheanaerobicchamber.Pelletthecellsat3,000xgfor15minutes.Washthecellstwicein1x
PBS,fullyresuspendingthecellpelleteachtime.
5.Incubateovernightat4Ctoaidinlysisofvegetativeandmothercells.
6.Incubateat70Cfor20mintokillanyresidualvegetativecells.
7.Todeterminecolonyformingunits(CFU)permilliliter,seriallydiluteeachsporepreparationin1xPBSandplateonBHIS+0.1%sodium
taurocholate.Incubateplatesforaminimumof24hrbeforeenumeratingcolonies.
8.Sporescanbestoredin1xPBSateitherroomtemperatureor4Cforlongtermstorage.Ifstoredat4C,itmaybeusefultoreheatthespore
preparationat55Cfor15mintorestoreefficientgermination.

RepresentativeResults

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AnexampleofC.difficilegrownonBHISandColumbiaanaerobicsheepbloodagarmediacanbeseeninFigure2.C.difficileformsirregularcolonies
thatareflatandpossessagroundglassappearancewhichisevidentonbothmedia.Here,anerythromycinsensitiveclinicalisolateofC.difficile,
630E30,isgrownonBHISagar,anenriched,nonselectivemedium,for24hrat37C(Figure2A).ColoniesonColumbiaanaerobicsheepbloodagar
appearsimilartothosegrownonBHISunderwhitelight(Figure2B)however,theuseofthismediumalsoprovidesfordetectionofthegreenishor
chartreusefluorescenceexhibitedbyC.difficileunderlongwaveultraviolet(UV)light15(Figure2C).C.difficilecoloniesonTCCFAagarlooksimilar
togrowthonBHISagar.BecauseofthepresenceoftwoantibioticsinTCCFAmedium,atimeperiodof48hrgrowthisnecessarybeforeenumerating
colonies.

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Figure1.TheCoyLaboratoriesTypeCvinylchamberanditscomponents.(A)ACoyLaboratoriesTypeCvinylchamberwhichprovides
workspaceforasingleindividualatonetime(42in.x32in.).Itcontainsacatalystfanbox(backleftcorner)whichcirculatesandheatstheair,andholds
theStakPakcontainingthepalladiumcatalystrequiredtoreduceanyoxygencontamination.(B)Theairlockservesasaninterchangeandprovidesa
mechanismforthetransferofmaterialsinandoutofthechamberwhilepreventingsignificantoxygencontaminationwithintheanaerobicenvironment.
Theairlockhastwodoors:oneprovidingaccesstotheoutsideoftheairlockandtheotherprovidingaccesstotheinterioroftheanaerobicchamber.The
airlockisprogrammableandallowsforcustomizedcyclesforentryintothechamber.Itisoperableinautomatic,semimanualandmanualmodes.(C)
Attached,flexiblelatexglovesareprovidedwhichallowfullrangeofmotionandreachwithinthechamber.Theglovesaresecuredtoaspecializedcuff
attachedtothevinylsleeveswithvinyladhesive,permittingreplacementofgloveswithoutdisruptingtheanaerobicatmosphereofthechamber.
Neopreneglovesarealsoavailable.(D)TheModel10GasAnalyzercontinuouslymonitorsbothoxygenandhydrogenlevelsprovidingan
instantaneousreadoutoftheatmospherewithinthechamber.Thisunitallowsforimmediatealertsifaleakoccurs,anincorrectgasmixisusedor
additionalproblemsariseviaaudiblealarmsandaflashingLEDlight.

Figure2.TheappearanceofC.difficilecoloniesonvariousmedia.Thecharacteristicflat,irregular,groundglassappearanceisevidentwithan
erythromycinsensitiveclinicalisolateofC.difficile,630E30,grownonBHISagarfor24hr(A)andColumbiaanaerobicsheepbloodagarfor48hr
underwhitelight(B)andlongwaveultravioletlight(C).

