14/03/2016 LaboratoryMethods>BAM:<i>Salmonella</i>

U.S.FoodandDrugAdministration
ProtectingandPromotingYourHealth

BAM:Salmonella
NOTICE:
IfyouarelookingforBAMChapter5:Salmonella(December2007Edition)
thatisincorporatedbyreferencein21CFRParts16and118:Federal
RegisterFinalRule
(/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/Eg
gs/ucm170615.htm)(July9,2009,74FR33030):PreventionofSalmonella
EnteritidisinShellEggsDuringProduction,Storage,andTransportation,
pleaseusetheseversionsoftheBAMSalmonellaChapter
(/downloads/Food/FoodScienceResearch/UCM244774.pdf)(PDF,189
Kb)andAppendix1:RapidMethodsforDetectingFoodbornePathogens
(/downloads/Food/FoodScienceResearch/UCM244777.pdf)(PDF,195Kb).
These2documentsarealsoavailableasacombinedfile
(/downloads/Food/FoodScienceResearch/UCM309839.pdf)(PDF,382Kb).

ThemostrecentEditionofBAMChapter5:Salmonellaisavailablebelow
thisnotice.

May2014Version

BacteriologicalAnalyticalManual
Chapter5
Salmonella

Authors:WallaceH.Andrews,AndrewJacobson,andThomasHammack
(mailto:Thomas.Hammack@fda.hhs.gov)

RevisionHistory:

December2015AsectionfortheStatensSerumInstituteProcedurewasaddedtoSectionE:
IdentificationofSalmonella.
May2014TheVitekmethodofPresumptivegenericidentificationofSalmonellawasupdated.
February2014SectiononDetectionandisolationofSalmonellafromshelleggswasreplaced,and
validationdataandadditonalreferenceswereaddedinanAppendix.
August2012,November2011MadeavailableinPDFformatversionsofChapter5:Salmonellaand
Appendix1(archived)from2009whichwereincorporatedbyreferencein21CFRParts16and118:
FederalRegisterFinalRule(July9,2009,74FR33030):PreventionofSalmonellaEnteritidisinShell

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14/03/2016 LaboratoryMethods>BAM:<i>Salmonella</i>

EggsDuringProduction,Storage,andTransportation.
November2011AdditiontoSectionC:PreparationoffoodsforisolationofSalmonella:Leafygreen
vegetablesandherbs.
February2011RemovedlinktoAppendix1:RapidMethodsforDetectingFoodbornePathogens
(nowarchived).
December2007Mameypulpmethodadded,andSectionDrevised.
June2006Eggsmethodrevisedforshelleggsandliquidwholeeggs.
April2003Froglegsmethod,Lacticcasein,Rennetcasein,SodiumcaseinateandRabbitcarcass
methodsrevised,topearsandotherdogchewtoysadded.RemovedsectionA.25,Mechanicalshaker.
October25,2001ExtensionoftheapplicabilityoftheorangejuicemethodinsectionC.19toapple
juiceandapplecider.
1999DEC,2000MAR,and2000AUGFinalrevisionon2000NOV14(seetheIntroductionfora
summaryofchanges).

Toobtainacopyofapriorversionnotcurrentlyposted,pleasecontactThomasHammack
(mailto:Thomas.Hammack@fda.hhs.gov)

ChapterContents
Introduction
EquipmentandMaterials
MediaandReagents
PreparationoffoodsforisolationofSalmonella
IsolationofSalmonella
IdentificationofSalmonella
References

Introduction
SeveralchangesarebeingintroducedinthiseditionofBAM(8thEdition).Thefirstchangeinvolvesthe
expandeduseofRappaportVassiliadis(RV)medium
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063568.htm)forfoodswithbothhighandlow
levelsofcompetitivemicroflora.Inthepreviousedition,RVmediumwasrecommendedonlyforthe
analysisofshrimp.BasedonthecompletionofAOACprecollaborative(5,6)andcollaborative(7,8)
studies,RVmediumisnowbeingrecommendedfortheanalysisofhighmicrobialandlowmicrobialload
foods.RVmediumreplacesselenitecystine(SC)brothfortheanalysisofallfoods,exceptguargum.In
addition,RVmediumreplaceslauryltryptosebrothforusewithdryactiveyeast.Tetrathionate(TT)
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063686.htm)brothcontinuestobeusedasthe
secondselectiveenrichmentbroth.However,TTbrothistobeincubatedat43Cfortheanalysisofhigh
microbialloadfoodsandat35Cfortheanalysisoflowmicrobialloadfoods,includingguargum.

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Thesecondchangeinvolvestheoptionofrefrigeratingincubatedpreenrichmentsandselective
enrichmentsoflowmoisturefoodsforupto72h.Withthisoption,sampleanalysescanbeinitiatedaslate
asWednesdayorThursdaywithoutweekendworkbeinginvolved.

Thethirdchangeinvolvesreducingtheperiodofincubationofthelysineironagar(LIA)
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064484.htm)slants.Intheformeredition
(BAM7),triplesugarironagar(TSI)
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063699.htm)andLIAslantswereincubatedat
35Cfor242hand482h,respectively.Unpublisheddatahavedemonstratedthatthe48hreadingof
LIAslantsiswithoutdiagnosticvalue.Of193LIAslantsexamined,allgavedefinitiveresultswithin242h
ofincubation.Nosignificantchangesalteredthefinaltestresultwhentheslantswereincubatedan
additional24h.Thus,boththeTSIandLIAslantsarenowincubatedfor242h.

Thefourthchangeinvolvestheprocedureforsurfacedisinfectionofshelleggs.Inthepreviousedition
(BAM7),eggshellsweresurfacedisinfectedbysoakingin0.1%mercuricchloridesolutionfor1hfollowed
bysoakingin70%ethanolfor30min.Mercuricchlorideisclassifiedasahazardouswaste,andis
expensivetodisposeofaccordingtoEnvironmentalProtectionAgencyguidelines.Inthisedition(BAM8)
eggshellsarenowsurfacedisinfectedbysoakingforatleast10secina3:1solutionconsistingof3parts
of70%alcohol(ethylorisopropyl)to1partofiodine/potassiumiodidesolution.

Thefifthchangeinvolvesthesamplepreparationofeggs.Eggcontents(yolkandalbumen)arethoroughly
mixedbeforeanalysis.Aftermixingtheeggcontents,25g(ml)areaddedto225mltrypticase(tryptic)soy
brothsupplementedwithferroussulfate.

Amethodfortheanalysisofguargumhasbeenincluded.Whenguargumispreenrichedata1:9
sample/brothratio,ahighlyviscous,nonpipettablemixtureresults.Additionoftheenzymecellulasetothe
preenrichmentmedium,however,resultsinareadilypipettablemixture.

Amethodfororangejuice(pasteurizedandunpasteurized)hasbeenincludedduetorecentorangejuice
relatedoutbreaks.

Thedirectionsforpickingcoloniesfromtheselectiveplatingagarshavebeenmademoreexplicittoreflect
theintentofthemethod.Intheabsenceoftypicalorsuspectcoloniesontheselectiveplatingagars,itis
recommendedthatatypicalcoloniesbepickedtoTSIandLIAslants.Thisrecommendationisbasedonthe
factthatupto4%ofallSalmonellaculturesisolatedbyFDAanalystsfromcertainfoods,especially
seafoods,duringthepastseveralyearshavebeenatypical.

Finally,sincethepublicationofBAM7,a6waycomparisonwasconductedoftherelativeeffectivenessof
thethreeselectiveplatingagarsrecommendedintheBAM(bismuthsulfite
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063348.htm),Hektoenenteric
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064342.htm),andxyloselysine
desoxycholateagars(/Food/FoodScienceResearch/LaboratoryMethods/ucm062986.htm))and
threerelativelynewagars(EF18,xyloselysineTergitol4,andRambachagars).Ourresults(9)indicated
noadvantageinreplacinganyoftheBAMrecommendedagarswithoneormoreoftheneweragars.
Thus,thecombinationofselectiveplatingagarsrecommendedinBAM7remainsunchanged.

ReturntoChapterContents

A.EquipmentandMaterials

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1.Blenderandsterileblenderjars(seeBAMChapter1
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063335.htm))
2.Sterile,16oz(500ml)widemouth,screwcapjars,sterile500mlErlenmeyerflasks,sterile250
mlbeakers,sterileglassorpaperfunnelsofappropriatesize,and,optionally,containersof
appropriatecapacitytoaccommodatecompositedsamples
3.Sterile,bentglassorplasticspreaderrods
4.Balance,withweights2000gcapacity,sensitivityof0.1g
5.Balance,withweights120gcapacity,sensitivityof5mg
6.Incubator,352C
7.Refrigeratedincubatororlaboratoryrefrigerator,42C
8.Waterbath,491C
9.Waterbath,circulating,thermostaticallycontrolled,430.2C
10.Waterbath,circulating,thermostaticallycontrolled,420.2C
11.Sterilespoonsorotherappropriateinstrumentsfortransferringfoodsamples
12.Sterileculturedishes,15100mm,glassorplastic
13.Sterilepipets,1ml,with0.01mlgraduations5and10ml,with0.1mlgraduations
14.Inoculatingneedleandinoculatingloop(about3mmidor105l),nichrome,platinumiridium,
chromelwire,orsterileplastic
15.Steriletestorculturetubes,16150mmand20150mmserologicaltubes,1075mmor13
100mm
16.Testorculturetuberacks
17.Vortexmixer
18.Sterileshears,largescissors,scalpel,andforceps
19.Lamp(forobservingserologicalreactions)
20.FisherorBunsenburner
21.pHtestpaper(pHrange68)withmaximumgraduationsof0.4pHunitspercolorchange
22.pHmeter
23.Plasticbags,2837cm,sterile,withresealabletape.(Items2324areneededintheanalysisof
froglegsandrabbitcarcasses.)
24.Plasticbeakers,4liter,autoclavable,forholdingplasticbagduringshakingandincubation.
25.Sponges,nonbactericidal(Nascocat#B01299WA),orequivalent.
26.Swabs,nonbactericidal,cottontipped.
B.Media(/Food/FoodScienceResearch/LaboratoryMethods/ucm055778.htm)andReagents
(/Food/FoodScienceResearch/LaboratoryMethods/ucm055791.htm)
Forpreparationofmediaandreagents,refertoMethods967.25967.28inOfficialMethodsofAnalysis
(1).

