BAM:SalmonellaNOTICE:IfyouarelookingforBAMChapter5:Salmonella(December2007Edition)thatisincorporatedbyreferencein21CFRParts16and118:Federal Register Final Rule1(July9,2009,74FR33030):PreventionofSalmonellaEnteritidisinShellEggsDuringProduction,Storage,andTransportation,pleaseusetheseversionsoftheBAMSalmonella Chapter2(PDF,189Kb)andAppendix 1: Rapid Methods for Detecting Foodborne Pathogens3(PDF,195Kb).ThemostrecentEditionofBAMChapter5:Salmonellaisavailablebelowthisnotice.November2011VersionBacteriologicalAnalyticalManualChapter5SalmonellaAuthors:WallaceH.AndrewsandThomas HammackRevisionHistory: November2011-AdditiontoSectionC:PreparationoffoodsforisolationofSalmonella:Leafygreenvegetablesandherbs. February2011RemovedlinktoAppendix1:RapidMethodsforDetectingFoodbornePathogens(nowarchived). December2007Mameypulpmethodadded,andSectionDrevised. June2006Eggsmethodrevisedforshelleggsandliquidwholeeggs. April2003Froglegsmethod,Lacticcasein,Rennetcasein,SodiumcaseinateandRabbitcarcassmethodsrevised,topearsandotherdogchewtoysadded.RemovedsectionA.25,Mechanicalshaker. October25,2001ExtensionoftheapplicabilityoftheorangejuicemethodinsectionC.19toapplejuiceandapplecider. 1999-DEC,2000-MAR,and2000-AUGFinalrevisionon2000-NOV-14(seetheIntroductionforasummaryofchanges).Toobtainacopyofapriorversionnotcurrentlyposted,pleasecontactThomas HammackChapterContents Introduction Equipment and Materials Media and Reagents Preparation of foods for isolation of Salmonella Isolation of Salmonella Identification of Salmonella ReferencesIntroductionSeveralchangesarebeingintroducedinthiseditionofBAM(8thEdition).ThefirstchangeinvolvestheexpandeduseofRappaport-Vassiliadis (RV) medium4forfoodswithbothhighandlowlevelsofcompetitivemicroflora.Inthepreviousedition,RVmediumwasrecommendedonlyfortheanalysisofshrimp.BasedonthecompletionofAOACprecollaborative(5, 6)andcollaborative(7, 8)studies,RVmediumisnowbeingrecommendedfortheanalysisofhighmicrobialandlowmicrobialloadfoods.RVmediumreplacesselenitecystine(SC)brothfortheanalysisofallfoods,exceptguargum.Inaddition,RVmediumreplaceslauryltryptosebrothforusewithdryactiveyeast.Tetrathionate (TT)5brothcontinuestobeusedasthesecondselectiveenrichmentbroth.However,TTbrothistobeincubatedat43Cfortheanalysisofhighmicrobialloadfoodsandat35Cfortheanalysisoflowmicrobialloadfoods,includingguargum.Thesecondchangeinvolvestheoptionofrefrigeratingincubatedpreenrichmentsandselectiveenrichmentsoflow-moisturefoodsforupto72h.Withthisoption,sampleanalysescanbeinitiatedaslateasWednesdayorThursdaywithoutweekendworkbeinginvolved.Thethirdchangeinvolvesreducingtheperiodofincubationofthelysine iron agar (LIA)6slants.Intheformeredition(BAM-7),triple sugar iron agar (TSI)7andLIAslantswereincubatedat35Cfor242hand482h,respectively.Unpublisheddatahavedemonstratedthatthe48hreadingofLIAslantsiswithoutdiagnosticvalue.Of193LIAslantsexamined,allgavedefinitiveresultswithin242hofincubation.Nosignificantchangesalteredthefinaltestresultwhentheslantswereincubatedanadditional24h.Thus,boththeTSIandLIAslantsarenowincubatedfor242h.Thefourthchangeinvolvestheprocedureforsurfacedisinfectionofshelleggs.Inthepreviousedition(BAM-7),eggshellsweresurface-disinfectedbysoakingin0.1%mercuricchloridesolutionfor1hfollowedbysoakingin70%ethanolfor30min.Mercuricchlorideisclassifiedasahazardouswaste,andisexpensivetodisposeofaccordingtoEnvironmentalProtectionAgencyguidelines.Inthisedition(BAM-8)eggshellsarenowsurface-disinfectedbysoakingforatleast10secina3:1solutionconsistingof3partsof70%alcohol(ethylorisopropyl)to1partofiodine/potassiumiodidesolution.Thefifthchangeinvolvesthesamplepreparationofeggs.Eggcontents(yolkandalbumen)arethoroughlymixedbeforeanalysis.Aftermixingtheeggcontents,25g(ml)areaddedto225mltrypticase(tryptic)soybrothsupplementedwithferroussulfate.Amethodfortheanalysisofguargumhasbeenincluded.Whenguargumispreenrichedata1:9sample/brothratio,ahighlyviscous,nonpipettablemixtureresults.Additionoftheenzymecellulasetothepreenrichmentmedium,however,resultsinareadilypipettablemixture.Amethodfororangejuice(pasteurizedandunpasteurized)hasbeenincludedduetorecentorangejuice-relatedoutbreaks.Thedirectionsforpickingcoloniesfromtheselectiveplatingagarshavebeenmademoreexplicittoreflecttheintentofthemethod.Intheabsenceoftypicalorsuspectcoloniesontheselectiveplatingagars,itisrecommendedthatatypicalcoloniesbepickedtoTSIandLIAslants.Thisrecommendationisbasedonthefactthatupto4%ofallSalmonellaculturesisolatedbyFDAanalystsfromcertainfoods,especiallyseafoods,duringthepastseveralyearshavebeenatypical.Finally,sincethepublicationofBAM-7,a6-waycomparisonwasconductedoftherelativeeffectivenessofthethreeselectiveplatingagarsrecommendedintheBAM(bismuth sulfite8,Hektoen enteric9,andxylose lysine desoxycholate agars10)andthreerelativelynewagars(EF-18,xyloselysineTergitol4,andRambachagars).Ourresults(9)indicatednoadvantageinreplacinganyoftheBAM-recommendedagarswithoneormoreoftheneweragars.Th