美国FDA细菌学分析手册第八版弯曲菌

1、俺BAM: Ca案mpyloba扒cter HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm 白Return 爸to BAM 柏table o笆f conte翱nts艾1澳January隘 2001办Bacteri绊ologica叭l Analy唉tical M八anual瓣Chapter稗 7矮Campylo稗bacter拜Authors俺:叭 Jan M.昂 Hunt, HYPERLINK mailto:carlos.abeyta 昂Carlos

2、按Abeyta稗 and To翱ny Tran熬Revisio澳n Histo盎ry:暗Updated澳 and re昂vised: 案2000-DE埃C-29 隘Media I罢nstruct哀ions Mo翱dified 靶on 2001笆-MAR-08挨 俺Isolati坝on of 爸Campylo爱bacter扒 Specie隘s from 哎Food an霸d Water办Campylo埃bacter 盎is cons阿idered 霸by many般 to be 安the lea板ding ca爸use of 霸enteric澳 illnes癌s in th敖e 扮United

3、哀States啊 (20,26白). 碍Campylo啊bacter般 specie罢s can c柏ause mi坝ld to s般evere d瓣iarrhea昂, with 矮loose, 皑watery 百stools 柏often f矮ollowed氨 by blo佰ody dia俺rrhea (笆7,20). 熬C捌. 安jejuni白, 皑C唉. 阿coli艾, and 暗C靶. 靶lari摆 accoun版t for m跋ore tha扮n 99% o八f the h扒uman is爸olates 昂(搬C捌. 傲jejuni搬 90%). 按Other s盎pecies 半h

4、ave be胺en asso扮ciated 搬with hu伴man ill哎ness in柏 recent哀 years 稗(7,17,1罢8,23,26拌,27).瓣Campylo般bacter肮 specie熬s are h傲ighly i板nfectiv耙e. The 傲infecti拔ve dose笆 of 扮C昂. 班jejuni芭 ranges唉 from 5鞍00 to 1拌0,000 c捌ells, d柏ependin霸g on th罢e strai佰n, dama八ge to c绊ells fr暗om envi柏ronment阿al stre岸sses, a败nd the

5、摆suscept芭ibility把 of the盎 host (班4,6,7,2板0,27). 柏Only th半e mesop百hilic 盎C佰. 板fetus叭 is nor癌mally i拜nvasive背. Therm笆ophilic安 specie埃s (opti白mum 澳42C绊) such 艾as 跋C搬. 颁jejuni班 are oc疤casiona暗lly inv哀asive. 佰The inf靶ections叭 are ma拌nifeste爸d as me胺ningiti版s, pneu般monia, 埃miscarr扒iage, a拜nd a se坝vere fo板

6、rm of G耙uillain案-Barr 捌syndrom笆e (6,20八). Ther哀motoler俺ant str凹ains of澳 耙C拜. 罢fetus袄 that g佰row at 扒42C隘 have b扳een iso凹lated f芭rom pat跋ients (跋17).办Campylo绊bacters按 are ca碍rried i哎n the i昂ntestin挨al trac安t of a 疤wide va八riety o凹f wild 熬and dom安estic a颁nimals,拜 especi凹ally bi皑rds. Th拜ey can 暗establi

7、澳sh a te唉mporary笆 asympt安omatic 巴carrier疤 state,巴 as wel搬l as il袄lness, 跋in huma氨ns. Thi案s is es版peciall哎y preva案lent in澳 develo罢ping co叭untries霸 (20). 斑Consump百tion of爱 food a靶nd wate班r conta白minated罢 with u扮ntreate案d anima碍l or hu霸man was盎te acco艾unts fo瓣r 70% o百f 阿Campylo背bacter傲-relate耙d illne败s

8、ses ea搬ch year颁. The f挨oods in安clude u扳npasteu敖rized m安ilk, me阿ats, po暗ultry, 吧shellfi啊sh, fru傲its, an白d veget按ables, 白(1,8-11邦,17,19,捌20,22, 哎25,26).般C哀. 吧jejuni巴 can su颁rvive 2澳-4 week哀s under按 moist,白 reduce坝d-oxyge鞍n condi板tions a胺t 癌4C隘, often佰 outlas安ting th隘e shelf氨 life o斑f the p芭roduct 坝(ex

9、cept埃 in raw爸 milk p熬roducts柏). They胺 can al霸so surv跋ive 2-5哀 months熬 at 巴-20C傲, but o扮nly a f埃ew days按 at roo爱m tempe柏rature 氨(5,8-11班,20). E半nvironm俺ental s扳tresses扮, such 袄as expo胺sure to颁 air, d肮rying, 案low pH,耙 heatin敖g, free唉zing, a皑nd prol靶onged s埃torage,板 damage昂 cells 爸and hin岸der rec唉overy

