美国FDA细菌学分析手册第八版BAM弯曲菌Word文档下载推荐.docx
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2000-DEC-29
∙MediaInstructionsModifiedon2001-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-Barré
syndrome(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(1.5-5µ
m),withcorkscrewmotility.Theyoftenjointoformzigzagshapes(20,24).Campylobacterspp.arecurrentlyidentifiedbytestsdescribedbyHarvey(13)andBarretetal.(3).PCRgenusandspeciesidentificationmethodshavebeenpublished(12,18,30).
ForadditionalinformationcontactCarlosAbeyta,FDA,PRLNW,Bothell,WA98041-3012.Phonenumber:
(425)483-4890.
SAMPLEPREPARATIONFLOWCHARTFORVARIOUSFOODSANDWATER(SECTIONC.2.a-h)
*forsamplesproducedorprocessed<
10dayspreviously.
†forfrozensamplesorsamplesproducedorprocessed≥10dayspreviously.
A.Equipmentandmaterials
1.Balances,6000gcapacity,accurateto0.1g;
and200gcapacity,accurateto0.0001g
2.Sterilestomacherbags,400and3500mlbagsand400mlfilterbags(otherbagtypesandsizesdescribedinrefs.16,21,and22)
3.Whirl-pakbagracksandstainlesssteelbaskets
4.Benchtopshaker
5.Centrifuge,refrigerated,capableof20,000xg
6.Polypropyleneorstainlesssteel250mlcentrifugebottlesand50mlcentrifugetubes,sterile
7.Largefunnelswithcheeseclothlinings,sterile(forwholeseafoodandmeatsamplesoriffilterbagsareunavailable)
8.Whiteororangegreasepencilstomarkblood-freeagarplates
9.50mlsterileconicalcentrifugetubes
10.Plastic5-10mltubeswithscrewcaplids,sterile
11.Cryotubes,1ml,sterile
12.Phase-contrastmicroscope,with100Xoilimmersionobjectiveordark-fieldmicroscopewith63Xobjectiveorlightmicroscopewith1000Xobjective
13.Microscopeslides,1cmsqcoverslipsandimmersionoil
14.Gastankassembly(5%O2,10%CO2,85%N2)andvacuumsource(Fig.1)
15.Microaerobiccontainersystem
a.Anæ
robejarsandbags:
1.Jarswithvacuum-pressuregaugeandSchradervalves.Theymaybeusedwitheitherthegastank/vacuumassemblyorwithgas-generatingenvelopes.3.4L,(Difco1950-30-2orOxoidHP11)maybeusedwitheitherthegastank/vacuumassemblyorwithgas-generatingenvelopes.
2.Jarswithoutagaugeorvalves(2.5literBBLorEMDiagnostics[Remel,Lenexa,KS66215]and9.5literBBL)anaerobejars.Theseareusedwithgas-generatingenvelopes(2.5litertypesuchasOxoidN025AortheBBLandEMgaspaks).
3.Rectangularjars2.5and5.5literanaeroberectangularjars(InternationalBioproducts,800-729-7611orMitsubishiGasChemicalAmerica,212-752-4620).
4.Anæ
robepouchesorrectangularjars,0.4L,for2-plateincubation(InternationalBioproductsorMitsubishi).Pouchesfor1-plateincubationareavailablefromEMDiagnostics.
5.Air-tightplasticbags(4milwt),12"
X16"
orlargerthatcanbeclosedbyheatsealingortapecanbeusedasanincubationchamber.
b.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.
c.AnAnæ
robegaspak(1only)canbeusedwitha9.5literBBLanæ
robejar.Asinglepakinalargecontainerreducestheoxygenlevelto~5%andproducesothergasespromotingCampylbactergrowth.Eithergaspaktype(does/doesnotrequirewater)canbeused.
16.Airincubators,25±
2,30±
2,37±
2,and42±
1°
C.
17.Waterbath,preferablyshakertype,range30-42°
Corcoliformbathsetto37and42°
C.Shakerwaterbathshouldhaveflaskclamps,250or500mlifgassedflasksareused.Shakerwaterbathmaybeusedeitherwithbubblersystemorgassedflasksystem.Staticwaterbathcanbeusedonlywiththebubblersystem.
18.Shakerairincubatororairincubatorwithshakerplatform(alternativetoshakerwaterbath)
19.Shakinggassedflaskorbagsystem(Fig.2)
a.Bags,seeFig.2.Metalizedpolypouches,AssociatedBagCompany,Milwaukee,Wi.,800-926-6100.Use6"
X8"
for100mlenrichmentsand8"
X10"
bagsfor250mlenrichments.Largerbagsalsoareavailable.Bagsarenotsterilebutcanberadiationsterilized.Ifusingnon-sterilebags,includeabagcontrolusingBoltonbrothorListeriaEnrichmentbrothwithoutantibiotics.
b.Vacuumflasks,250or500ml,withrubberstopperandfoam-pluggedvacuumtubingonthesidearm,sterilized(seeFig.2).FurtherinformationonassemblyanduseiscontainedinBAM,7thed.,1992,chapter7.
20.Bubblersystem(Fig.3).Twogasdeliveryvalvesystemsareavailable.
a.Evaporator/concentratormanifoldwithY-connector,availablein6or12positionsets(AFCInternational,Inc.,DownersGrove,IL;
800-952-3293)SeesectionsD-3foruseandF-1forassemblyinstructions.
1.Plasticluer-lockstopcocks.Stopcockslockintotheoutletvalves.
2.Plasticaquariumtubing,3/16"
innerdiameter
b.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.
c.Enrichmentbrothcontainer(useeither):
1.400mlorlargerstomacherorstomacherfilterbags,twisttiesandstainlesssteelbaskets
2.250or500mlErlenmeyerflasks,foampluggedwithfoilwrapandsterilized.Two-inchsqParafilmpiecesandweightedringsoraplatformwithclamps.Forinstructionsforassemblyanduse,seefig.2andBAM,7thed.,1992,chapt.7.
d.Plastic1mlsterilepipets.
21.Wateranalysisapparatus
a.Zetaporfilters,45µ
m(Cuno,Meriden,CT,800-243-6894;
nosubstitutions),47-293mm(dependingonfilteringunitsize),autoclavedseparatelyfromfilteringunit
b.For47mmfilterapparatus:
1.Teflon-facedborosilicateglass47mmholder(s)andfilterclampforceps,sterilized
2.Manifold,6-12place,formultiplesubsamples
3.Vacuumflask,1-4L,andifamanifoldisused,arubberstopperwitha6to8-inchplastictubeinsertedandahoseconnectingtheplastictubetothemanifold.Anotherhoseconnectstheflasksidearmtoavacuumsource.
c.For90,142,or293mmfilterapparatus
1.90,142,or293mmfilteringunitwith3fthoseattachedattop,sterilized
2.Vacuumflask,4-6L,setupasinb-3above,exceptthehoseattachedtothevacuumflask'
sstoppertubeconnectstothefilteringunitoutletport
B.Media2,biochemicalsandreagents3(sectionG,exceptwhereotherwiseindicated)
1.Media
a.CampylobacterEnrichmentBroth(Boltonformula,OxoidAM7526orMalthusDiagnosticsLAB-135,MalthusDiagnostics,NorthRidgeville,OH;
216-327-2585)withlysedhorsebloodandantibioticsupplement(OxoidNDX131orMalthusDiagnosticsX131).Alternatively,antibioticsupplementmaybepreparedfromindividualcomponents(G-1).[M28a4]
b.Campylobacter