1.Lactosebroth(M74(/Food/FoodScienceResearch/LaboratoryMethods/ucm064414.htm))
2.Nonfatdrymilk(reconstituted)(M111

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(/Food/FoodScienceResearch/LaboratoryMethods/ucm064144.htm))
3.Selenitecystine(SC)broth(M134
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063597.htm))
4.Tetrathionate(TT)broth(M145
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063686.htm))
5.RappaportVassiliadis(RV)medium(M132
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063568.htm)).NOTE:RVmedium
mustbemadefromitsindividualingredients.Commercialformulationsarenotacceptable.
6.Xyloselysinedesoxycholate(XLD)agar(M179
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062986.htm))
7.Hektoenenteric(HE)agar(M61
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064342.htm))
8.Bismuthsulfite(BS)agar(M19
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063348.htm))
9.Triplesugarironagar(TSI)(M149
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063699.htm))
10.Tryptone(tryptophane)broth(M164
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063867.htm))
11.Trypticase(tryptic)soybroth(M154
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063805.htm))
12.Trypticasesoytryptosebroth(M160
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063862.htm))
13.MRVPbroth(M104(/Food/FoodScienceResearch/LaboratoryMethods/ucm064068.htm))
14.Simmonscitrateagar(M138
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063639.htm))
15.Ureabroth(M171(/Food/FoodScienceResearch/LaboratoryMethods/ucm062935.htm))
16.Ureabroth(rapid)(M172
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062967.htm))
17.Malonatebroth(M92(/Food/FoodScienceResearch/LaboratoryMethods/ucm064500.htm))
18.Lysineironagar(LIA)(EdwardsandFife)(M89
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064484.htm))
19.Lysinedecarboxylasebroth(M87)
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064474.htm)
20.Motilitytestmedium(semisolid)(M103
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064059.htm))
21.Potassiumcyanide(KCN)broth(M126
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063514.htm))
22.Phenolredcarbohydratebroth(M121
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063494.htm))
23.Purplecarbohydratebroth(M130
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063529.htm))

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24.MacConkeyagar(M91(/Food/FoodScienceResearch/LaboratoryMethods/ucm064496.htm))
25.Nutrientbroth(M114(/Food/FoodScienceResearch/LaboratoryMethods/ucm064169.htm))
26.Brainheartinfusion(BHI)broth(M24
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063362.htm))
27.Papainsolution,5%(M56a
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063490.htm))
28.Cellulasesolution,1%(M187
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064568.htm))
29.Tryptosebloodagarbase(M166
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062871.htm))
30.Universalpreenrichmentbroth(M188
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062927.htm))
31.Universalpreenrichmentbroth(withoutferricammoniumcitrate)(M188a
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062933.htm))
32.Bufferedpeptonewater(M192
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063366.htm))
33.DeyEngleybroth(M193
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064244.htm))
34.Potassiumsulfitepowder,anhydrous
35.Chlorinesolution,200ppm,containing0.1%sodiumdodecylsulfate(R12a
(/Food/FoodScienceResearch/LaboratoryMethods/ucm061212.htm))
36.Ethanol,70%(R23(/Food/FoodScienceResearch/LaboratoryMethods/ucm061906.htm))
37.Kovacs'reagent(R38(/Food/FoodScienceResearch/LaboratoryMethods/ucm062242.htm))
38.VogesProskauer(VP)testreagents(R89
(/Food/FoodScienceResearch/LaboratoryMethods/ucm061693.htm))
39.Creatinephosphatecrystals
40.Potassiumhydroxidesolution,40%(R65
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062972.htm))
41.1NSodiumhydroxidesolution(R73
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063321.htm))
42.1NHydrochloricacid(R36
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062240.htm))
43.Brilliantgreendyesolution,1%(R8
(/Food/FoodScienceResearch/LaboratoryMethods/ucm061201.htm))
44.Bromcresolpurpledyesolution,0.2%(R9
(/Food/FoodScienceResearch/LaboratoryMethods/ucm061203.htm))
45.Methylredindicator(R44
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062248.htm))
46.Steriledistilledwater
47.TergitolAnionic7(R78(/Food/FoodScienceResearch/LaboratoryMethods/ucm063332.htm))

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48.TritonX100(R86(/Food/FoodScienceResearch/LaboratoryMethods/ucm061652.htm))
49.Physiologicalsalinesolution,0.85%(sterile)(R63
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062966.htm))
50.Formalinizedphysiologicalsalinesolution(R27
(/Food/FoodScienceResearch/LaboratoryMethods/ucm061921.htm))
51.Salmonellapolyvalentsomatic(O)antiserum
52.Salmonellapolyvalentflagellar(H)antiserum
53.Salmonellasomaticgroup(O)antisera:A,B,C1,C2,C3,D1,D2,E1,E2,E3,E4,F,G,H,I,Vi,and
othergroups,asappropriate
54.SalmonellaSpicerEdwardsflagellar(H)antisera

ReturntoChapterContents

C.PreparationoffoodsforisolationofSalmonella

NOTICE:
IfyouarelookingforBAMChapter5:Salmonella(December2007
Edition)thatisincorporatedbyreferencein21CFRParts16and118:
FederalRegisterFinalRule
(/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/
Eggs/ucm170615.htm)(July9,2009,74FR33030):Preventionof
SalmonellaEnteritidisinShellEggsDuringProduction,Storage,and
Transportation,pleaseusetheseversionsoftheBAMSalmonella
Chapter(/downloads/Food/FoodScienceResearch/UCM244774.pdf)
(PDF,189Kb)andAppendix1:RapidMethodsforDetectingFoodborne
Pathogens(/downloads/Food/FoodScienceResearch/UCM244777.pdf)
(PDF,195Kb).These2documentsarealsoavailableasacombinedfile
(/downloads/Food/FoodScienceResearch/UCM309839.pdf)(PDF,382
Kb).

ThemostrecentEditionofBAMChapter5:Salmonellacontinuesbelow
thisnotice.

Thefollowingmethodsarebasedontheanalysisofa25ganalyticalunitata1:9sample/brothratio.
Dependingontheextentofcompositing,addenoughbrothtomaintainthis1:9ratiounlessotherwise
indicated.Forsamplesnotanalyzedonanexactweightbasis,e.g.,froglegs,refertothespecific
methodforinstructions.

1.Driedeggyolk,driedeggwhites,driedwholeeggs,liquidmilk(skimmilk,2%fatmilk,
whole,andbuttermilk),andpreparedpowderedmixes(cake,cookie,doughnut,biscuit,
andbread),infantformula,andoralortubefeedingscontainingegg.Preferably,donot
thawfrozensamplesbeforeanalysis.Iffrozensamplemustbetemperedtoobtainanalytical
portion,thawsuitableportionasrapidlyaspossibletominimizeincreaseinnumberofcompeting
organismsortoreducepotentialofinjuringSalmonellaorganisms.Thawbelow45Cfor15min
withcontinuousagitationinthermostaticallycontrolledwaterbathorthawwithin18hat25C.

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Asepticallyweigh25gsampleintosterile,widemouth,screwcapjar(500ml)orother
appropriatecontainer.Fornonpowderedsamples,add225mlsterilelactosebroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064414.htm).Ifproductispowdered,
addabout15mlsterilelactosebrothandstirwithsterileglassrod,spoon,ortonguedepressorto
smoothsuspension.Add3additionalportionsoflactosebroth,10,10,and190ml,fortotalof225
ml.Stirthoroughlyuntilsampleissuspendedwithoutlumps.Capjarsecurelyandletstand605
minatroomtemperature.MixwellbyswirlinganddeterminepHwithtestpaper.AdjustpH,if
necessary,to6.80.2withsterile1NNaOHor1NHCl.Capjarsecurelyandmixwellbefore
determiningfinalpH.Loosenjarcapabout1/4turnandincubate242hat35C.Continueasin
D,111,below.
2.Eggs