10、 t按o a gre埃ater de扒gree th笆an for 笆most ba跋cteria.碍 Older 矮and str奥essed o捌rganism熬s gradu拔ally be暗come co肮ccoidal翱 and in叭creasin叭gly dif敖ficult 敖to cult霸ure (5,拜20). Ox笆ygen qu爸enching唉 agents靶 in med扮ia such碍 as hem巴in and 阿charcoa澳l as we百ll as a八 microa胺erobic 芭atmosph癌ere and盎 preenr摆ichment盎

11、can si按gnifica败ntly im半prove r耙ecovery般 (2,14-绊16,21,2柏5,28).颁Campylo瓣bacters捌 are mi扒croaero稗philic,扒 very s办mall, c蔼urved, 唉thin, G八ram-neg柏ative r半ods (1.捌5-5 m)爸, with 靶corkscr肮ew moti斑lity. T半hey oft爱en join班 to for般m zigza般g shape巴s (20,2盎4). 袄Campylo佰bacter敖 spp. a八re curr靶ently i伴dentifi哎ed

12、by t奥ests de扒scribed阿 by Har跋vey (13碍) and B稗arret e鞍t al.(3板). PCR 百genus a伴nd spec碍ies ide埃ntifica坝tion me挨thods h岸ave bee柏n publi八shed (1鞍2,18,30艾).疤For add芭itional袄 inform伴ation c疤ontact HYPERLINK mailto:carlos.abeyta 癌Carlos 肮Abeyta鞍, FDA, 哀PRLNW, 柏Bothell版, 绊WA爱 霸98041-3疤012伴. Phone埃 number皑:

13、(425)哀 483-48扳90.拌SAMPLE 绊PREPARA板TION FL跋OWCHART扒 FOR VA颁RIOUS F吧OODS AN百D WATER疤 (SECTI疤ON C.2.拌a-h)半*for sa般mples p班roduced版 or pro芭cessed 背10 day哎s previ吧ously.叭for fr办ozen sa拜mples o版r sampl伴es prod唉uced or摆 proces啊sed 1邦0 days 坝previou颁sly.熬Equipme阿nt and 哀materia颁ls 捌Balance熬s, 肮6000 g氨 capac

14、i澳ty, acc颁urate t绊o 啊0.1 g肮; and 哎200 g哎 capaci唉ty, acc笆urate t熬o 笆0.0001 胺g半 摆Sterile挨 stomac暗her bag般s, 400 扳and 350癌0 ml ba瓣gs and 佰400 ml 爸filter 绊bags (o挨ther ba百g types扒 and si埃zes des傲cribed 靶in refs柏. 16, 2笆1, and 啊22) 鞍Whirl-p扮ak bag 柏racks a搬nd stai翱nless s柏teel ba鞍skets 艾Bench t哀op shak叭e

15、r 稗Centrif昂uge, re巴frigera颁ted, ca芭pable o芭f 20,00叭0 x 爸g阿 爱Polypro隘pylene 阿or stai艾nless s扮teel 25熬0 ml ce昂ntrifug翱e bottl皑es and 捌50 ml c霸entrifu埃ge tube按s, ster八ile 暗Large f暗unnels 芭with ch岸eese cl爱oth lin案ings, s阿terile 拔(for wh傲ole sea笆food an氨d meat 百samples背 or if 柏filter 把bags ar稗e unava隘ila

16、ble)拔 肮White o摆r orang伴e greas挨e penci爱ls to m捌ark blo叭od-free扒 agar p笆lates 扳50 ml s蔼terile 瓣conical靶 centri搬fuge tu巴bes 哎Plastic昂 5-10 m颁l tubes俺 with s白crew ca胺p lids,伴 steril叭e 哀Cryotub耙es, 1 m摆l, ster昂ile 澳Phase-c捌ontrast伴 micros袄cope, w艾ith 100白X oil i邦mmersio柏n objec佰tive or啊 dark-f癌ield mi罢c

17、roscop柏e with 叭63X obj版ective 坝or ligh伴t micro挨scope w安ith 100班0X obje澳ctive 奥Microsc鞍ope sli傲des, 阿1 cm稗 sq cov邦er slip按s and i斑mmersio鞍n oil 搬Gas tan般k assem矮bly (5%耙 O哎2绊, 10% C拌O吧2皑, 85% N伴2芭) and v绊acuum s八ource (败Fig. 1)隘 坝Microae版robic c八ontaine办r syste岸m 艾Anrobe班 jars a瓣nd bags拌: 拜Jars wi澳th

18、 vacu矮um-pres拌sure ga鞍uge and盎 Schrad袄er valv半es. The按y may b笆e used 巴with ei笆ther th捌e gas t扳ank/vac柏uum ass懊embly o半r with 哎gas-gen搬erating奥 envelo办pes. 扒3.4 L耙, (Difc跋o 1950-哎30-2 or俺 Oxoid 拜HP11) m捌ay be u敖sed wit扒h eithe岸r the g案as tank捌/vacuum耙 assemb矮ly or w唉ith gas绊-genera阿ting en暗velopes耙.

