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1、BAM: SaalmoneellaNOTICE:If you are llookinng forr BAM Chaptter 5: Salmmonellla (Deecembeer 20007 Ediition) thatt is iincorpporateed by referrence in 211 CFR Partss 16 aand 1118: Feederall Regiister Finall Rulee1 (Jully 9, 2009, 74 FFR 330030): Preveentionn of SSalmonnella Enterritidiis in Shelll Egg
2、ss Duriing Prroducttion, Storaage, aand Trranspoortatiion, ppleasee use theseeverssions of thhe BAMMSalmmonellla Chaapter22 (PDFF, 1899 Kb)andAAppenddix 1: Rapiid Metthods for DDetectting FFoodboorne PPathoggens3 (PDFF, 1955 Kb).The mosst reccentEEditioon of BAM CChapteer 5: Salmoonellaa is aavailaa
3、ble bbelow this noticce.Novembeer 20111 VerrsionBacteriiologiical AAnalyttical ManuaalChappter 55SalmoonellaaAuthorss: Walllace H. Anndrewss and Thomaas HammmackRevisioon Hisstory: Novembeer 20111 - AAdditiion too Secttion CC: Preeparattion oof fooods foor isoolatioon of Salmoonellaa: Leaafy grreen
4、vvegetaables and hherbs. Februarry 20111 Removved liink too Appeendix 1: Raapid MMethodds forr Deteectingg Fooddbornee Pathhogenss (noww archhived). Decembeer 20007 MMamey pulp methood addded, aand Seectionn D reevisedd. June 20006 Eggs methood revvised for sshell eggs and lliquidd wholle egggs. Apr
5、il 22003 Frogg legss methhod, LLacticc caseein, RRennett caseein, SSodiumm caseeinatee and Rabbiit carrcass methoods reevisedd, topp earss and otherr dog chew toys addedd. Remmoved sectiion A.25, MMechannical shakeer. Octoberr 25, 2001 Exttensioon of the aappliccabiliity off the orangge juiice meeth
6、od in seectionn C.199 to aapple juicee and applee cideer. 1999-DEEC, 20000-MAAR, annd 20000-AUGG Finaal revvisionn on 22000-NNOV-144 (seee the Introoductiion foor a ssummarry of changges).To obtaain a copy of a priorr verssion nnot cuurrenttly poosted, pleaase coontactt Thommas HaammackkChapterr Con
7、ttents Introduuctionn Equipmeent annd Matterialls Media aand Reeagentts Preparaation of fooods ffor issolatiion off Salmmonellla Isolatiion off Salmmonellla Identifficatiion off Salmmonellla ReferenncesIntroduuctionnSeverall channges aare beeing iintrodduced in thhis edditionn of BBAM (88th Ediition
8、). Thee firsst chaange iinvolvves thhe exppandedd use of Raappapoort-Vaassiliiadis (RV) mediuum4 for foodss withh bothh highh and low llevelss of ccompettitivee micrroflorra. Inn the previious eeditioon, RVV mediium waas reccommennded oonly ffor thhe anaalysiss of sshrimpp. Bassed onn the complletio
9、nn of AAOAC pprecolllaborrativee (5, 6) annd colllaborrativee (7, 8) sttudiess, RV mediuum is now bbeing recommmendeed forr the analyysis oof higgh miccrobiaal andd low microobial load foodss. RV mediuum repplacess seleenite cystiine (SSC) brroth ffor thhe anaalysiss of aall fooods, exceppt guaar gu
10、mm. In addittion, RV meedium replaaces llauryll trypptose brothh for use wwith ddry acctive yeastt. Tettrathiionatee (TT)5 brotth conntinuees to be ussed ass the seconnd sellectivve enrrichmeent brroth. Howevver, TTT brooth iss to bbe inccubateed at 43C ffor thhe anaalysiss of hhigh mmicrobbial lloa
11、d ffoods and aat 35C forr the analyysis oof loww micrrobiall loadd foodds, inncludiing guuar guum.The seccond cchangee invoolves the ooptionn of rrefriggeratiing inncubatted prreenriichmennts annd sellectivve enrrichmeents oof loww-moissture foodss for up too 72 hh. Witth thiis opttion, samplle anaa