a.Shelleggs[10,11].Eggswithchipped,cracked,orbrokenshellsarenotincludedinthe
sample.Removeanyadherentmaterialfromtheeggshellsurface.Disinfecteggsurfacewith
asolutionconsistingof3partsof70%alcohol(ethylorisopropyl)to1partiodine/potassium
iodidesolution.Prepare70%alcoholsolutioneitherbydiluting700ml100%alcoholwith
steriledistilledwaterforafinalvolumeof1,000mlorbydiluting700ml95%alcoholwith
steriledistilledwaterforafinalvolumeof950ml.Prepareiodine/potassiumiodidesolutionby
dissolving100gpotassiumiodidein200300mlsteriledistilledwater.Add50giodineand
heatgentlywithconstantmixinguntiltheiodineisdissolved.Dilutetheiodine/potassium
iodidesolutionto1,000mlwithsteriledistilledwater.Storeiodine/potassiumiodidesolutionin
anamberglassstopperedbottleinthedarkifnotusedimmediately.Preparethedisinfection
solutionbyadding250mliodine/potassiumiodidesolutionto750ml70%alcoholsolution
andmixwell.Submergeeggsindisinfectionsolutionfor10seconds(makesurenotlessthan
10seconds).Removeeggsfromthesolutionandallowtoairdry.Eachsampleshallconsist
oftwenty(20)eggs,foratotaloffifty(50)samplesperpoultryhouse.Eggsarecracked
asepticallyintoa4Lsterilebeakerorothersuitablecontainerbyglovedhands,withachange
ofglovesbetweensamples.Mixsamplesthoroughlywithasteriletoolbyglovedhandsuntil
yolksarecompletelymixedwiththealbumen,withachangeofglovesbetweensamples.
Preenrichthe20eggsamplebyadding2LsterileTrypticasesoybroth(TSBroom
temperature)andmixwellwithasteriletool.Coversecurelyandincubate242hat35C.
ContinueasinD,111,below.
SeeSalmonellaAppendix
(/Food/FoodScienceResearch/LaboratoryMethods/ucm386988.htm)forvalidationdata
b.Liquidwholeeggs(homogenized).Combinefifteen(15)25mltestportionsintoa375ml
compositecontainedina6literErlenmeyerflask.Compositesareheldatroomtemperature
(2024C)for962h.After962h,add3,375mlsterileTSBsupplementedwith
ferroussulfate(/Food/FoodScienceResearch/LaboratoryMethods/ucm063816.htm),
asdescribedabove,andmixwellbyswirling.Letstand605minatroomtemperature.Mix
wellbyswirlinganddeterminepHwithtestpaper.AdjustpH,ifnecessary,to6.80.2.
Incubate242hat35C.ContinueasinD,111,below.
c.Hardboiledeggs(chicken,duck,andothers).Iftheeggshellsarestillintact,disinfectthe
shellsasdescribedaboveandasepticallyseparatetheshellsfromtheeggs.Pulverizethe
eggs(eggyolksolidsandeggwhitesolids)asepticallyandweigh25gintoasterile500ml
Erlenmeyerflaskorotherappropriatecontainer.Add225mlTSB
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063805.htm)(withoutferrous
sulfate)andmixwellbyswirling.Continueasdescribedabove.
3.Nonfatdrymilk

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a.Instant.Asepticallyweigh25gsampleintosterilebeaker(250ml)orotherappropriate
container.Usingsterileglassorpaperfunnel(madewithtapetowithstandautoclaving),pour
25ganalyticalunitgentlyandslowlyoversurfaceof225mlbrilliantgreenwatercontainedin
sterile500mlErlenmeyerflaskorotherappropriatecontainer.Alternatively,25ganalytical
unitsmaybecompositedandpouredoverthesurfaceofproportionatelylargervolumesof
brilliantgreenwater.Preparebrilliantgreenwaterbyadding2ml1%brilliantgreendye
solution(/Food/FoodScienceResearch/LaboratoryMethods/ucm061201.htm)per1000
mlsteriledistilledwater.Letcontainerstandundisturbedfor605min.Incubateloosely
cappedcontainer,withoutmixingorpHadjustment,for242hat35C.ContinueasinD,1
11,below.
b.NonInstant.Examineasdescribedforinstantnonfatdrymilk,exceptthatthe25ganalytical
unitsmaynotbecomposited.
4.Drywholemilk.Examineasdescribedforinstantnonfatdrymilk,exceptthatthe25ganalytical
unitsmaynotbecomposited.
5.Casein

a.Lacticcasein.Asepticallyweigh25gsampleintosterilebeaker(250ml)orother
appropriatecontainer.Usingsterileglassorpaperfunnel(madewithtapetowithstand
autoclaving),pour25ganalyticalunitgentlyandslowlyoverthesurfaceof225mlUniversal
Preenrichmentbrothcontainedinsterile500mlErlenmeyerflaskorotherappropriate
container.Analyticalunits(25g)maybecomposited.Letcontainerstandundisturbed605
min.Incubatelooselycappedcontainer,withoutmixingorpHadjustment,for242hat
35C.ContinueasinD,111,below.
b.Rennetcasein.Asepticallyweigh25gsampleintosterilebeaker(250ml)orother
appropriatecontainer.Usingsterileglassorpaperfunnel(madewithtapetowithstand
autoclaving),pour25ganalyticalunitgentlyandslowlyoverthesurfaceof225mllactose
brothcontainedinsterile500mlErlenmeyerflaskorotherappropriatecontainer.Analytical
units(25g)maybecomposited.Letcontainerstandundisturbed605min.Incubateloosely
cappedcontainer,withoutmixingorpHadjustment,for242hat35C.ContinueasinD,1
11,below.
c.Sodiumcaseinate.Asepticallyweigh25gsampleintosterile,widemouth,screwcapjar
(500ml)orotherappropriatecontainer.Add225mlsterilelactosebrothandmixwell.
Analyticalunitsmaybecomposited.Letstand60minatroomtemperaturewithjarsecurely
capped.MixwellbyswirlinganddeterminepHwithtestpaper.AdjustpH,ifnecessary,to6.8
0.2.Loosenjarabout1/4turnandincubate242hat35C.ContinueasinD,111,
below.
6.Soyflour.Examineasdescribedforrennetcasein,except25ganalyticalunits(25g)maynotbe
composited.
7.Eggcontainingproducts(noodles,eggrolls,macaroni,spaghetti),cheese,dough,
preparedsalads(ham,egg,chicken,tuna,turkey),fresh,frozen,ordriedfruitsand
vegetables,nutmeats,crustaceans(shrimp,crab,crayfish,langostinos,lobster),and
fish.Preferably,donotthawfrozensamplesbeforeanalysis.Iffrozensamplemustbetempered
toobtainanalyticalportion,thawbelow45Cfor<15minwithcontinuousagitationin
thermostaticallycontrolledwaterbathorthawwithin18hat25C.
Asepticallyweigh25gsampleintosterileblendingcontainer.Add225mlsterilelactosebroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064414.htm)andblend2min.
Asepticallytransferhomogenizedmixturetosterile,widemouth,screwcapjar(500ml)orother

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appropriatecontainerandletstand605minatroomtemperaturewithjarsecurelycapped.Mix
wellbyswirlinganddeterminepHwithtestpaper.AdjustpH,ifnecessary,to6.80.2.Mixwell
andloosenjarcapabout1/4turn.Incubate242hat35C.ContinueasinD,111,below.
8.Driedyeast(activeandinactiveyeast).Asepticallyweigh25gsampleintosterile,widemouth,
screwcapjar(500ml)orotherappropriatecontainer.Add225mlsteriletrypticasesoybroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063805.htm).Mixwelltoform
smoothsuspension.Letstand605minatroomtemperaturewithjarsecurelycapped.Mixwell
byswirlinganddeterminepHwithtestpaper.AdjustpH,ifnecessary,to6.80.2,mixingwell
beforedeterminingfinalpH.Loosenjarcap1/4turnandincubate242hat35C.Continueas
inD,111,below.
9.Frostingandtoppingmixes.Asepticallyweigh25gsampleintosterile,widemouth,screwcap
jar(500ml)orotherappropriatecontainer.Add225mlnutrientbroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064169.htm)andmixwell.Capjar
securelyandletstand605minatroomtemperature.MixwellbyswirlinganddeterminepH
withtestpaper.AdjustpH,ifnecessary,to6.80.2.Loosenjarcapabout1/4turnandincubate
242hat35C.ContinueasinD,111,below.
10.Spices

a.Blackpepper,whitepepper,celeryseedorflakes,chilipowder,cumin,paprika,
parsleyflakes,rosemary,sesameseed,thyme,andvegetableflakes.Asepticallyweigh
25gsampleintosterile,widemouth,screwcapjar(500ml)orotherappropriatecontainer.
Add225mlsteriletrypticasesoybroth(TSB)
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063805.htm)andmixwell.Cap
jarsecurelyandletstand605minatroomtemperature.Mixwellbyswirlinganddetermine
pHwithtestpaper.AdjustpH,ifnecessary,to6.80.2.Loosenjarcapaboutl/4turnand
incubate242hat35C.ContinueasinD,111,below.
b.Onionflakes,onionpowder,garlicflakes.Asepticallyweigh25gsampleintosterile,
widemouth,screwcapjar(500ml)orotherappropriatecontainer.PreenrichsampleinTSB
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063805.htm)withaddedK2SO3
(5gK2SO3per1000mlTSB,resultinginfinal0.5%K2SO3concentration).AddK2SO3to
brothbeforeautoclaving225mlvolumesin500mlErlenmeyerflasksat121Cfor15min.
Afterautoclaving,asepticallydetermineand,ifnecessary,adjustfinalvolumeto225ml.Add
225mlsterileTSBwithaddedK2SO3tosampleandmixwell.ContinueasinC10a.
c.Allspice,cinnamon,cloves,andoregano.Atthistimetherearenoknownmethodsfor
neutralizingthetoxicityofthese4spices.Dilutethembeyondtheirtoxiclevelstoexamine
them.Examineallspice,cinnamon,andoreganoat1:100sample/brothratio,andclovesat
1:1000sample/brothratio.Examineleafycondimentsatsample/brothratiogreaterthan1:10
becauseofphysicaldifficultiesencounteredbyabsorptionofbrothbydehydratedproduct.
ExaminethesespicesasdescribedinC10a,above,maintainingrecommended
sample/brothratios.
11.Candyandcandycoating(includingchocolate).Asepticallyweigh25gsampleintosterile
blendingcontainer.Add225mlsterile,reconstitutednonfatdrymilk
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064144.htm)andblend2min.
Asepticallytransferhomogenizedmixturetosterile,widemouth,screwcapjar(500ml)orother
appropriatecontainerandletstand605minatroomtemperaturewithjarsecurelycapped.Mix
wellbyswirlinganddeterminepHwithtestpaper.AdjustpH,ifnecessary,to6.80.2.Add0.45
ml1%aqueousbrilliantgreendyesolutionandmixwell.Loosenjarcaps1/4turnandincubate24
2hat35C.ContinueasinD,111,below.

