美國(guó)FDA細(xì)菌學(xué)分析手冊(cè)第八版BAM彎曲菌模板

BAM:Campylobacter

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1

January

BacteriologicalAnalyticalManual

Chapter7

Campylobacter

Authors:JanM.Hunt,

CarlosAbeyta

andTonyTran

RevisionHistory:

Updatedandrevised:-DEC-29

MediaInstructionsModifiedon-MAR-08

IsolationofCampylobacterSpeciesfromFoodandWater

CampylobacterisconsideredbymanytobetheleadingcauseofentericillnessintheUnitedStates(20,26).Campylobacterspeciescancausemildtoseverediarrhea,withloose,waterystoolsoftenfollowedbybloodydiarrhea(7,20).C.jejuni,C.coli,andC.lariaccountformorethan99%ofthehumanisolates(C.jejuni90%).Otherspecieshavebeenassociatedwithhumanillnessinrecentyears(7,17,18,23,26,27).

Campylobacterspeciesarehighlyinfective.TheinfectivedoseofC.jejunirangesfrom500to10,000cells,dependingonthestrain,damagetocellsfromenvironmentalstresses,andthesusceptibilityofthehost(4,6,7,20,27).OnlythemesophilicC.fetusisnormallyinvasive.Thermophilicspecies(optimum42°C)suchasC.jejuniareoccasionallyinvasive.Theinfectionsaremanifestedasmeningitis,pneumonia,miscarriage,andasevereformofGuillain-Barrésyndrome(6,20).ThermotolerantstrainsofC.fetusthatgrowat42°Chavebeenisolatedfrompatients(17).

Campylobactersarecarriedintheintestinaltractofawidevarietyofwildanddomesticanimals,especiallybirds.Theycanestablishatemporaryasymptomaticcarrierstate,aswellasillness,inhumans.Thisisespeciallyprevalentindevelopingcountries(20).Consumptionoffoodandwatercontaminatedwithuntreatedanimalorhumanwasteaccountsfor70%ofCampylobacter-relatedillnesseseachyear.Thefoodsincludeunpasteurizedmilk,meats,poultry,shellfish,fruits,andvegetables,(1,8-11,17,19,20,22,25,26).

C.jejunicansurvive2-4weeksundermoist,reduced-oxygenconditionsat4°C,oftenoutlastingtheshelflifeoftheproduct(exceptinrawmilkproducts).Theycanalsosurvive2-5monthsat-20°C,butonlyafewdaysatroomtemperature(5,8-11,20).Environmentalstresses,suchasexposuretoair,drying,lowpH,heating,freezing,andprolongedstorage,damagecellsandhinderrecoverytoagreaterdegreethanformostbacteria.Olderandstressedorganismsgraduallybecomecoccoidalandincreasinglydifficulttoculture(5,20).Oxygenquenchingagentsinmediasuchasheminandcharcoalaswellasamicroaerobicatmosphereandpreenrichmentcansignificantlyimproverecovery(2,14-16,21,25,28).

Campylobactersaremicroaerophilic,verysmall,curved,thin,Gram-negativerods(1.5-5μm),withcorkscrewmotility.Theyoftenjointoformzigzagshapes(20,24).Campylobacterspp.arecurrentlyidentifiedbytestsdescribedbyHarvey(13)andBarretetal.(3).PCRgenusandspeciesidentificationmethodshavebeenpublished(12,18,30).

Foradditionalinformationcontact

CarlosAbeyta

,FDA,PRLNW,Bothell,WA98041-3012.Phonenumber:(425)483-4890.

SAMPLEPREPARATIONFLOWCHARTFORVARIOUSFOODSANDWATER(SECTIONC.2.a-h)

*forsamplesproducedorprocessed<10dayspreviously.

?forfrozensamplesorsamplesproducedorprocessed≥10dayspreviously.