19、把Jars wi百thout a隘 gauge 澳or valv碍es (2.5挨 liter 懊BBL or 唉EM Diag傲nostics俺 Remel败, Lenex摆a, KS 6半6215 a艾nd 9.5 肮liter B敖BL) ana笆erobe j唉ars. Th氨ese are班 used w摆ith gas般-genera澳ting en岸velopes挨 (2.5 l搬iter ty案pe such隘 as Oxo班id N澳025A敖 or the板 BBL an盎d EM ga拔s paks)盎. 耙Rectang鞍ular ja澳rs 2.5 皑and 5.5靶

20、 liter 扳anaerob背e recta笆ngular 罢jars (I癌nternat班ional B按ioprodu吧cts, 80百0-729-7隘611 or 坝Mitsubi拌shi Gas邦 Chemic跋al Amer隘ica, 21扒2-752-4般620). 拌Anrobe傲 pouche肮s or re奥ctangul吧ar jars氨, 俺0.4 L绊, for 2爸-plate 芭incubat八ion (In捌ternati埃onal Bi昂oproduc版ts or M安itsubis摆hi). Po皑uches f靶or 1-pl罢ate inc暗ubati

21、on佰 are av摆ailable埃 from E蔼M Diagn蔼ostics.靶 斑Air-tig版ht plas哀tic bag艾s (4 mi伴l wt), 挨12 X 1伴6 or l扮arger t爱hat can半 be clo芭sed by 哎heat se版aling o氨r tape 奥can be 岸used as哀 an inc般ubation昂 chambe哎r. 凹Campy g邦as-gene拜rating 蔼envelop哀es or p耙ouches:矮 for 3.碍4 liter百 jars, 百Oxoid B哎R56 or 般CN安035A稗; for

22、 2懊.5 lite扒r and 9搬.5 lite鞍r jars,扒 Oxoid 澳CN稗025A绊, Difco皑 1956-2白4-4, BB把L 71040奥 or 710凹34 or E敖M Diagn案ostics 隘5301367挨8; for 稗rectang案ular ja跋rs, Mit啊subishi绊 10-04;懊 for 2-罢plate, 斑Mitsubi芭shi 20-阿04 and 败1-plate盎, EM Di氨agnosti般cs, 53-昂13699. 摆The Oxo白id CNO把25A袄 and th袄e Mitsu安bishi a稗nd EM

23、e坝nvelope唉s are u敖sed wit办hout wa氨ter.捌 CAUTI盎ON: ONL百Y GAS P澳ACS USE熬D WITHO俺UT WATE笆R ARE C办OMPATIB般LE WITH靶 THE IT绊EMS IN 巴A.3) AN绊D A.4).熬 THE RE癌CTANGUL碍AR JARS百 CAN EX肮PLODE I凹F USED 按WITH TH皑E OXOID氨 BR56, 隘BBL OR 啊DIFCO E凹NVELOPE捌S.啊An 俺Anrobe安 邦gas pak把 啊(1 only绊) 摆can be 颁used wi胺th a 扮9.5

24、 lit皑er BBL 癌anrobe芭 jar. 搬A singl胺e pak i扒n a lar霸ge cont巴ainer r鞍educes 盎the oxy靶gen lev拌el to 矮 5% and拌 produc懊es othe鞍r gases翱 promot把ing Cam阿pylbact扒er grow癌th. Eit肮her gas耙 pak ty癌pe (doe伴s/does 扒not req盎uire wa摆ter) ca俺n be us矮ed. 班Air inc盎ubators巴, 25 俺2, 30 跋 2, 37 扒 2, an芭d 42 罢1C败. 俺Water

25、 b凹ath, pr蔼eferabl啊y shake矮r type,八 range 盎30埃-42C跋 or col蔼iform b芭ath set拔 to 37 耙and 案42C蔼. Shake按r water挨 bath s拌hould h碍ave fla皑sk clam八ps, 250跋 or 500跋 ml if 爸gassed 拌flasks 瓣are use搬d. Shak隘er wate奥r bath 版may be 爱used ei鞍ther wi氨th bubb氨ler sys吧tem or 霸gassed 癌flask s氨ystem. 般Static 唉water b爸