12、lysess can be innitiatted ass latee as WWednessday oor Thuursdayy withhout wweekennd worrk beiing innvolveed.The thiird chhange invollves rreduciing thhe perriod oof inccubatiion off the lysinne iroon agaar (LIIA)6 slannts. IIn thee formmer edditionn (BAMM-7), triplle suggar irron aggar (TTSI)7 and
13、LIA sslantss weree incuubatedd at 335C ffor 244 2 h andd 48 2 h, resppectivvely. Unpubblisheed datta havve demmonstrrated that the 448 h rreadinng of LIA sslantss is wwithouut diaagnosttic vaalue. Of 1993 LIAA slannts exxamineed, alll gavve deffinitiive reesultss withhin 244 2 h of incubbationn. No
14、signiificannt chaanges alterred thhe finnal teest reesult when the sslantss weree incuubatedd an aadditiional 24 h. Thuss, botth thee TSI and LLIA sllants are nnow inncubatted foor 24 2 hh.The fouurth cchangee invoolves the pproceddure ffor suurfacee disiinfecttion oof sheell egggs. IIn thee prevvio
15、us editiion (BBAM-7), eggg shellls weere suurfacee-disiinfectted byy soakking iin 0.11% merrcuricc chlooride soluttion ffor 1 h folllowedd by ssoakinng in 70% eethanool forr 30 mmin. MMercurric chhloridde is classsifiedd as aa hazaardouss wastte, annd is expennsive to diisposee of aaccordding tto En
16、vvironmmentall Prottectioon Ageency gguidellines. In tthis eeditioon (BAAM-8) egg sshellss are now ssurfacce-dissinfeccted bby soaaking for aat leaast 100 sec in a 3:1 ssolutiion coonsistting oof 3 pparts of 700% alccohol (ethyyl or isoprropyl) to 11 partt of iiodinee/potaassiumm iodiide soolutioon.
17、The fiffth chhange invollves tthe saample prepaaratioon of eggs. Egg conteents (yolk and aalbumeen) arre thooroughhly miixed bbeforee anallysis. Afteer mixxing tthe eggg conntentss, 25 g (mll) aree addeed to 225 mml tryypticaase (ttryptiic) sooy brooth suupplemmentedd withh ferrrous ssulfatte.A meth
18、ood forr the analyysis oof guaar gumm has been incluuded. When guar gum iis preeenricched aat a 11:9 saample/brothh ratiio, a highlly visscous, nonppipetttable mixtuure reesultss. Addditionn of tthe ennzyme celluulase to thhe preeenricchmentt mediium, hhoweveer, reesultss in aa readdily ppipetttable
19、 mixtuure.A methood forr orannge juuice (pasteeurizeed andd unpaasteurrized) has been incluuded ddue too receent orrange juicee-relaated ooutbreeaks.The dirrectioons foor piccking colonnies ffrom tthe seelectiive pllatingg agarrs havve beeen madde morre expplicitt to rreflecct thee inteent off the m
20、ethood. Inn the absennce off typiical oor susspect colonnies oon thee seleectivee platting aagars, it iis reccommennded tthat aatypiccal cooloniees be pickeed to TSI aand LIIA slaants. This recommmendaation is baased oon thee factt thatt up tto 4% of alll Sallmonellla cuulturees isoolatedd by FFDA a
21、nnalystts froom cerrtain foodss, esppeciallly seeafoodds, duuring the ppast sseveraal yeaars haave beeen attypicaal.Finallyy, sinnce thhe pubblicattion oof BAMM-7, aa 6-waay commparisson waas connducteed of the rrelatiive efffectiivenesss of the tthree selecctive platiing aggars rrecommmendedd in tt
22、he BAAM (biismuthh sulffite8, Hekttoen eenteriic9, andd xyloose lyysine desoxxychollate aagars110) annd thrree reelativvely nnew aggars (EF-188, xyllose llysinee Terggitol 4, annd Rammbach agarss). Ouur ressults (9) iindicaated nno advvantagge in replaacing any oof thee BAM-recommmendeed agaars wiit
23、h onne or more of thhe newwer aggars. Thus, the combiinatioon of selecctive platiing aggars rrecommmendedd in BBAM-7 remaiins unnchangged.A. Equipmeent annd Matterialls 1. Blenderr and steriile bllenderr jarss (seee Chaptter 1111) 2. Sterilee, 16 oz (5500 mll) widde-mouuth, sscrew-cap jjars, steriil