Equipmentandmaterials

Balances,6000gcapacity,accurateto0.1g;and200gcapacity,accurateto0.0001g

Sterilestomacherbags,400and3500mlbagsand400mlfilterbags(otherbagtypesandsizesdescribedinrefs.16,21,and22)

Whirl-pakbagracksandstainlesssteelbaskets

Benchtopshaker

Centrifuge,refrigerated,capableof20,000xg

Polypropyleneorstainlesssteel250mlcentrifugebottlesand50mlcentrifugetubes,sterile

Largefunnelswithcheeseclothlinings,sterile(forwholeseafoodandmeatsamplesoriffilterbagsareunavailable)

Whiteororangegreasepencilstomarkblood-freeagarplates

50mlsterileconicalcentrifugetubes

Plastic5-10mltubeswithscrewcaplids,sterile

Cryotubes,1ml,sterile

Phase-contrastmicroscope,with100Xoilimmersionobjectiveordark-fieldmicroscopewith63Xobjectiveorlightmicroscopewith1000Xobjective

Microscopeslides,1cmsqcoverslipsandimmersionoil

Gastankassembly(5%O2,10%CO2,85%N2)andvacuumsource(Fig.1)

Microaerobiccontainersystem

An?robejarsandbags:

Jarswithvacuum-pressuregaugeandSchradervalves.Theymaybeusedwitheitherthegastank/vacuumassemblyorwithgas-generatingenvelopes.3.4L,(Difco1950-30-2orOxoidHP11)maybeusedwitheitherthegastank/vacuumassemblyorwithgas-generatingenvelopes.

Jarswithoutagaugeorvalves(2.5literBBLorEMDiagnostics[Remel,Lenexa,KS66215]and9.5literBBL)anaerobejars.Theseareusedwithgas-generatingenvelopes(2.5litertypesuchasOxoidN025AortheBBLandEMgaspaks).

Rectangularjars2.5and5.5literanaeroberectangularjars(InternationalBioproducts,800-729-7611orMitsubishiGasChemicalAmerica,212-752-4620).

An?robepouchesorrectangularjars,0.4L,for2-plateincubation(InternationalBioproductsorMitsubishi).Pouchesfor1-plateincubationareavailablefromEMDiagnostics.

Air-tightplasticbags(4milwt),12"X16"orlargerthatcanbeclosedbyheatsealingortapecanbeusedasanincubationchamber.

Campygas-generatingenvelopesorpouches:for3.4literjars,OxoidBR56orCN035A;for2.5literand9.5literjars,OxoidCN025A,Difco1956-24-4,BBL71040or71034orEMDiagnostics53013678;forrectangularjars,Mitsubishi10-04;for2-plate,Mitsubishi20-04and1-plate,EMDiagnostics,53-13699.TheOxoidCNO25AandtheMitsubishiandEMenvelopesareusedwithoutwater.

§CAUTION:ONLYGASPACSUSEDWITHOUTWATERARECOMPATIBLEWITHTHEITEMSINA.3)ANDA.4).THERECTANGULARJARSCANEXPLODEIFUSEDWITHTHEOXOIDBR56,BBLORDIFCOENVELOPES.

AnAn?robegaspak(1only)canbeusedwitha9.5literBBLan?robejar.Asinglepakinalargecontainerreducestheoxygenlevelto~5%andproducesothergasespromotingCampylbactergrowth.Eithergaspaktype(does/doesnotrequirewater)canbeused.

Airincubators,25±2,30±2,37±2,and42±1°C.

Waterbath,preferablyshakertype,range30-42°Corcoliformbathsetto37and42°C.Shakerwaterbathshouldhaveflaskclamps,250or500mlifgassedflasksareused.Shakerwaterbathmaybeusedeitherwithbubblersystemorgassedflasksystem.Staticwaterbathcanbeusedonlywiththebubblersystem.

Shakerairincubatororairincubatorwithshakerplatform(alternativetoshakerwaterbath)

Shakinggassedflaskorbagsystem(Fig.2)

Bags,seeFig.2.Metalizedpolypouches,AssociatedBagCompany,Milwaukee,Wi.,800-926-6100.Use6"X8"for100mlenrichmentsand8"X10"bagsfor250mlenrichments.Largerbagsalsoareavailable.Bagsarenotsterilebutcanberadiationsterilized.Ifusingnon-sterilebags,includeabagcontrolusingBoltonbrothorListeriaEnrichmentbrothwithoutantibiotics.

Vacuumflasks,250or500ml,withrubberstopperandfoam-pluggedvacuumtubingonthesidearm,sterilized(seeFig.2).FurtherinformationonassemblyanduseiscontainedinBAM,7thed.,1992,chapter7.

Bubblersystem(Fig.3).Twogasdeliveryvalvesystemsareavailable.