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12.Coconut.Asepticallyweigh25gsampleintosterile,widemouth,screwcapjar(500ml)orother
appropriatecontainer.Add225mlsterilelactosebroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064414.htm),shakewell,andlet
stand605minatroomtemperaturewithjarsecurelycapped.Mixwellbyswirlingand
determinepHwithtestpaper.AdjustpH,ifnecessary,to6.80.2.Addupto2.25mlsteamed
(15min)TergitolAnionic7
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063332.htm)andmixwell.
Alternatively,usesteamed(15min)TritonX100
(/Food/FoodScienceResearch/LaboratoryMethods/ucm061652.htm).Limituseofthese
surfactantstominimumquantityneededtoinitiatefoaming.ForTritonX100thisquantitymaybe
aslittleas2or3drops.Loosenjarcapaboutl/4turnandincubate242hat35C.Continueas
inD,111,below.
13.Fooddyesandfoodcoloringsubstances.FordyeswithpH6.0orabove(10%aqueous
suspension),usemethoddescribedfordriedwholeeggs(Cl,above).Forlakeddyesordyes
withpHbelow6.0,asepticallyweigh25gsampleintosterile,widemouth,screwcapjar(500ml)
orotherappropriatecontainer.Add225mltetrathionatebroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063686.htm)withoutbrilliantgreen
dye.Mixwellandletstand605minatroomtemperaturewithjarsecurelycapped.UsingpH
meter,adjustpHto6.80.2.Add2.25ml0.1%brilliantgreendyesolution
(/Food/FoodScienceResearch/LaboratoryMethods/ucm061201.htm)andmixthoroughlyby
swirling.Loosenjarcapabout1/4turnandincubate242hat35C.ContinueasinD,311,
below.
14.Gelatin.Asepticallyweigh25gsampleintosterile,widemouth,screwcapjar(500ml)orother
appropriatecontainer.Add225mlsterilelactosebroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064414.htm)and5ml5%aqueous
papainsolution(/Food/FoodScienceResearch/LaboratoryMethods/ucm063490.htm)and
mixwell.Capjarsecurelyandincubateat35Cfor605min.Mixwellbyswirlinganddetermine
pHwithtestpaper.AdjustpH,ifnecessary,to6.80.2.Loosenjarcapabout1/4turnand
incubate242hat35C.ContinueasinD,111,below.
15.Meats,meatsubstitutes,meatbyproducts,animalsubstances,glandularproducts,and
meals(fish,meat,bone).Asepticallyweigh25gsampleintosterileblendingcontainer.Add225
mlsterilelactosebroth(/Food/FoodScienceResearch/LaboratoryMethods/ucm064414.htm)
andblend2min.Asepticallytransferhomogenizedmixturetosterilewidemouth,screwcapjar
(500ml)orotherappropriatecontainerandletstand605minatroomtemperaturewithjar
securelycapped.Ifmixtureispowderorisgroundorcomminuted,blendingmaybeomitted.For
samplesthatdonotrequireblending,addlactosebrothandmixthoroughlyletstandfor605
minatroomtemperaturewithjarsecurelycapped.
MixwellbyswirlinganddeterminepHwithtestpaper.AdjustpH,ifnecessary,to6.80.2.Add
upto2.25mlsteamed(15min)TergitolAnionic7andmixwell.Alternatively,usesteamed(15
min)TritonX100.Limituseofthesesurfactantstominimumquantityneededtoinitiatefoaming.
Actualquantitywilldependoncompositionoftestmaterial.Surfactantswillnotbeneededin
analysisofpowderedglandularproducts.Loosenjarcaps1/4turnandincubatesamplemixtures
242hat35C.ContinueasinD,111,below.
16.Froglegs.(Thismethodisusedforalldomesticandimportedfroglegs.)Place15pairsoffrog
legsintosterileplasticbagandcoverwithsterilelactosebrothata1:9sampletobroth(g/ml)ratio
(seeA,2324,above).Ifsinglelegsareestimatedtoaverage25gormore,examineonlyoneleg
ofeachof15pairs.Placebaginlargeplasticbeakerorothersuitablecontainer.Mixwellandlet
stand605minatroomtemperature.MixwellbyswirlinganddeterminepHwithtestpaper.

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AdjustpH,ifnecessary,to6.80.2.Placeplasticbagcontainingthefroglegsandlactosebroth
intoplasticbeakerorothersuitablecontainer.Incubate242hat35C.Continueexamination
asinD,111,below.
17.Rabbitcarcasses.(Thismethodisusedforalldomesticandimportedrabbitcarcasses.)Place
rabbitcarcassintosterileplasticbag.Placebaginbeakerorothersuitablecontainer.Addsterile
lactosebrothata1:9sampletobroth(g/ml)ratiotocovercarcass(seeA,2324,above).Mixwell
byswirlingandletstand605minatroomtemperature.MixwellbyswirlinganddeterminepH
withtestpaper.AdjustpH,ifnecessary,to6.80.2.Incubate242hat35C.Continue
examinationasinD,111,below.
18.Guargum.Asepticallyweigh25gsampleintosterilebeaker(250ml)orotherappropriate
container.Preparea1.0%cellulasesolution(add1gcellulaseto99mlsteriledistilledwater).
Dispenseinto150mlbottles.(Cellulasesolutionmaybestoredat25Cforupto2weeks).Add
225mlsterilelactosebroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064414.htm)and2.25mlsterile1%
cellulasesolutiontosterile,widemouth,screwcapjar(500ml)orotherappropriatecontainer.
Whilevigorouslystirringthecellulase/lactosebrothwithmagneticstirrer,pour25ganalyticalunit
quicklythroughsterileglassfunnelintothecellulase/lactosebroth.Capjarsecurelyandletstand
605minatroomtemperature.IncubatelooselycappedcontainerwithoutpHadjustment,for24
2hat35C.ContinueasinD,111,below.
19.Orangejuice(pasteurizedandunpasteurized),applecider(pasteurizedand
unpasteurized),andapplejuice(pasteurized).Asepticallyadd25mlsampleto225ml
Universalpreenrichmentbroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062927.htm)inasterile,widemouth,
screwcappedjar(500ml)orotherappropriatecontainer.Swirltheflaskcontentsthoroughly.Cap
jarsecurelyandletstand605minatroomtemperature.DonotadjustpH.Incubateloosely
cappedcontainerfor242hat35C.ContinueasinD,111,below(treatasalowmicrobial
loadfood).
20.Pigearsandothertypesofdogchewpieces.Place1piece(or23piecesifsmallersizes)
fromeachsampleunitintosterileplasticbag.Placebagintolargebeakerorothersuitable
container.Addsterilelactosebrothata1:9sampletobroth(g/ml)ratiotocoverpieces(seeA,
2324,above).Mixwellbyswirlingandletstand605minatroomtemperature.Mixwellby
swirlinganddeterminepHwithtestpaper.AdjustpH,ifnecessary,to6.80.2.Addeither
steamed(15min)TergitolAnionic7orsteamed(15min)TritonX100uptoa1%concentration.
Forexample,if225mllactosebrothisadded,themaximumvolumeofaddedsurfactantis2.25
ml.Limituseofthesesurfactantstominimumquantitytoinitiatefoaming.Incubate242hat35
C.ContinueexaminationasinD,111,below.
21.Cantaloupes.Preferably,donotthawfrozensamplesbeforeanalysis.Iffrozensamplemustbe
temperedtoobtainanalyticalportion,thawbelow45Cfor<15minwithcontinuousagitationin
thermostaticallycontrolledwaterbathorthawwithin18hat25C.
Forcomminutedorcutfruit,asepticallyweigh25gsampleintosterileblendingcontainer.Add225
mlsterileUniversalpreenrichmentbroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062927.htm)(UP)andblend2min.
Asepticallytransferhomogenizedmixturetosterile,widemouth,screwcapjar(500ml)orother
appropriatecontainerandletstand605minatroomtemperaturewithjarsecurelycapped.Do
notadjustpH.Mixwellandloosenjarcapabout1/4turn.Incubate242hat35C.Continueas
inD,111,below.
Forwholecantaloupes,donotrinseevenifthereisvisibledirt.Examinethecantaloupes"asis".