26、ath can半 be use艾d only 摆with th搬e bubbl昂er syst捌em. 傲Shaker 疤air inc矮ubator 艾or air 傲incubat疤or with背 shaker碍 platfo艾rm (alt敖ernativ傲e to sh胺aker wa按ter bat澳h)安Shaking八 gassed板 flask 碍or bag 瓣system 拌(Fig. 2拔) 芭Bags, s叭ee Fig.拌 2. Met拔alized 拜poly po捌uches, 澳Associa埃ted Bag摆 Compan昂y, 鞍Milwauk扮ee跋,

27、Wi., 吧800-926败-6100. 摆Use 6 肮X 8 fo败r 100 m唉l enric敖hments 巴and 8 把X 10 b板ags for阿 250 ml搬 enrich按ments. 颁Larger 氨bags al坝so are 败availab耙le. Bag挨s are n邦ot ster昂ile but爱 can be哀 radiat肮ion ste办rilized皑. If us白ing non扒-steril版e bags,般 includ哀e a bag敖 contro按l using疤 扮Bolton斑 broth 岸or List般eria En袄r

28、ichmen胺t broth哎 withou摆t antib坝iotics.跋 扒Vacuum 熬flasks,败 250 or靶 500 ml埃, with 案rubber 颁stopper哀 and fo摆am-plug霸ged vac昂uum tub跋ing on 霸the sid凹e arm, 把sterili矮zed (se肮e Fig.2俺). Furt安her inf凹ormatio奥n on as案sembly 挨and use跋 is con坝tained 皑in BAM,稗 7th ed按., 1992哀, chapt傲er 7. 爱Bubbler哀 system安 (Fi

29、g. 邦3). Two扳 gas de胺livery 艾valve s艾ystems 敖are ava癌ilable.按 熬Evapora稗tor/con般centrat碍or mani哀fold wi板th Y-co按nnector爱, avail搬able in爸 6 or 1扳2 posit埃ion set哎s (AFC 捌Interna翱tional,背 Inc., 爸Downers搬 Grove隘, 昂IL伴; 800-9奥52-3293板) See s肮ections摆 D-3 fo叭r use a皑nd F-1 般for ass捌embly i胺nstruct芭ions. 版Pl

30、astic笆 luer-l鞍ock sto啊pcocks.靶 Stopco懊cks loc鞍k into 艾the out叭let val奥ves. 盎Plastic爱 aquari稗um tubi昂ng, 3/1哎6 inne吧r diame澳ter 爸Nupro S疤-series艾 fine m芭etering百 valves耙 with 1绊/8 Swa版ge-lok 耙compres俺sion fi哎ttings 版and 1/8安 teflo摆n or po艾ly-flo 办tubing.般 Two to啊 four i奥nch lon邦g piece昂s of 3/癌16 inn

31、绊er-diam袄eter aq案uarium 绊tubing 傲(equal 班to numb澳er of v岸alves).板 See se盎cts. D-巴3 & F-2白 for in佰structi白ons on 芭operati般ng and 败assembl碍ing thi熬s bubbl碍er unit八. These岸 compon翱ents ar袄e avail按able fr把om loca埃l valve搬 suppli扒ers or 哎contact哀 Indian芭apolis 巴Valve a奥nd Fitt俺ing Co,靶 Indian肮apolis,捌 IN

32、; 31斑7-248-2凹468, fo挨r infor碍mation 叭on the 懊nearest胺 suppli捌er. 熬Enrichm安ent bro鞍th cont佰ainer (拌use eit板her): 八400 ml 叭or larg啊er stom败acher o佰r stoma袄cher fi般lter ba懊gs, twi案st ties背 and st癌ainless岸 steel 氨baskets俺 啊250 or 霸500 ml 芭Erlenme办yer fla扮sks, fo巴am plug挨ged wit鞍h foil 瓣wrap an唉d steri稗l

33、ized. 办Two-inc吧h sq Pa伴rafilm 跋pieces 把and wei搬ghted r爸ings or唉 a plat澳form wi奥th clam奥ps. For按 instru扳ctions 暗for ass稗embly a扳nd use,巴 see fi霸g. 2 an耙d BAM, 胺7th ed.爸, 1992,芭 chapt.阿 7. 巴Plastic芭 1 ml s俺terile 袄pipets.霸 奥Water a袄nalysis班 appara拔tus 哎Zetapor拜 filter奥s, 45 靶m (Cuno熬, 案Meriden扮, 暗CT笆,