Evaporator/concentratormanifoldwithY-connector,availablein6or12positionsets(AFCInternational,Inc.,DownersGrove,IL;800-952-3293)SeesectionsD-3foruseandF-1forassemblyinstructions.

Plasticluer-lockstopcocks.Stopcockslockintotheoutletvalves.

Plasticaquariumtubing,3/16"innerdiameter

NuproS-seriesfinemeteringvalveswith1/8"Swage-lokcompressionfittingsand1/8"teflonorpoly-flotubing.Twotofourinchlongpiecesof3/16"inner-diameteraquariumtubing(equaltonumberofvalves).Seesects.D-3&F-2forinstructionsonoperatingandassemblingthisbubblerunit.ThesecomponentsareavailablefromlocalvalvesuppliersorcontactIndianapolisValveandFittingCo,Indianapolis,IN;317-248-2468,forinformationonthenearestsupplier.

Enrichmentbrothcontainer(useeither):

400mlorlargerstomacherorstomacherfilterbags,twisttiesandstainlesssteelbaskets

250or500mlErlenmeyerflasks,foampluggedwithfoilwrapandsterilized.Two-inchsqParafilmpiecesandweightedringsoraplatformwithclamps.Forinstructionsforassemblyanduse,seefig.2andBAM,7thed.,1992,chapt.7.

Plastic1mlsterilepipets.

Wateranalysisapparatus

Zetaporfilters,45μm(Cuno,Meriden,CT,800-243-6894;nosubstitutions),47-293mm(dependingonfilteringunitsize),autoclavedseparatelyfromfilteringunit

For47mmfilterapparatus:

Teflon-facedborosilicateglass47mmholder(s)andfilterclampforceps,sterilized

Manifold,6-12place,formultiplesubsamples

Vacuumflask,1-4L,andifamanifoldisused,arubberstopperwitha6to8-inchplastictubeinsertedandahoseconnectingtheplastictubetothemanifold.Anotherhoseconnectstheflasksidearmtoavacuumsource.

For90,142,or293mmfilterapparatus

90,142,or293mmfilteringunitwith3fthoseattachedattop,sterilized

Vacuumflask,4-6L,setupasinb-3above,exceptthehoseattachedtothevacuumflask'sstoppertubeconnectstothefilteringunitoutletport

Media

2,biochemicalsand

reagents

3(sectionG,exceptwhereotherwiseindicated)

Media

CampylobacterEnrichmentBroth(Boltonformula,OxoidAM7526orMalthusDiagnosticsLAB-135,MalthusDiagnostics,NorthRidgeville,OH;216-327-2585)withlysedhorsebloodandantibioticsupplement(OxoidNDX131orMalthusDiagnosticsX131).Alternatively,antibioticsupplementmaybepreparedfromindividualcomponents(G-1).[

M28a

4]

Campylobacterisolationagars(useeither)

Abeyta-Hunt-Bark(AHB)agar(G-2)[

M29a

5]

ModifiedCampyblood-freeagar(mCCDA)(G-2)[

M30a

6]

Abeyta-Hunt-Barkagar(G-2),withoutantibiotics

Heartinfusionagar(HIA)slants(

M59

7)

0.1%Peptonediluent(

R56

8)

Semi-solidmedium,modified,forbiochemicalidentification(G-5)

Neutralred(NR)solution,glycine,NaCl,cysteineHCl,KNO3

Triplesugariron(TSI)agarslants(

M149

9)

O-Fglucose(

M116

10),modified;preparehalfthetubeswithglucoseandhalfwithout.

MacConkeyagar(

M91

11)

Cultureshippingmedia

Cary-Blairmedium(

M31

12)orA-Hslantswith5%filteredfetalbovineserumorlysedhorseblood,w/oantibiotics(G-2)

Culturestoragemedia

Semi-solidmedium,modified,forshort-termculturestorage(G-4)[

M30c

13]

Culturefreezingmedia(G-3)forlong-termstorage(

M30b

14)

Biochemicalsand

reagents

15

Hippurate(

R33

16)andninhydrin(

R47

17)reagents

Nalidixicacidandcephalothinantibioticdisks(Difco)

Hydrogenperoxide,3%

Fetalbovineserum(FBS),filtered(0.22μm)

Oxidasereagent,liquidtypepreferred(

R54

18)

Gramstainreagents(

R32

19);counterstainwith0.5%carbolfuchsin(Difco)

NitratedetectionreagentsAandB(

R48

20)

Leadacetatestrips(Difco)

DryspotCampyTest(Oxoid,DR150availablefromHardyDiagnostics800-266-2222[

.com

21

\o"DisclaimerIcon"

22])orAlertforCampylobacter(Cat.No.9800[94tests]or9801[22tests],NeogenCorporation,800-234-5333or

.com

23

\o"DisclaimerIcon"

24).