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Placethecantaloupeintoasterileplasticbag.AddenoughUP
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062927.htm)brothtoallowthe
cantaloupetofloat.ThevolumeofUP
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062927.htm)brothmaybe1.5times
theweightofthecantaloupes.Forexample,cantaloupesweighing1500gwillprobablyneeda
volumeofapproximately2250mlUP
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062927.htm)brothtofloat.Addmore
broth,ifnecessary.Placetheplasticbag,withcantaloupesandUP
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062927.htm)broth,intoa5liter
beaker,orotherappropriatecontainer,forsupportduringincubation.Allowtheopenendflapof
theplasticbagto"foldover"soastoformasecure,butnotairtight,closureduringincubation.
Letstandfor605minatroomtemperature.DonotadjustpH.Incubateslightlyopenedbag,
containingcantaloupe,for242hat35C.ContinueasinD,111,below.
22.Mangoes.Preferably,donotthawfrozensamplesbeforeanalysis.Iffrozensamplemustbe
temperedtoobtainanalyticalportion,thawbelow45Cfor<15minwithcontinuousagitationin
thermostaticallycontrolledwaterbathorthawwithin18hat25C.
Forcomminutedorcutfruit,asepticallyweigh25gsampleintosterileblendingcontainer.Add225
mlsterilebufferedpeptonewater(BPW)
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063366.htm)andblend2min.
Asepticallytransferhomogenizedmixturetosterile,widemouth,screwcapjar(500ml)orother
appropriatecontainerandletstand605minatroomtemperaturewithjarsecurelycapped.Mix
wellbyswirlinganddeterminepHwithtestpaper.AdjustpH,ifnecessary,to6.80.2.Mixwell
andloosenjarcapabout1/4turn.Incubate242hat35C.ContinueasinD,111,below.
Forwholemangoes,donotrinseevenifthereisvisibledirt.Examinethemangoes"asis".
Placethemangointoasterileplasticbag.AddenoughBPW
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063366.htm)toallowthemangoto
float.ThevolumeofBPW
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063366.htm)maybe1.0timesthe
weightofthemangoes.Forexample,mangoesweighing500gwillprobablyneedavolumeof
approximately500mlBPW
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063366.htm)brothtofloat.Addmore
broth,ifnecessary.Placetheplasticbag,withmangoesandBPW
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063366.htm)broth,intoa5liter
beaker,orotherappropriatecontainer,forsupportduringincubation.
Letstandfor605minatroomtemperature.AdjustpHto6.80.2,ifnecessary.Incubate
slightlyopenedbagfor242hat35C.ContinueasinD,111,below.
23.Tomatoes.Forcomminutedorcutfruit,asepticallyweigh25gsampleintosterileblending
container.Add225mlsterilebufferedpeptonewaterandblend2min.Asepticallytransfer
homogenizedmixturetosterile,widemouth,screwcapjar(500ml)orotherappropriate
containerandletstand605minatroomtemperaturewithjarsecurelycapped.Mixwellby
swirlinganddeterminepHwithtestpaper.AdjustpH,ifnecessary,to6.80.2.Mixwelland
loosenjarcapabout1/4turn.Incubate242hat35C.ContinueasinD,111,below.
Forwholetomatoes,donotrinseevenifthereisvisibledirt.Examinethetomatoes"asis".
Placethetomatointoasterileplasticbagorothersuitablecontainer(sterilefoilcoveredbeaker
canbeused).AddenoughUP
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062927.htm)brothtoallowthe

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tomatotofloat.ThevolumeofUP
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062927.htm)brothmaybe1.0times
theweightofthetomato.Forexample,tomatoesweighing300gwillprobablyneedavolumeof
approximately300mlUP
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062927.htm)brothtofloat.Add
more,ifnecessary.Placetheplasticbag(ifused),withtomatoandUP
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062927.htm)broth,intoasterile
beaker(beakersizeisdependentonthesizeofthetomato),orotherappropriatecontainer,for
supportduringincubation.Allowtheopenendflapoftheplasticbagto"foldover"soastoforma
secure,butnotairtight,closureduringincubation.
Letstandfor605minatroomtemperature.DonotadjustpH.Incubateslightlyopenedbagfor
242hat35C.ContinueasinD,111,below.
24.Environmentaltesting.Sampleenvironmentalsurfaceswithsterileswabsorsponges.Place
theswab/spongeinasterileWhirlpakbag,orequivalent,thatcontainsenoughDeyEngley(DE)
broth(/Food/FoodScienceResearch/LaboratoryMethods/ucm064244.htm)tocoverthe
swab/sponge.
Transportswabs/spongesinaninsulatedtransportcontainerwithfrozengelpackstokeepthe
samplescold,butnotfrozen.Ifsamplescannotbeprocessedimmediately,refrigerateat42C.
Startsampleanalysiswithin482hofcollection.
Addswab/spongeto225mllactosebrothinasterile,widemouth,screwcappedjar(500ml)or
otherappropriatecontainer.Swirltheflaskcontentsthoroughly.Capjarsecurelyandletstand60
5minatroomtemperature.MixwellbyswirlinganddeterminepHwithtestpaper.AdjustpH,if
necessary,to6.80.2.Incubate242hat35C.ContinueexaminationasinD,111,below.
25.Alfalfaseedsandmungbeans.Asepticallyweigh25galfalfaseedsormungbeansintoasterile
500mLErlenmeyerflask.Asepticallyadd225mLlactosebrothtothetestportionandswirlthe
Erlenmeyerflask.CoverthemouthoftheErlenmeyerflaskwithsterilealuminumfoilandallow
contentstostandatroomtemperaturefor605min.AdjustthepHofthecultureto6.80.2,if
necessary.Incubatefor242hat352C.ContinueasinD,111,below(treatashigh
microbialloadfood).
26.Mameypulp.Iffrozen,samplemustbetemperedtoobtainanalyticalportion.Thawbelow45C
for<15minwithcontinuousagitationinthermostaticallycontrolledwaterbathorthawwithin18h
at25C.
Formameypulp,suspectedtobecontaminatedwithS.Typhi,asepticallyweigh25gsampleinto
sterile,widemouth,screwcapjar(500ml)orotherappropriatecontainer.Add225mlsterile
UniversalPreenrichmentbrothwithoutferricammoniumcitrate
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062933.htm),mixbyswirling,andlet
stand605minatroomtemperaturewithjarsecurelycapped.DonotadjustpH.Mixwelland
loosenjarcapabout1/4turn.Incubate242hat35C.ContinueasinD,111,below.Treatas
alowmicrobialloadfood.
Formameypulp,NOTsuspectedtobecontaminatedwithS.Typhi,asepticallyweigh25g
sampleintosterile,widemouth,screwcapjar(500ml)orotherappropriatecontainer.Add225
mlsterileUniversalPreenrichmentbroth,mixbyswirling,andletstand605minatroom
temperaturewithjarsecurelycapped.DonotadjustpH.Mixwellandloosenjarcapabout1/4
turn.Incubate242hat35C.ContinueasinD,111,below.

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27.Leafygreenvegetablesandherbs(babyspinach,Romainelettuce,cilantro,curlyparsley,
Italianparsley,culantro,cabbage,andbasil).Asepticallyweigh25gintoasterilewidemouth
Erlenmeyerflaskorotherappropriatecontainer.Add225mLlactosebrothandmanuallymix
contentsbyvigorouslyswirlingtheflask25timesclockwiseand25timescounterclockwise.Allow
theflasktostandatroomtemperaturefor605.0minutes,measurethepHandadjustitto6.8
0.2with1NNaOHor1NHCl,ifnecessary.Incubateat352.0Cfor242.0hoursand
continueasinD,111,below.

ReturntoChapterContents
D.IsolationofSalmonella

1.Tightenlidandgentlyshakeincubatedsample.
GuargumandfoodssuspectedtobecontaminatedwithS.Typhi.Transfer1mlmixtureto
10mlselenitecystine(SC)broth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063597.htm)andanother1ml
mixtureto10mlTTbroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063686.htm).Vortex.
Allotherfoods.Transfer0.1mlmixtureto10mlRappaportVassiliadis(RV)medium
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063568.htm)andanother1ml
mixtureto10mltetrathionate(TT)broth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063686.htm).Vortex.
2.Incubateselectiveenrichmentmediaasfollows:
Foodswithahighmicrobialload.IncubateRVmedium242hat420.2C(circulating,
thermostaticallycontrolled,waterbath).IncubateTTbroth242hat430.2C(circulating,
thermostaticallycontrolled,waterbath).
Foodswithalowmicrobialload(exceptguargumandfoodssuspectedtobe
contaminatedwithS.Typhi).IncubateRVmedium242hat420.2C(circulating,
thermostaticallycontrolled,waterbath).IncubateTTbroth242hat352.0C.
GuargumandfoodssuspectedtobecontaminatedwithS.Typhi.IncubateSCandTT
broths242hat35C.
3.Mix(vortex,iftube)andstreak3mmloopful(10l)incubatedTTbrothonbismuthsulfite(BS)
agar(/Food/FoodScienceResearch/LaboratoryMethods/ucm063348.htm),xyloselysine
desoxycholate(XLD)agar
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062986.htm),andHektoenenteric
(HE)agar(/Food/FoodScienceResearch/LaboratoryMethods/ucm064342.htm).Prepare
BSplatesthedaybeforestreakingandstoreindarkatroomtemperatureuntilstreaked.
4.Repeatwith3mmloopful(10l)ofRVmedium(forsamplesofhighandlowmicrobialload
foods)andofSCbroth(forguargum).
5.Referto994.04inOfficialMethodsofAnalysis(1)foroptionofrefrigeratingincubatedsample
preenrichmentsandincubatedsampleselectiveenrichments(SCandTTbrothsonly)oflow
moisturefoods.ThisoptionallowssampleanalysestobeinitiatedaslateasThursdaywhilestill
avoidingweekendwork.
6.Incubateplates242hat35C.
7.ExamineplatesforpresenceofcoloniesthatmaybeSalmonella.

