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

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

《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》由会员分享，可在线阅读，更多相关《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（33页珍藏版）》请在冰豆网上搜索。

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