Sterilewater,1-2liters;70%ethanolor1000ppmhypochloritesolution(G-6)

Samplepreparation.

Backgroundinformation

Campylobacterspp.cansurvive,butnotmultiply,infoodatrefrigerationtemperaturesfor1-3weeks,especiallyiffoods(exceptrawmilk)areinairtightcontainers.Theirnumbersdecrease2logsuponfreezingat-20°C,butthesurvivingorganismscanberecovered≥5months.SamplesshouldbeanalyzedforCampylobacterassoonasasamplepackageisopened;introductionoffreshoxygenaddssignificantstresstoalreadyweakenedorganisms.

Productionofoxygen-neutralizingenzymesisdecreasedinmicroaerophiles,especiallywhencellsareunderstress.Tocombatthisproblemoxygen-quenchingcompounds,suchasFBP,hemin,bloodand/orcharcoal,areaddedtothemedia.Preparedmediaabsorboxygenduringstorage;usefreshlypreparedmediawheneverpossible.Alternatively,ifpreparedbrothbaseisstoredintightlyclosedcontainersawayfromlight(heminislightsensitive),itcanbeusedforupto2months.ProtectagarcontainingFPBfromlightandrefrigeratewhennotinuse.

Boththeinitialsamplepreparationanda1:10dilutionareoftenneededforenrichmentwhenhighnumbersofbackgroundflora(withbroadspeciesdiversity)arepresent.Withthesampledilution,antibioticsperformmoreeffectivelyandcampylobactercellscanutilizethelow-oxygenatmospheremoreefficiently.Ifheavybackgroundcontaminationissuspected,add1:10dilutionenrichment.Thefollowinginstructionsincludemandatorydilutionenrichmentsforshellfishandeggs.

PreparationofSamples

Add2rehydratedvialsofBoltonantibioticadditivesand50mllysedhorsebloodto1000mlBoltonbrothbase.Alternatively,antibioticadditivescanbepreparedfromindividualcomponents(G-1).

Allsampletypesexceptthoselistedinfollowingsect.2(b-h)

Placefilterbaginwirepetridishholder(typeusedinanaerobejars).Holdbaglininginplacewithmetalbindercliptopreventcollapseduringfilling.Weigh25gsample(50giffruitorvegetables)intobag,andadd100mlenrichmentbroth.Removebagfromholder,keepingclipattachedandwraptwist-tiearoundtop.Placebag(s)inbasketorwhirl-pakrack.Shakegentlyfor5min.orplaceonatable-topshakersetat25rpm.

Aftertherinsingstep,hold5min.Removefilterliningandallowittodrainafewseconds.Iffilterbagisnotavailable,rinsesampleinasterilebag,andpourcontentsthroughasterile,cheesecloth-linedfunnelintotheincubationbagorflask.Whenusingmetalizedpolypouchesforthegassedbagincubation,placefilterlinerfromastomacherbagintothepouchbeforeweighinginthesample.Note:Whenanalyzingacidicfoods,suchaschickensalad,adjustbrothpHto7.4with2NNaOHaftertherinsingstep.

Lobstertailorcrabclaws.Weigh50-100gintoafilter-linedbagandrinseasina,above.

Wholemeatcarcassorsamplethatcannotbeeasilyreducedto25g(e.g.,wholerabbit,lobsterorlargerpieceofgamemeat)

Placesampleinto3500mlstomacherorothersterilebag.Add200ml0.1%peptonewater.Twistbagtoseal,andswirlcontentsfor2-3min.Tiltbag,andholdbackfoodpiecestoletrinseliquidflowtoonecorner.Sanitizeabottombagcornerwith1000ppmhypochloritesolutionor70%ethanol;thenrinsewithsterilewater.Asepticallycutcornerofbag,andpourrinsethroughsterilecheesecloth-linedfunnelintoa250mlcentrifugebottle.Centrifugeat16,000xgfor15min.Discardsupernatant,andresuspendpelletin10ml0.1%peptonewater.Transfer3mlpelletmixtureto100mlbroth.