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TYPICALSalmonellaCOLONYMORPHOLOGY
Pick2ormorecoloniesofSalmonellafromeachselectiveagarafter242hincubation.Typical
Salmonellacoloniesareasfollows:

a.Hektoenenteric(HE)agar.Bluegreentobluecolonieswithorwithoutblackcenters.Many
culturesofSalmonellamayproducecolonieswithlarge,glossyblackcentersormayappear
asalmostcompletelyblackcolonies.
b.Xyloselysinedesoxycholate(XLD)agar.Pinkcolonieswithorwithoutblackcenters.
ManyculturesofSalmonellamayproducecolonieswithlarge,glossyblackcentersormay
appearasalmostcompletelyblackcolonies.
c.Bismuthsulfite(BS)agar.Brown,gray,orblackcoloniessometimestheyhaveametallic
sheen.Surroundingmediumisusuallybrownatfirst,butmayturnblackintimewith
increasedincubation,producingthesocalledhaloeffect.
IftypicalcoloniesarepresentontheBSagarafter242hincubation,thenpick2ormore
colonies.IrrespectiveofwhetherornotBSagarplatesarepickedat242h,reincubateBSagar
platesanadditional242h.After482hincubation,pick2ormoretypicalcolonies,ifpresent,
fromtheBSagarplates,onlyifcoloniespickedfromtheBSagarplatesincubatedfor242h
giveatypicalreactionsintriplesugarironagar(TSI)andlysineironagar(LIA)thatresultinculture
beingdiscardedasnotbeingSalmonella.SeesectionsD.9andD.10,below,fordetailsin
interpretingTSIandLIAreactions.
ATYPICALSalmonellaCOLONYMORPHOLOGY
IntheabsenceoftypicalorsuspiciousSalmonellacolonies,searchforatypicalSalmonella
coloniesasfollows:

a.HEandXLDagars.AtypicallyafewSalmonellaculturesproduceyellowcolonieswithor
withoutblackcentersonHEandXLDagars.IntheabsenceoftypicalSalmonellacolonieson
HEorXLDagarsafter242hincubation,thenpick2ormoreatypicalSalmonellacolonies.
b.BSagar.Atypicallysomestrainsproducegreencolonieswithlittleornodarkeningofthe
surroundingmedium.IftypicalorsuspiciouscoloniesarenotpresentonBSagarafter242
h,thendonotpickanycoloniesbutreincubateanadditional242h.Iftypicalorsuspicious
coloniesarenotpresentafter482hincubation,thenpick2ormoreatypicalcolonies.
SUGGESTEDCONTROLCULTURES
Inadditiontothepositivecontrolcultures(typicalSalmonella),3additionalSalmonellacultures
arerecommendedtoassistintheselectionofatypicalSalmonellacolonymorphologyonselective
agars.Theseculturesarealactosepositive,H2SpositiveS.diarizonae(ATCC12325)anda
lactosenegative,H2SnegativeS.abortusequi(ATCC9842)ORalactosepositive,H2S
negativeS.diarizonae(ATCC29934).TheseculturesmaybeobtainedfromtheAmericanType
CultureCollection(http://www.atcc.org),10801UniversityBoulevard,Manassas,VA20110
2209.
8.Lightlytouchtheverycenterofthecolonytobepickedwithsterileinoculatingneedleand
inoculateTSIslantbystreakingslantandstabbingbutt.Withoutflaming,inoculateLIAslantby
stabbingbutttwiceandthenstreakingslant.Sincelysinedecarboxylationreactionisstrictly
anaerobic,theLIAslantsmusthavedeepbutt(4cm).Storepickedselectiveagarplatesat58C.
9.IncubateTSIandLIAslantsat35Cfor242h.Captubeslooselytomaintainaerobicconditions
whileincubatingslantstopreventexcessiveH2Sproduction.Salmonellainculturetypically
producesalkaline(red)slantandacid(yellow)butt,withorwithoutproductionofH2S(blackening

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ofagar)inTSI.InLIA,Salmonellatypicallyproducesalkaline(purple)reactioninbuttoftube.
Consideronlydistinctyellowinbuttoftubeasacidic(negative)reaction.Donoteliminatecultures
thatproducediscolorationinbuttoftubesolelyonthisbasis.MostSalmonellaculturesproduce
H2SinLIA.SomenonSalmonellaculturesproduceabrickredreactioninLIAslants.
10.AllculturesthatgiveanalkalinebuttinLIA,regardlessofTSIreaction,shouldberetainedas
potentialSalmonellaisolatesandsubmittedforbiochemicalandserologicaltests.Culturesthat
giveanacidbuttinLIAandanalkalineslantandacidbuttinTSIshouldalsobeconsidered
potentialSalmonellaisolatesandshouldbesubmittedforbiochemicalandserologicaltests.
CulturesthatgiveanacidbuttinLIAandanacidslantandacidbuttinTSImaybediscardedas
notbeingSalmonella.Testretained,presumedpositiveTSIculturesasdirectedinD11,below,
todetermineiftheyareSalmonellaspecies,includingS.arizonae.IfTSIculturesfailtogive
typicalreactionsforSalmonella(alkalineslantandacidbutt)pickadditionalsuspiciouscolonies
fromselectivemediumplatenotgivingpresumedpositivecultureandinoculateTSIandLIAslants
asdescribedinD8,above.
11.Applybiochemicalandserologicalidentificationteststo:

a.ThreepresumptiveTSIculturesrecoveredfromsetofplatesstreakedfromRVmedium(or
SCbrothforguargum),ifpresent,and3presumptiveTSIagarculturesrecoveredfrom
platesstreakedfromTTbroth,ifpresent.
b.If3presumptivepositiveTSIculturesarenotisolatedfromonesetofagarplates,testother
presumptivepositiveTSIagarcultures,ifisolated,bybiochemicalandserologicaltests.
Examineaminimumof6TSIculturesforeach25ganalyticalunitoreach375gcomposite.
ReturntoChapterContents
E.IdentificationofSalmonella

1.Mixedcultures.StreakTSIagarculturesthatappeartobemixedonMacConkeyagar
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064496.htm),HEagar
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064342.htm),orXLDagar
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062986.htm).Incubateplates242
hat35C.ExamineplatesforpresenceofcoloniessuspectedtobeSalmonella.

a.MacConkeyagar.Typicalcoloniesappeartransparentandcolorless,sometimeswithdark
center.ColoniesofSalmonellawillclearareasofprecipitatedbilecausedbyotherorganisms
sometimespresent.
b.Hektoenenteric(HE)agar.SeeD7a,above.
c.Xyloselysinedesoxycholate(XLD)agar.SeeD7b,above.Transferatleast2colonies
suspectedtobeSalmonellatoTSIandLIAslantsasdescribedinD7,above,andcontinue
asinD9,above.
2.Purecultures

a.Ureasetest(conventional).Withsterileneedle,inoculategrowthfromeachpresumed
positiveTSIslantcultureintotubesofureabroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062935.htm).Sinceoccasional,
uninoculatedtubesofureabrothturnpurplered(positivetest)onstanding,include
uninoculatedtubeofthisbrothascontrol.Incubate242hat35C.

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b.Optionalureasetest(rapid).Transfertwo3mmloopfulsofgrowthfromeachpresumed
positiveTSIslantcultureintotubesofrapidureabroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062967.htm).Incubate2hin37
0.5Cwaterbath.Discardallculturesgivingpositivetest.Retainforfurtherstudyall
culturesthatgivenegativetest(nochangeincolorofmedium).
3.Serologicalpolyvalentflagellar(H)test

a.Performthepolyvalentflagellar(H)testatthispoint,orlater,asdescribedinE5,below.
InoculategrowthfromeachureasenegativeTSIagarslantintoeither1)BHIbroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063362.htm)andincubate46h
at35Cuntilvisiblegrowthoccurs(totestonsameday)or2)trypticasesoytryptose
broth(/Food/FoodScienceResearch/LaboratoryMethods/ucm063862.htm)andincubate
242hat35C(totestonfollowingday).Add2.5mlformalinizedphysiologicalsaline
solutionto5mlofeitherbrothculture.
b.BDDIFCOProcedure.Select2formalinizedbrothculturesandtestwithSalmonella
polyvalentflagellar(H)antiserapermanufacturer'sinstructions.Place0.5mlofappropriately
dilutedSalmonellapolyvalentflagellar(H)antiserumin1075mmor13100mm
serologicaltesttube.Add0.5mlantigentobetested.Preparesalinecontrolbymixing0.5ml
formalinizedphysiologicalsalinesolutionwith0.5mlformalinizedantigen.Incubatemixtures
in4850Cwaterbath.Observeat15minintervalsandreadfinalresultsin1h.
Positiveagglutinationintestmixtureandnoagglutinationincontrol.
Negativenoagglutinationintestmixtureandnoagglutinationincontrol.
Nonspecificagglutinationinbothtestmixtureandcontrol.Testtheculturesgivingsuch
resultswithSpicerEdwardsantisera.
c.StatensSerumInstituteProcedure.Performthepolyvalentflagellar(H)testusingStatens
SerumInstituteSalmonellapolyvalentflagellar(H)antisera.TheSalmonellaisgrownover
nightonanonselectiveagarmedium.Swarmagaristhebestsuitedmediumforgrowing
culturesforHtyping,butHantigenscanbeserotypedfromanonselectiveagarmediumif
theHantigensarewellexpressed.Addasmalldropofantiserum(approx.20L)onaglass
slideorplasticpetridish(15100mm).Transfercultureusinganinoculatingloopfrom
severalcoloniestothedropofantiserumandmixwell.Theamountofcultureshouldbe
sufficienttogiveadistinctmilkyturbidity.Tilttheslideorpetridishfor510seconds.A
positivereactionisseenasvisibleagglutination,whereasanegativereactionisseenas
homogeneousmilkyturbidity.Alateorweakagglutinationshouldbeconsiderednegative.
Physiologicalsaline(0.85%,pH7.4)isusedasanegativecontrolandmustbenegative.
4.SpicerEdwardsserologicaltest.Usethistestasanalternativetothepolyvalentflagellar(H)
test.Itmayalsobeusedwithculturesgivingnonspecificagglutinationinpolyvalentflagellar(H)
test.PerformSpicerEdwardsflagellar(H)antiseratestasdescribedinE,3b,above.Perform
additionalbiochemicaltests(E,5ac,below)onculturesgivingpositiveflagellartestresults.If
bothformalinizedbrothculturesarenegative,performserologicaltestson4additionalbroth
cultures(E,3a,above).Ifpossible,obtain2positiveculturesforadditionalbiochemicaltestingE,
5ac,below).IfallureasenegativeTSIculturesfromsamplegivenegativeserologicalflagellar
(H)testresults,performadditionalbiochemicaltestsE,5ac,below).
5.Testingofureasenegativecultures