34、 800-2澳43-6894白; no su安bstitut扮ions), 吧47吧-293 mm耙 (depen拌ding on翱 filter瓣ing uni哎t size)盎, autoc翱laved s啊eparate版ly from佰 filter岸ing uni啊t 稗For 埃47 mm暗 filter安 appara般tus: 佰Teflon-唉faced b埃orosili扳cate gl俺ass 霸47 mm隘 holder哎(s) and昂 filter叭 clamp 半forceps哀, steri吧lized 蔼Manifol板d, 6-12俺 place,盎 for

35、 mu版ltiple 艾subsamp熬les 伴Vacuum 翱flask, 邦1叭-4 L爱, and i把f a man哎ifold i颁s used,案 a rubb岸er stop唉per wit阿h a 6 t版o 8-inc岸h plast坝ic tube扮 insert翱ed and 巴a hose 奥connect蔼ing the蔼 plasti凹c tube 爸to the 般manifol叭d. Anot盎her hos俺e conne澳cts the矮 flask 拌side ar百m to a 笆vacuum 艾source.胺 靶For 90,鞍 142, o靶r

36、俺293 mm疤 filter敖 appara叭tus 凹90, 142半, or 昂293 mm摆 filter按ing uni把t with 百3 ft凹 hose a背ttached佰 at top肮, steri办lized 昂Vacuum 挨flask, 柏4斑-6 L盎, set u板p as in拜 b-3 ab耙ove, ex懊cept th胺e hose 颁attache啊d to th碍e vacuu把m flask袄s stop岸per tub靶e conne懊cts to 芭the fil蔼tering 傲unit ou颁tlet po安rt HYPERLINK /Fo

37、od/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm055778.htm 按Media皑2哎, bioch碍emicals坝 and HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm055791.htm 疤reagent颁s扮3班 (secti按on G, e跋xcept w瓣here ot唉herwise胺 indica巴ted) Media 笆Campylo绊bact

38、er 背Enrichm翱ent Bro版th (Bol阿ton for昂mula, O按xoid AM肮7526 or板 Malthu半s Diagn疤ostics 袄LAB-135邦, Malth般us Diag稗nostics斑, 八North R稗idgevil吧le笆, 熬OH矮; 216-3安27-2585澳) with 案lysed h摆orse bl跋ood and埃 antibi阿otic su吧pplemen翱t (Oxoi扳d NDX13般1 or Ma八lthus D隘iagnost坝ics X13碍1). Alt邦ernativ板ely, an扒tibioti背c su

39、ppl岸ement m哎ay be p矮repared佰 from i碍ndividu盎al comp鞍onents 伴(G-1). HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm063368.htm 背M28a斑4皑 把Campylo扮bacter邦 isolat岸ion aga柏rs (use傲 either按) 绊Abeyta-捌Hunt-Ba扮rk (AHB瓣) agar 拌(G-2) HYPERLINK /Food/ScienceResearch/Labo

40、ratoryMethods/BacteriologicalAnalyticalManualBAM/ucm061209.htm 胺M29a颁5把 坝Modifie澳d Campy胺 blood-懊free ag疤ar (mCC凹DA)(G-2颁) HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm064002.htm 敖M30a懊6皑 俺Abeyta-唉Hunt-Ba奥rk agar柏 (G-2),岸 withou斑t antib柏iotics 扮Heart i班nfus

41、ion岸 agar (唉HIA) sl皑ants ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm064330.htm 岸M59颁7碍) 傲0.1% Pe半ptone d埃iluent 岸( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm062265.htm 疤R56熬8俺) 矮Semi-so搬lid med绊ium, mo把difie

42、d,矮 for 阿biochem懊ical id瓣entific凹ation稗 (G-5) 挨Neutral拜 red (N爸R) solu安tion, g懊lycine,拌 NaCl, 盎cystein皑e HCl, 胺KNO耙3暗 扮Triple 隘sugar i蔼ron (TS绊I) agar疤 slants爱 ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm063699.htm 阿M149板9鞍) 绊O-F glu芭cose ( HYPERLINK /Fo

43、od/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm064230.htm 版M116八10癌), modi挨fied; p邦repare 拜half th伴e tubes阿 with g胺lucose 柏and hal傲f witho阿ut. 芭MacConk扮ey agar斑 ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm064496.htm 笆M91坝11搬)

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46、calAnalyticalManualBAM/ucm064309.htm 百M30b颁14懊) 稗Biochem板icals a罢nd HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm055791.htm 昂reagent芭s百15笆 矮Hippura哎te ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm062230.htm 暗R33背

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