Liquideggyolkorwholeeggmixture

Dividesampleintocompositesoftwosubsamplespercomposite,25gpersub.Weigh25gofeachcompositeinto125mlbroth.Aftergentlymixing,transfer25mltoanother100mlbroth.Analyzeboththe1:6and1:48dilutions.

Shellfish,shucked

Ingeneral,aminimumof12shellfishshallbetakeninordertoobtainarepresentativesample(APHA1970,RecommendedProceduresfortheExaminationofSeaWaterandShellfish).Dependingonthesizeofthespecies,thiswillyieldanapproximately100to200gcompositeofshellliquorandmeat.Collecttheappropriatequantitiesofshellliquorandmeatsinasterileblenderorothersuitablesterilecontainer.Blendatlowspeedorstomachfor60s.Remove25gshellfishhomogenateforsampleanalysistoaStomacherbagor500mlflask.

Add225mlenrichmentbroth.Transfer25mlofthemixturetoasecond225mlenrichmentbroth.Analyzeboththe1:10and1:100enrichments.

Ifenrichmentsarebubbledduringincubation,leavetheminbagsor500mlflasks.Ifincubatingingassedbagorflaskshakersystem,use6x10-inchmetalizedpolypouchesor500mlvacuumflasks.Ifincubatinginanaerobejars,reducevolume/flaskorbagto125mlbydividingeachenrichmentintotwoparts.Thegasdoesnotpenetrateintoalargervolumesufficientlytoprovidepropergrowthofcampylobacters.

Water

Requestinvestigatorscollect2-4litersforanalysis.Whencollected,5mlof1Msodiumthiosulfateshouldbeaddedperliterofchlorinatedwatersample.

Filtersmallervolumesamplesthrough45μmZetaporfilters,47mmdiameter.Thesefiltershaveapositivecharge.ThenegativelychargedGram-negativeorganismsaremoreeffectivelyretainedinthefilter.Filterlargervolumes,especiallythosethatareturbid,through90mmorlargerdiameterfilters.

Placefilterunitintoautoclavablepan.Iffilterclogs,wearsterileglovesandopenfilterholderunittoasepticallyremovefilterwithsterileforceps.Placefilterintoenrichmentbroth(seebelow).Placeanothersterilefilterinunit,reassemble,andcontinuefiltering.Useasmanyfiltersasneededpersubsample.Whenanalyzingseaorothersaltwater,flushexcesssaltofffilterbyrunning100-1,000ml(dependingonfiltersize)sterilephosphatebufferthroughthefilterasthelastofthesampleisgoingthroughthefilter.Dothiswitheveryfilterusedforsaltwateranalyses.Donotletfilterbecomecompletelydry.Immediatelytransferfinishedfiltertobroth.Campylobactersareverysensitivetodryingandhighsaltconcentrations.

Placefilter(s)inbrothintheenrichmentcontainer.Whenusinglargefilters,fragmentwithasterilepipet.Besurethebrothcoversthefilter(s).

EnrichmentsincubatedinCampygasinanaerobejarsshouldbe125mlorless.Largervolumesshouldbedividedintosmalleramounts,asepticallydividingthefilters.

Swabs

Pipet10mlenrichmentbrothintosterile50mlErlenmeyerflaskswithfoiltops.Placeoneswabintoeachflask,asepticallybreakingoffthesticksbelowthetopoftheflask.Replacecoversloosely.Placeflasksinanaerobejar.Tofittwolayersofflasksinjar,placeacardboardcircleoverbottomlayer,leavingspacearoundthecardboard'sedgeforgascirculation.

Milk,frozendairyproducts

Rawmilk.InstructtheinvestigatortotestrawmilkatthecollectionsitebyusingasterilepipettetoplacetestportionontopHtestpaper(pH6-8range).IfthepHisbelow7.6,addsterile1-2NNaOHandgentlytoadjustitto7.5±0.2.Immediatelyuponreceiptinthelaboratory,testthepHofthedairysamplewithpHtestpaperandadjusttopH7.5±0.2withsterile1-2NNaOHifnecessary.Centrifugea50gportionat20,000xgfor40minutes.Discardsupernatantanddissolvepellet(notfatlayer)in10mlenrichmentbroth.Transferpelletto90mlenrichmentbroth.