24、e 5000 ml Erlennmeyerr flassks, ssterille 2500 ml bbeakerrs, stterilee glasss or paperr funnnels oof apppropriiate ssize, and, optioonallyy, conntaineers off apprropriaate caapacitty to accommmodatte commpositted saampless 3. Sterilee, bennt glaass orr plasstic sspreadder roods 4. Balancee, witth we
25、iights; 20000 g caapacitty, seensitiivity of 0.1 g 5. Balancee, witth weiights; 120 g cappacityy, sennsitivvity oof 5 mmg 6. Incubattor, 335 22 C 7. Refrigeeratedd incuubatorr or llaboraatory refriigerattor, 44 2C 8. Water bbath, 49 1C 9. Water bbath, circuulatinng, thhermosstaticcally-contrrolledd,
26、 43 0.22C 10. Water bbath, circuulatinng, thhermosstaticcally-contrrolledd,42 0.2C 11. Sterilee spooons orr otheer apppropriiate iinstruumentss for transsferriing foood saampless 12. Sterilee cultture ddishess, 15 x 1000 mm, glasss or pplastiic 13. Sterilee pipeets, 11 ml, with 0.01 ml grraduattions
27、; 5 annd 10 ml, wwith 00.1 mll gradduatioons 14. Inoculaating needlle andd inocculatiing looop (aabout 3 mm iid or 10 5ll), niichromme, pllatinuum-iriidium, chroomel wwire, or stterilee plasstic 15. Sterilee testt or cculturre tubbes, 116 x 1150 mmm and 20 x 150 mmm; seerologgical tubess, 10 x 75 mm
28、 orr 13 xx 100 mm 16. Test orr cultture ttube rracks 17. Vortex mixerr 18. Sterilee sheaars, llarge scisssors, scalppel, aand foorcepss 19. Lamp (ffor obbserviing seerologgical reacttions) 20. Fisher or Buunsen burneer 21. pH testt papeer (pHH rangge 6-88) witth maxximum graduuationns of 0.4 ppH uni
29、its peer collor chhange 22. pH meteer 23. Plastiic baggs, 288 x 377 cm, steriile, wwith rreseallable tape. (Iteems 233-24 aare neeeded in thhe anaalysiss of ffrog llegs aand raabbit carcaasses.) 24. Plasticc beakkers, 4 litter, aautocllavablle, foor hollding plasttic baag durring sshakinng andd incu
30、ubatioon. 25. Spongess, nonn-bacttericiidal (Nascoo cat # B011299WAA), orr equiivalennt. 26. Swabs, non-bbacterricidaal, cootton-tippeed.B. Media112 andd Reaggents113For preeparattion oof meddia annd reaagentss, reffer too Methhods 9967.255-967.28 inn Officcial MMethodds of Analyysis (1).1. Lactosee
31、 brotth (M77414) 2. Nonfat dry mmilk (reconnstituuted) (M111115) 3. Selenitte cysstine (SC) brothh (M133416) 4. Tetrathhionatte (TTT) brooth (MM145177) 5. Rappapoort-Vaassiliiadis (RV) mediuum (M113218). NNOTE: RV meedium must be maade frrom itts inddividuual inngrediients. Commmerciaal forrmulattio
32、ns are nnot accceptaable. 6. Xylose lysinne dessoxychholatee (XLDD) agaar (M117919) 7. Hektoenn enteeric (HE) aagar (M6120) 8. Bismuthh sulffite (BS) aagar (M1921) 9. Triple sugarr ironn agarr (TSII) (M114922) 10. Tryptonne (trryptopphane) brotth (M116423) 11. Trypticcase (trypttic) ssoy brroth (M15
33、4244) 12. Trypticcase ssoy brroth wwith fferrouus sullfate (M186625) 13. Trypticcase ssoy-trryptosse brooth (MM160266) 14. MR-VP bbroth (M104427) 15. Simmonss citrrate aagar (M138288) 16. Urea brroth (M171299) 17. Urea brroth (rapidd) (M117230) 18. Malonatte brooth (MM9231) 19. Lysine iron agar (LIA
34、) (Edwwards and FFife) (M8932) 20. Lysine decarrboxyllase bbroth (M87)33 21. Motilitty tesst meddium (semissolid) (M100334) 22. Potassiium cyyanidee (KCNN) brooth (MM126355) 23. Phenol red ccarbohhydratte brooth (MM121366) 24. Purple carboohydraate brroth (M130377) 25. MacConkkey aggar (MM9138) 26.