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a.Lysinedecarboxylasebroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064474.htm).IfLIAtestwas
satisfactory,itneednotberepeated.Uselysinedecarboxylasebrothforfinaldeterminationof
lysinedecarboxylaseifculturegivesdoubtfulLIAreaction.Inoculatebrothwithsmallamount
ofgrowthfromTSIslantsuspiciousforSalmonella.Replacecaptightlyandincubate482
hat35Cbutexamineat24hintervals.Salmonellaspeciescausealkalinereactionindicated
bypurplecolorthroughoutmedium.Negativetestisindicatedbyyellowcolorthroughout
medium.Ifmediumappearsdiscolored(neitherpurplenoryellow)addafewdropsof0.2%
bromcresolpurpledyeandrereadtubereactions.
b.Phenolreddulcitolbroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063494.htm)orpurplebroth
base(/Food/FoodScienceResearch/LaboratoryMethods/ucm063529.htm)with0.5%
dulcitol.InoculatebrothwithsmallamountofgrowthfromTSIculture.Replacecaploosely
andincubate482hat35C,butexamineafter24h.MostSalmonellaspeciesgivepositive
test,indicatedbygasformationininnerfermentationvialandacidpH(yellow)ofmedium.
Productionofacidshouldbeinterpretedasapositivereaction.Negativetestisindicatedby
nogasformationininnerfermentationvialandred(withphenolredasindicator)orpurple
(withbromcresolpurpleasindicator)colorthroughoutmedium.
c.Tryptone(ortryptophane)broth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063867.htm).Inoculatebroth
withsmallgrowthfromTSIagarculture.Incubate242hat35Candproceedasfollows:

1.Potassiumcyanide(KCN)broth.
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063514.htm)Transfer3mm
loopfulof24htryptophanebrothculturetoKCNbroth.Heatrimoftubesothatgood
sealisformedwhentubeisstopperedwithwaxcoatedcork.Incubate482hat35C
butexamineafter24h.Interpretgrowth(indicatedbyturbidity)aspositive.Most
Salmonellaspeciesdonotgrowinthismedium,asindicatedbylackofturbidity.
2.Malonatebroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm064500.htm).Transfer3
mmloopfulof24htryptonebrothculturetomalonatebroth.Sinceoccasional
uninoculatedtubesofmalonatebrothturnblue(positivetest)onstanding,include
uninoculatedtubeofthisbrothascontrol.Incubate482hat35C,butexamineafter
24h.MostSalmonellaspeciesculturesgivenegativetest(greenorunchangedcolor)in
thisbroth.
3.Indoletest.Transfer5mlof24htryptophanebrothculturetoemptytesttube.Add0.2
0.3mlKovacs'reagent
(/Food/FoodScienceResearch/LaboratoryMethods/ucm062242.htm).Most
Salmonellaculturesgivenegativetest(lackofdeepredcoloratsurfaceofbroth).
Recordintermediateshadesoforangeandpinkas.
4.Serologicalflagellar(H)testsforSalmonella.Ifeitherpolyvalentflagellar(H)test(E
3,above)ortheSpicerEdwardsflagellar(H)testtubetest(E4,above)hasnotalready
beenperformed,eithertestmaybeperformedhere.
5.DiscardasnotSalmonellaanyculturethatshowseitherpositiveindoletestandnegative
serologicalflagellar(H)test,orpositiveKCNtestandnegativelysinedecarboxylasetest.
6.Serologicalsomatic(O)testsforSalmonella.
(PretestallantiseratoSalmonellawithknowncultures.)

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a.Polyvalentsomatic(O)test.Usingwaxpencil,markoff2sectionsabout12cmeachon
insideofglassorplasticpetridish(15100mm).Commerciallyavailablesectionedslides
maybeused.Emulsify3mmloopfulofculturefrom2448hTSIslantor,preferably,tryptose
bloodagarbase(withoutblood)with2ml0.85%saline.Add1dropofculturesuspensionto
upperportionofeachrectangularcrayonmarkedsection.Add1dropofsalinesolutionto
lowerpartofonesectiononly.Add1dropofSalmonellapolyvalentsomatic(O)antiserumto
othersectiononly.Withcleansteriletransferlooporneedle,mixculturesuspensionwith
salinesolutionforonesectionandrepeatforothersectioncontainingantiserum.Tiltmixtures
inbackandforthmotionfor1minandobserveagainstdarkbackgroundingoodillumination.
Consideranydegreeofagglutinationapositivereaction.Classifypolyvalentsomatic(O)test
resultsasfollows:
Positiveagglutinationintestmixturenoagglutinationinsalinecontrol.
Negativenoagglutinationintestmixturenoagglutinationinsalinecontrol.
Nonspecificagglutinationintestandincontrolmixtures.Performfurtherbiochemicaland
serologicaltestsasdescribedinEdwardsandEwing'sIdentificationofEnterobacteriaceae
(2).
b.Somatic(O)grouptests.TestasinE6a,above,usingindividualgroupsomatic(O)
antiseraincludingVi,ifavailable,inplaceofSalmonellapolyvalentsomatic(O)antiserum.
ForspecialtreatmentofculturesgivingpositiveViagglutinationreaction,refertosec.
967.28BinOfficialMethodsofAnalysis(1).Recordculturesthatgivepositiveagglutination
withindividualsomatic(O)antiserumaspositiveforthatgroup.Recordculturesthatdonot
reactwithindividualsomatic(O)antiserumasnegativeforthatgroup.
7.Additionalbiochemicaltests.ClassifyasSalmonellathosecultureswhichexhibittypical
Salmonellareactionsfortests111,showninTable1.IfoneTSIculturefrom25ganalyticalunit
isclassifiedasSalmonella,furthertestingofotherTSIculturesfromthesame25ganalyticalunit
isunnecessary.CulturesthatcontaindemonstrableSalmonellaantigensasshownbypositive
Salmonellaflagellar(H)testbutdonothavebiochemicalcharacteristicsofSalmonellashouldbe
purified(El,above)andretested,beginningwithE2,above.
PerformthefollowingadditionaltestsonculturesthatdonotgivetypicalSalmonellareactionsfor
tests111inTable1andthatconsequentlydonotclassifyasSalmonella.

a.Phenolredlactosebroth
(/Food/FoodScienceResearch/LaboratoryMethods/ucm063494.htm)orpurplelactose
broth(/Food/FoodScienceResearch/LaboratoryMethods/ucm063529.htm).

1.Inoculatebrothwithsmallamountofgrowthfromunclassified2448hTSIslant.
Incubate482hat35C,butexamineafter24h.
Positiveacidproduction(yellow)andgasproductionininnerfermentationvial.
Considerproductionofacidonlyaspositivereaction.MostculturesofSalmonellagive
negativetestresult,indicatedbynogasformationininnerfermentationvialandred(with
phenolredasindicator)orpurple(withbromcresolpurpleasindicator)throughout
medium.
2.DiscardasnotSalmonella,culturesthatgivepositivelactosetests,exceptculturesthat
giveacidslantsinTSIandpositivereactionsinLIA,orculturesthatgivepositive
malonatebrothreactions.Performfurthertestsontheseculturestodetermineiftheyare
S.arizonae.
b.Phenolredsucrosebroth

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(/Food/FoodScienceResearch/LaboratoryMethods/ucm063494.htm)orpurplesucrose
broth(/Food/FoodScienceResearch/LaboratoryMethods/ucm063529.htm).Follow
proceduredescribedinE,7a1,above.DiscardasnotSalmonella,culturesthatgivepositive
sucrosetests,exceptthosethatgiveacidslantsinTSIandpositivereactionsinLIA.
c.MRVPbroth(/Food/FoodScienceResearch/LaboratoryMethods/ucm064068.htm).
InoculatemediumwithsmallamountofgrowthfromeachunclassifiedTSIslantsuspectedto
containSalmonella.Incubate482hat35C.

1.PerformVogesProskauer(VP)testatroomtemperatureasfollows:Transfer1ml48h
culturetotesttubeandincubateremainderofMRVPbrothanadditional48hat35C.
Add0.6mlnaphtholandshakewell.Add0.2ml40%KOHsolutionandshake.To
intensifyandspeedreaction,addafewcrystalsofcreatine.Readresultsafter4h
developmentofpinktorubyredcolorthroughoutmediumispositivetest.Mostcultures
ofSalmonellaareVPnegative,indicatedbyabsenceofdevelopmentofpinktoredcolor
throughoutbroth.
2.Performmethylredtestasfollows:To5mlof96hMRVPbroth,add56dropsof
methylredindicator.Readresultsimmediately.MostSalmonellaculturesgivepositive
test,indicatedbydiffuseredcolorinmedium.Adistinctyellowcolorisnegativetest.
Discard,asnotSalmonella,culturesthatgivepositiveKCNandVPtestsandnegative
methylredtest.
d.Simmonscitrateagar.Inoculatethisagar,usingneedlecontaininggrowthfromunclassified
TSIagarslant.Inoculatebystreakingslantandstabbingbutt.Incubate962hat35C.
Readresultsasfollows:
Positivepresenceofgrowth,usuallyaccompaniedbycolorchangefromgreentoblue.
MostculturesofSalmonellaarecitratepositive.
Negativenogrowthorverylittlegrowthandnocolorchange.
8.Classificationofcultures.Classify,asSalmonella,culturesthathavereactionpatternsofTable
l.Discard,asnotSalmonella,culturesthatgiveresultslistedinanysubdivisionofTable2.
PerformadditionaltestsdescribedinEdwardsandEwing'sIdentificationofEnterobacteriaceae
(2)toclassifyanyculturethatisnotclearlyidentifiedasSalmonellabyclassificationschemein
TablelornoteliminatedasnotbeingSalmonellabytestreactionsinTable2.Ifneitherof2TSI
culturescarriedthroughbiochemicaltestsconfirmstheisolateasSalmonella,performbiochemical
tests,beginningwithE5,onremainingureasenegativeTSIculturesfromsame25ganalytical
unit.