Discussion

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ThemethodsdescribedhereallowforsimpleandquickrecoveryofC.difficilefromavarietyoffecalsamples,includinghumans,miceandhamsters,as
wellasthelongtermstorageofC.difficileasglycerolorsporestocks.C.difficilecanbeadifficultorganismtocultivate,butcarefulmaintenanceofan
anaerobicenvironmentandtheapplicationofaseptictechniquescanprovideforrobustgrowthandareductionincontamination.
Anaerobicchambers:ConsiderationsandMaintenance
Therearetwotypesofanaerobicchambers:rigidchambersorvinylchambers.Rigidchambersaretypicallymadeofaluminumorarigidpolymer(e.g.
Plexiglasoracrylicmaterials),allowingtheuseofcausticchemicals.Rigidchamberscanbeconvertedtoaglovelessstyleandarelessproneto
punctureshowever,leaksaredifficulttodetectandfindandrigidchambersrequiresomemethodtocompensateforgasdisplacementduringuse.The
polymerunitsoftenareequippedwithapurgeonlyairlock,whichmaycostmoretooperate.Formaintenanceofstringentatmosphericconditions,cost,
flexibility,laboratoryspacerequirementsandeasymaintenance,vinylanaerobicchambersarerecommended(CoyLaboratoryProducts,Figure1A).
Thisanaerobicchamberiscomprisedofavinylgloveboxthatissupportedbyatubularaluminumstructureandisconnectedtoanairlockwhich
functionsasaninterchangeforthetransferofmaterialsinandoutofthechamber(Figure1B),usinglatexorneopreneglovesattachedtovinylsleeves
(Figure1C).Itisimportanttopreciselyfollowmanufacturer'sinstructionsforpropersetupoftheanaerobicchamber.Itmaybetemperaturecontrolled
withone(ormore,dependingonthesizeofthechamber)heaterboxunitswhichprovideaircirculationthroughapalladiumcatalyst.Thepalladium
catalystservestoreduceoxygencontaminationintroducedthroughtheinterchangebyconvertinghydrogenandoxygenintowater.Ideally,bothoxygen
andhydrogenlevelsaremonitoredusingagasanalyzer(Figure1D),displayingoxygenconcentrationinpartspermillion(ppm)andhydrogen
concentrationasapercentage.
Liquidandsolidmediashouldbeprereducedintheanaerobicchamberbeforeuse.Petridishes(100mmindiameter)canbereducedforonlytwohours
priortouse31however,liquidmediashouldbereducedovernight.Importantly,mediashouldbecooledpriortotransferintothechambertoprevent
liquidflashingduringairlockevacuation.Plasticconsumables,suchas96wellplates,shouldbeprereducedovernightbeforeuse,asplasticisporous
andcanstoreoxygenmolecules32.Dependingontheapplication,someplasticsmustbeprereducedforupto48hrbeforeuse33.Biohazardwaste
shouldbedisposedofappropriately.Asmallbiohazardbagmaybekeptinsidethechamber,butshouldbereplacedfrequently.
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Ahydrogenconcentrationofatleast3%mustbemaintainedinsidethechamber,asthiswillensurebothgoodgrowthofC.difficileandefficientoxygen
removal.Ifthehydrogenconcentrationdropsbelow3%orifthechamberhasnotbeenusedforseveraldays,achambercycleshouldbeexecutedto
restorethehydrogenconcentrationtodesirablelevels.Here,approximatelyathirdofthegasinthechamberisvacuumedout(thevinylboxwilldeflate
significantly)andreplacedwithfreshgasmix.Ensurethattheairlockisanaerobicbeforebeginning.Pumpingtoomuchgasintothechambershouldbe
avoided,asthisnotonlyplacesunnecessarystressonthevinylbag,butwillalsoprecludetheuserfromreachingintothebackofthechamber.Always
performthisprocedureinawellventilatedarea.Takeadditionalprecautionsifthechamberiskeptinasmall,poorlyventilatedroom.Openanydoorsto
theroomasthegascontentsinsidethechambercanasphyxiatetheuser.
Additionalmaintenanceincludesregenerationofthepalladiumcatalysttoensureefficientoxygenremovalwithinthechamber.Asoxygenandhydrogen
arereducedbythecatalyst,watercanaccumulateonthesurfaceofthepalladiumcoveredaluminumpellets,reducingtheirefficiencyovertime.To
overcomethis,thepalladiumcatalystsshouldberegeneratedatleastonceaweekbybakingatleast230Cforonehour.Additionally,asaresultofboth
oxygenreductionandevaporationofwaterfromplatesandmedia,wateraccumulationisacommonissuewithinanaerobicchambers.Toensure
comfortablemanipulationandincreasethehalflifeoftheequipmentwithinthechamber,desiccantpacksmaybeused,butshouldbedriedoutfrequently
followingthesameprocedureusedforthepalladiumcatalysts.Alternatively,adehumidifiermaybeusedwhichcirculatesairthroughametalblockthat
condensateswaterandiscollectedinacontainer.Thedevicepreventsoverflowofthewatercontainershowever,dehumidifiersmustbemonitoredand