Othermilktypesandicecream.AdjustpHasinrawmilk.Centrifugea50gportionat20,000xgfor40minutes.Discardsupernatantanddissolvepellet(notfatlayer)in10mlenrichmentbroth.Transferpelletto90mlenrichmentbroth.

Icecreamandotherfrozendairyproducts:meltandasepticallyremoveanycandyorothersolidsbeforeweighingout.

Cheese.Weigh50gintoafilterbagandadd50ml0.1%peptone.Stomach15-30s.Removelining,lettingitdrain5s,anddiscard.Centrifugeandremovepellettobrothasinrawmilk(h,1).

"Milksock"orstrainer(gauzepieceusedtofilteroutsolidsduringmilking).Place50gpiecein100mlbroth.

Preenrichmentandenrichment(modifiedParkandHumphreymethods)

Pre-enrichments

4hpre-enrichmentIftheageofthesampleisknowntobewithin10daysofproductionortimeofcontamination,orifthesampleisadairyproduct,pre-enrichat37°Cfor4h.Thepre-enrichmentshouldbeincubatedundermicroaerobicconditions.

5hpre-enrichmentUsethe5hmethodifanyproducthasbeenrefrigeratedfor≥10days.Allwaterorshellfishsamplesarepre-enrichedbythe5hmethod.

Incubateat30°Cfor3h,thenat37°Cfor2h.NOTE:Incubatemicroaerobicallyat30°Cunlessusinganon-shakingbath-bubblersystem(D-3).Bubblingstaticenrichmentsat30°Cfostersgrowthofanaerobes(D-3).Performthe37°Cincubationundermicroaerobicconditions.Thismethodyieldsgreaterrecoveryforseverelystressedorganisms.

Generalinformationconcerningbothmethods.Settheshakerspeedforbubblingenrichmentsto50-60rpmandto175-200rpmforgassedbagsorflasks.

Enrichment(microaerobic,D-3)

Afterpre-enrichment,raisethetemperatureinthewaterbathormovetoa42°Cincubator.IfanalyzingforC.fetus,keepthetemperatureat37°C,evenifathermotolerantstrain(growthat42°C)wasassociatedwiththesample.Incubateshakingenrichments23-24h,exceptshellfishsamples,whichareincubatedanextra4h.Dairysamplesareincubated48hrstotal.Incubatenon-shakingenrichments28-29h,exceptshellfish,whichareincubated48h.IncubatesamplesforC.fetusat48h(shaking)or52h(non-shaking).

Incubationandatmospheremodificationmethodsforenrichments

Analystsmaychoosefromthreemethodsforgeneratingmicroaerobicconditionsinenrichmentbroth.Theseare:bubblingthegasmixturethroughbroth,shakingenrichmentstoincorporatethegas,orincubatinginanaerobejarswithamodifiedatmosphere.

Thefirstmethodusesthebubblersystemthatalsocanincorporateshakingtheenrichmentsduringincubation.Thesecondusesheat-sealed,gassed,metalizedpolypouchesorevacuatedandgassedflasks.

Thethirdmethodistheevacuatedandgassedanaerobejar(orajarthatusesaCampygasenvelope).Choosethiswhenothersystemsarenotavailable.Exception:incubationofenrichmentsin50mlErlenmeyerflasks(i.e.,swabs),whichcanbeaccomplishedonlyinthejarsystem.

Thesystemsaredescribedasfollows:

Bubblersystem

Double-bagenrichmentstopreventbagsfromleaking(bagscantearduringshaking).Addabout10mlwatertotheouterbagforoptimumheattrasfertothebroth.Placestomacherbagsintostainlesssteelbaskets(4-6/basket).Fillexcessspaceinbasketwithwater-filleddilutionbottles.Place1mlplasticpipettipendintoeachbagandfastentightlywithatwist-tie.Insertthepluggedendofeachpipetintothetubingconnectedtothebubblervalves.

Openthemaingastankvalveandsetthepressureto4-6lbwiththeregulatoradjustingscrew.Thiswillgiveaflowrateof2-3bubblespersecintoeachbag(figs.1&3).Ensurethatthepipettipineachbagisinserted2/3intothebroth.Tiebubblertubingforeachenrichmentlooselytogetherabovethebasketstokeepthebagsstandingupright.Bringthewaterlevelofthebathupslightlyhigherthanthelevelofthebrothsinthebags.Replenishwaterasneededduringtheincubationperiod.