35、Nutriennt brooth (MM114399) 27. Brain hheart infussion (BHI) brothh (M24440) 28. Papain soluttion, 5% (MM56a411) 29. Cellulaase soolutioon, 1% (M188742) 30. Tryptosse bloood aggar baase (MM166433) 31. Universsal prreenriichmennt brooth (MM188444) 32. Universsal prreenriichmennt brooth (wwithouut fer
36、rric aammoniium ciitratee) (M1188a455) 33. Buffereed pepptone waterr (M199246) 34. Dey-Enggley bbroth (M193347) 35. Potassiium suulfitee powdder, aanhydrrous 36. Chlorinne sollutionn, 2000 ppm, conttaininng 0.11% soddium ddodecyyl sullfate (R12aa48) 37. Ethanoll, 70% (R23349) 38. Kovacs reaggent (R3
37、850) 39. Voges-PProskaauer (VP) ttest rreagennts (RR8951) 40. Creatinne phoosphatte cryystalss 41. Potassiium hyydroxiide soolutioon, 400% (R66552) 42. 1 N Soddium hhydroxxide ssolutiion (RR7353) 43. 1 N Hyddrochlloric acid (R3654) 44. Brilliaant grreen ddye soolutioon, 1% (R855) 45. Bromcreesol ppu
38、rplee dye soluttion, 0.2% (R956) 46. Methyl red iindicaator (R4457) 47. Sterilee disttilledd wateer 48. Tergitool Aniionic 7 (R77858) 49. Triton X-1000 (R86659) 50. Physiollogicaal salline ssolutiion, 00.85% (sterrile) (R6360) 51. Formaliinizedd physsiologgical salinne sollutionn (R27761) 52. Salmon
39、eella ppolyvaalent somattic (OO) anttiseruum 53. Salmoneella ppolyvaalent flageellar (H) aantiseerum 54. Salmoneella ssomatiic grooup (OO) anttiseraa: A, B, C11, C2, C3, D1, D2, E1, E2, E3, E4, F, G, H, I, VVi, annd othher grroups, as aapproppriatee 55. Salmoneella SSpicerr-Edwaards fflagelllar (H)
40、anntiserraC. Preparaation of fooods ffor issolatiion off SalmmonelllaThe folllowinng metthods are bbased on thhe anaalysiss of aa 25 gg anallyticaal uniit at a 1:99 sampple/brroth rratio. Depeendingg on tthe exxtent of coomposiiting, add enouggh brooth too mainntain this 1:9 rratio unlesss othherwis
41、se inddicateed. Foor sammples not aanalyzzed onn an eexact weighht bassis, ee.g., frog legs, refeer to the sspeciffic meethod for iinstruuctionns.1. Dried eegg yoolk, ddried egg wwhitess, driied whhole eeggs, liquiid millk (skkim miilk, 22% fatt milkk, whoole, aand buuttermmilk), and prepaared ppowd
42、erred miixes (cake, cookkie, ddoughnnut, bbiscuiit, annd breead), infannt forrmula, and oral or tuube feeedinggs conntainiing eggg.Preferaably, do noot thaaw froozen ssamplees beffore aanalyssis. IIf froozen ssamplee mustt be ttemperred too obtaain annalytiical pportioon, thhaw suuitablle porrtion a
43、s raapidlyy as ppossibble too miniimize increease iin nummber oof commpetinng orgganismms or to reeduce potenntial of innjurinng Sallmonellla orrganissms. TThaw bbelow 45C ffor 155 min with contiinuouss agittationn in tthermoostatiicallyy conttrolleed watter baath orr thaww withhin 188 h att 2-5C. A
44、ssepticcally weighh 25 gg sampple innto stterilee, widde-mouuth, sscrew-cap jjar (5500 mll) or otherr apprropriaate coontainner. FFor noonpowddered samplles, aadd 2225 ml steriile laactosee brotth65. Iff prodduct iis powwderedd, addd abouut 15 ml stterilee lacttose bbroth and sstir wwith ssterille g
45、laass rood, sppoon, or toongue depreessor to smmooth suspeensionn. Addd 3 addditioonal pportioons off lacttose bbroth, 10, 10, aand 1990 ml, for totall of 2225 mll. Stiir thooroughhly unntil ssamplee is ssuspennded wwithouut lummps. CCap jaar seccurelyy and let sstand 60 5 minn at rroom ttemperratur
46、ee. Mixx welll by sswirliing annd detterminne pH with test paperr. Adjjust ppH, iff neceessaryy, to 6.8 0.2 with steriile 1 N NaOOH or 1 N HHCl. CCap jaar seccurelyy and mix wwell bbeforee deteerminiing fiinal ppH. Looosen jar ccap abbout 11/4 tuurn annd inccubatee 24 2 h at 355C. CContinnue ass in
47、DD, 1-111 , bbelow.2. Eggs a. Shell eeggs. Removve anyy adheerent materrial ffrom tthe shhell ssurfacce. Diisinfeect egggs wiith 3:1 sollutionn conssistinng of 3 parrts off 70% alcohhol (eethyl or issoproppyl) tto 1 ppart iiodinee/potaassiumm iodiide soolutioon. Prreparee 70% alcohhol soolutioon eitther bby dillutingg 700 ml 1000% allcoholl withh sterrile ddistillled wwater for aa finaal vollume oof 1,0000 mll