containersshouldbeemptiedwhenclosetofull.Tocleanthevinylchamber,theuseofasoftclothandanycommerciallyavailablecleaner
recommendedforpolyvinylchloride(PVC)isadvised(e.g.PlasticMagicCleaner,partno.1600480,CoyLaboratories).Toavoidscratchingor
damagingthevinylchamber,donotusepapertowels,KimwipesorproductscontainingketonesorothercompoundsthatwilldamagePVC.
Finally,C.difficileproduceshydrogensulfidegas(H2S),whichisextremelyreactiveandcorrosive.Hydrogensulfidecanbedamagingto
instrumentation,thepalladiumcatalystandanyexposedmetals.Ifpossible,donotleaveanyinstrumentation,suchashomogenizersand
spectrophotometers,inthechamberforlongperiodsoftimestoavoidcorrosioncausedbyhydrogensulfide.Becauseoftheproductionofhydrogen
sulfide,itemssuchasthepalladiumcatalystandgasanalyzerwillneedtobeperiodicallyreplacedaspartofregularchambermaintenance.Alternatives
forreducinghydrogensulfidelevelswithinthechamber,suchasusingactivatedcharcoal,leadacetate,silverchlorideorsilversulfatetoabsorbor
chemicallyremovethehydrogensulfide,havebeendescribed34andmaybeutilizedtoslowdowncorrosionofequipment.
AdditionalPrecautions
Neveropenonedoortotheairlockwithoutensuringthattheotherdoorisclosedandlockedtopreventoxygencontamination.Additionally,avoiduseof
sharpitemswithinthechambertoreducetheriskofpuncturingthevinyl.
Itisimportanttonotethathydrogenisaflammablegasinthepresenceofoxygen.Takecarewhenintroducingitemsintothechamberthathighlevelsof
oxygencontaminationdonotoccur(greaterthan999ppm).Itisimportanttouseonlypremixednonflammableanaerobicgasmixandcloselymonitor
thegasanalyzerwithinthechamber,especiallywhenusinganewgastank.Ifbothhydrogenandoxygenconcentrationsabove4%occur,ensurethatthe
appropriateemergencyprocedures,whichshouldbeoutlinedinthelaboratory'sstandardoperatingprocedures(SOP),arefollowed.
TroubleshootingC.difficilegrowth
IfpoorgrowthofC.difficileculturesisobservedinrichmedium,thisismostoftenduetooxygencontaminationwithinthechamber.Theadditionofa
reducingagent(e.g.Lcysteineorthioglycolate)tothemediummayimprovegrowthhowever,theissueofoxygencontaminationwithinthechamber
wouldneedtobeaddressed.Monitoringoxygenandhydrogenlevelswithinthechamberusingagasanalyzercanquicklyalerttheusertoissuesbefore
adelayinprogressanddecreaseinproductivityoccurs.Ifoxygencontaminationoccurs,quicklyidentifyingthesourcewithagasleakdetector(available
fromCoyLaboratories)isadvised.Toincreasethechanceinfindingthelocationofaleak,aragsoakedinalcoholmaybeplacedwithinthechamber
sincethegasleakdetectorcanidentifyincreasedlevelsofhydrocarbonsaswellasincreasedlevelsofhydrogen.Oncethesourceoftheleakisfound,it
canberepairedusingglueorsiliconefollowingthemanufacturer'sinstructions.
Anumberoforganismsreadilygrowinanaerobicenvironments,includingothercommonClostridialspecies(e.g.Clostridiumperfringens)andthe
facultativeanaerobe,Escherichiacoli.Aseptictechniqueandotherstrategiescanreducetheriskofcontamination.Appropriateorganizationwithinthe
chamber,suchasplacingitemswithinreachtolessenthepotentialforspillsandpromptremovalofaccumulatedbiohazardwaste,canreduce
contaminationrisks.Additionally,papertowelsdampenedwithadilutebleachsolutioncanbeperiodicallybroughtintothechambertowipedown
surfacesandthelatexorneoprenegloves.Itiscriticaltonotleavebleachoralcoholsolutionsinsidethechamberforlongperiodsoftimeasthesecan
permeatetheatmosphereandsolidandliquidmedia,killingC.difficile,aswellasdamagingthevinyl34.Additionally,regularreplacementofthelatexor
neopreneglovescanalsoreducecontaminationrisk.Asmentionedabove,ifunsureifanorganismisC.difficileoracontaminant,atestforthepresence
ofthetdcBgeneusingPCRcanquicklydeterminewhethertheorganismisC.difficile21.Finally,ifcultivatingmultipleorganismswithinthesame
anaerobicchamber,itisimportanttonotethatC.difficilecanproducemetabolicbyproducts(e.g.hydrogensulfide)thatinhibitthegrowthofother
anaerobicorganismsandviceversa.

Disclosures

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Noconflictsofinterestdeclared.

Acknowledgments

Goto:

WewouldliketothankCoyLaboratoriesforkindlyprovidingpicturesoftheanaerobicchamber.ThisworkwassupportedbyNationalInstitutesof
HealthgrantDK087763(S.M.M.)andaSTEP/HHMICurriculumDevelopmentFellowship(A.N.E.).

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