RefertotheBAM,7thed.,1992,chap.7.forinstructionsonusingErlenmeyerflaskswiththebubbler.

Shakingflaskorbagsystem(usebagswithanairshakerincubator)

Shakerbagsystem.LoosentheringclampholdingtheSchraderchuckandclipvalveonthehoseatthegastank,andremovethevalve.Insertthetwo-wayconnectorattachedtoalengthof3/16"inner-diameteraquariumtubing.Openthemaingastankvalveandsettheregulatorto2lbwiththeregulatoradjustingscrew.Placeasterile1mlplasticpipetinotherendoftheaquariumtubing,andkeeppipettipsterilebyplacingthetipendinasterilebag(figures1&2).Useanewpipetforeachbag.

Heatsealeachfilledbag(metalizedpolypouch)withabagsealer.Cutaverysmallcornerfromthetopofthesealedend.Squeezeairfrombagbypressingareaaboveliquiduntiltheareaisflat.Insertpipetintoopencornerofbagandopenon-offvalveonthegashose.Fillareaabovethebrothwithgas.Repeatsqueezingandgassingeachbag2moretimes,endingwithagassingstep.Quicklyheatsealortapethecornerofthebagshut.Placegassedbagsintobasketslinedwithplasticbags.Setthebasket(s)ontoashakerincubatorplatform.Setshakerspeedto175-200rpm.

Shakerflasksystem.RefertoBAM,7thed.,1992,chap.7.

Gassedjarsystem.Placestomacherbagswiththetopslooselyclosedwithatwisttieinagasjar.Amountofbrothineachbagshouldnotbeover125ml.Whenusingthe5.5literrectangularjar,prepareadeeptrayfromfoilandtapetocontainthebottomsofthebagsinsidethejar.

Gaspakenvelopes.Use3BBLCampypak,PackPlusorEMAnaerocultCgas-generatingenvelopesper9.5literBBLjarand1persmalljar.Withthe3.4literDifcoandOxoidjars,useDifcoorOxoidgaspakenvelopes,whicharedesignedforusewitha3.4literjar.Gaspaksrequiringwaterneedtobeusedwithacatalyst.Witha2.5literrectangularjaruse1gaspak(typenotusedwithwater);3fora5.5literjar.Oruse1anaerobegaspakina9.5literBBLjar.

Gastankandvacuumsource.Aftertighteningjarlid,attachchuckandclipvalveofvacuumhosetovalveindicatedforvacuumonjarlid.Turnonvacuumandevacuatejarto15-20inchesofHg.Detachhoseandtightenlidslightlyifneeded.Openthemainvalveonthegastank.Adjustthepressureto6-8lbwithregulatoradjustingscrew(fig.1).Attachchuckandclipvalveonthetankhosetotheothervalveonthelid.Opentheregulatoron-offvalveandfilljarto5-10lb.Disconnectthehoseandclosetheregulatorvalve.Repeattheevacuationandgassingtwicemore,endingwithagassingstep.Whenopeningthejars,firstreleasepressurebypressingdownononeofthevalvestemswithaninoculatingloophandleorsimilarobject.

Guidelinesforstoringandmaintainingjars.Ifajarlidwithgaugesisknockedagainstahardsurface,agaugecanbecomemisaligned.Marknew"0"placeongaugeandadjustvacuumandgasreadingsaccordingly.

Storejarswithscrewclampsplacedinjarssothatoneendislyingoverlipofjarbottom.Proplidagainstclamptoallowfreeflowofairandpreventmoldbuild-upfromdampjar.Orcleanjarsbetweenuseswith70%alcoholanddrybeforestoring.

Ifajarwillnotholdvacuumorgaspressure,checkforthefollowing:cracksinthejarbottom,crackedormissingrubberringsorsealsinthelidsorafaultyvalvestem.ReplacementvalvestemsandaSchraderextractortoolareavailablefromthejars'distributorsorbicycleshops.Toreplacestems,placeprongsofextractorovervalvestemandturncounter-clockwiseuntilstemisremoved.Dropnewvalvestem(pin-headsideup)intovalveandturnclockwiseuntilmeetingresistance.

Positivecontrols

StoreCampylobacterculturesinfreezingmedium(G-)at-70°C.Ifculturesareusedoften,theycanbekeptatroomtemperatureinsemi-solidstoragemedia(G-4).Controlcult