Table1.BiochemicalandserologicalreactionsofSalmonella

# Result Salmonella
species
Testorsubstrate Positive Negative reaction(a)

1. Glucose(TSI) yellowbutt redbutt +

2. Lysinedecarboxylase(LIA) purplebutt yellowbutt +

3. H2 S(TSIandLIA) blackening noblackening +

4. Urease purpleredcolor nocolorchange

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4. Urease purpleredcolor nocolorchange

5. Lysinedecarboxylase purplecolor yellowcolor +

broth

6. Phenolreddulcitolbroth yellowcolorand/or nogasnocolorchange +(b)

gas

7. KCNbroth growth nogrowth

8. Malonatebroth bluecolor nocolorchange (c)

9. Indoletest redcoloratsurface yellowcoloratsurface

10. Polyvalentflagellartest agglutination noagglutination +

11. Polyvalentsomatictest agglutination noagglutination +

12. Phenolredlactosebroth yellowcolorand/or nogasnocolorchange (c)

gas

13. Phenolredsucrosebroth yellowcolorand/or nogasnocolorchange

gas

14. VogesProskauertest pinktoredcolor nocolorchange

15. Methylredtest diffuseredcolor diffuseyellowcolor +

16. Simmonscitrate growthbluecolor nogrowthnocolorchange v

a
+:90%ormorepositivein1or2days:90%ormorenegativein1or2days
v:variable.
b
MajorityofS.arizonaeculturesarenegative.
c
MajorityofS.arizonaeculturesarepositive.

Table2.CriteriafordiscardingnonSalmonellacultures

# Testorsubstrate Results

1. Urease positive(purpleredcolor)

2. Indoletestand positive(redcoloratsurface)
Polyvalentflagellar(H)test negative(noagglutination)
or
Indoletestand positive(redcoloratsurface)
SpicerEdwardsflagellartest negative(noagglutination)

3. Lysinedecarboxylaseand negative(yellowcolor)
KCNbroth positive(growth)

4. Phenolredlactosebroth positive(yellowcolorand/orgas)(a),(b)

5. Phenolredsucrosebroth positive(yellowcolorand/orgas)(b)

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5. Phenolredsucrosebroth positive(yellowcolorand/orgas)(b)

6. KCNbroth, positive(growth)
VogesProskauertest,and positive(pinktoredcolor)
Methylredtest negative(diffuseyellowcolor)

a TestmalonatebrothpositiveculturesfurthertodetermineiftheyareS.arizonae.
b
DonotdiscardpositivebrothculturesifcorrespondingLIAculturesgivetypical
SalmonellareactionstestfurthertodetermineiftheyareSalmonellaspecies.

9.PresumptivegenericidentificationofSalmonella.Asalternativetoconventionalbiochemical
tubesystem,useanyof5commercialbiochemicalsystems(API20E,EnterotubeII,
EnterobacteriaceaeII,MICROID,orVitek2GN)forpresumptivegenericidentificationof
foodborneSalmonella.Chooseacommercialsystembasedonademonstrationinanalyst'sown
laboratoryofadequatecorrelationbetweencommercialsystemandbiochemicaltubesystem
delineatedinthisidentificationsection.Commercialbiochemicalkitsshouldnotbeusedasa
substituteforserologicaltests(l).Assemblesuppliesandpreparereagentsrequiredforthekit.
InoculateeachunitaccordingtoMethod978.24(API20E,EnterotubeII,andEnterobacteriaceae
II),sec.989.12(MICROID),andMethod2011.17(Vitek2GN)inOfficialMethodsofAnalysis(1),
incubatingfortimeandtemperaturespecified.Addreagents,observe,andrecordresults.For
presumptiveidentification,classifycultures,accordingtoref.1,above,asSalmonellaornot
Salmonella.
ForconfirmationofculturespresumptivelyidentifiedasSalmonella,performtheSalmonella
serologicalsomatic(O)test(E6,above)andtheSalmonellaserologicalflagellar(H)test(E3,
above)ortheSpicerEdwardsflagellar(H)test(E4,above),andclassifyculturesaccordingto
thefollowingguidelines:

a.ReportasSalmonellathoseculturesclassifiedaspresumptiveSalmonellawithcommercial
biochemicalkitswhentheculturedemonstratespositiveSalmonellasomatic(O)testand
positiveSalmonella(H)test.
b.DiscardculturespresumptivelyclassifiedasnotSalmonellawithcommercialbiochemicalkits
whenculturesconformtoAOACcriteria(1)forclassifyingculturesasnotSalmonella.
c.Forculturesthatdonotconformtoaorb,classifyaccordingtoadditionaltestsspecifiedinE,
27,above,oradditionaltestsasspecifiedbyEwing(2),orsendtoreferencetyping
laboratoryfordefinitiveserotypingandidentification.
10.Treatmentofculturesgivingnegativeflagellar(H)test.Ifbiochemicalreactionsofcertain
flagellar(H)negativeculturestronglysuggestthatitisSalmonella,thenegativeflagellar
agglutinationmaybetheresultofnonmotileorganismsorinsufficientdevelopmentofflagellar
antigen.Proceedasfollows:Inoculatemotilitytestmediuminpetridish,usingsmallamountof
growthfromTSIslant.Inoculatebystabbingmediumonceabout10mmfromedgeofplateto
depthof23mm.Donotstabtobottomofplateorinoculateanyotherportion.Incubate24hat
35C.Iforganismshavemigrated40mmormore,retestasfollows:Transfer3mmloopfulof
growththatmigratedfarthesttotrypticasesoytryptosebroth.Repeateitherpolyvalentflagellar
(H)(E3,above)orSpicerEdwards(E4,above)serologicaltests.Ifculturesarenotmotileafter
thefirst24h,incubateanadditional24hat35Cifstillnotmotile,incubateupto5daysat25C.
Classifycultureasnonmotileifabovetestsarestillnegative.Ifflagellar(H)negativecultureis
suspectedofbeingaspeciesofSalmonellaonthebasisofitsbiochemicalreactions,FDA
laboratoriesshouldsubmitthecultureto

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14/03/2016 LaboratoryMethods>BAM:<i>Salmonella</i>

FDADenverLaboratory
AttentionSampleCustodian
DenverFederalCenter,Building20
6thAvenue&KiplingStreets
Denver,CO802250087

(AboveaddresseffectiveOctober1,2004)
forfurtheridentificationand/orserotyping.LaboratoriesotherthanFDAshouldmake
arrangementswithareferencelaboratoryfortheserotypingofSalmonellacultures.
11.Submissionofculturesforserotyping.Submit1isolateofeachsomaticgrouprecovered
fromeachanalyticalunit,unlessotherwiseinstructed.SubmitculturesonBHIagarslantsin
screwcaptubes(13100mmor16125mm)withcapssecuredtightly.Labeleachtubewith
samplenumber,subsample(analyticalunit)number,andcode,ifapplicable.Submitacopyofthe
CollectionReport,FD464,orImportSampleReport,FD784foreachsample.Placeculturesin
culturecontainerwithofficialFDAseal.Placeaccompanyingrecords(E11,above)inside
shippingcartonbutnotwithinofficiallysealedculturecontainer.Submitmemoorcoverletterfor
eachsamplenumbertoexpeditereportingofresults.Prepareculturesforshipmentaccordingto
requirementsforshipmentofetiologicalagents(3).Labelsecondaryshippingcontaineraccording
toref.4.Sendcontainerbymostrapidmailserviceavailable.Maintainduplicateculturesofthose
submittedforserotypingonlyonthosesamplesunderconsiderationforlegalaction.
MicrobiologyFieldlaboratoriesshouldfollowthefollowingguidanceinsendingSalmonella
isolatesforserotyping:
IsolatesfromNRL,WEAC,SRLandARLwillbeserotypedinARL:
ArkansasRegionalLaboratory
3900NCTRRoadBuilding26
Jefferson,AR72079
Attention:GwendolynAnderson
Tel#8705434621
Fax#8705434041
IsolatesfromSAN,PRLNW,PRLSWandDENwillbeserotypedinDEN:
DenverDistrictLaboratory
6thAvenue&KiplingStreet
DFCBuilding20
Denver,CO802250087
Attention:ShaunaMadson
Tel#3032369631
Fax#3032369675

ReturntoChapterContents

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16.Wang,H.,Gill,V.S.,Irvin,K.A.,Bolger,C.M.,Zheng,J.,Dickey,E.E.,Duvall,R.E.,Jacobson,A.P.,
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HypertextSource:BacteriologicalAnalyticalManual,8thEdition,RevisionA,1998.Chapter4.

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