National Standard of the People’s Republic of Chinatradechina.dairyaustralia.com.au/wp-content/uploads/2018/... · 2018-10-15 · National Standard of the People’s Republic of

GB4789.4-2016FoodMicrobiologicalExamination:Salmonella

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NationalStandardofthePeople’sRepublicofChina

GB4789.4-2016

NationalFoodSafetyStandard

FoodMicrobiologicalExamination:Salmonella

食品安全国家标准

食品微生物学检验沙门氏菌检验

• Releasedon2016-12-23

• Implementedon2017-06-23

• IssuedbyNHFPC&CFDA

DISCLAIMER: The English version is an unofficial translation of the original in Chinese forinformationandreferencepurposesonly. Incaseofadiscrepancy theChineseoriginal standardwillprevail.


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ForewordThisstandardreplacesGB4789.4-2010“FoodHygieneMicrobiologicalExamination:Salmonella”,SN0170-1992“Examination method of Salmonella (including Arizona) in Exported Food”, and SN/T 2552.5-2010“Microbiologicalexaminationofmilkanddairyproducts:Part5ExaminationofSalmonella”.ComparedtoGB4789.4-2010,majorchangesofthisstandardareasfollows:— Examinationprocedureandserologytestprocedurehasbeenrevised;— AnnexAandAnnexBhavebeenrevised.


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NationalFoodSafetyStandard

FoodMicrobiologicalExamination:Salmonella1. ScopeThisstandardspecifiesthetestingmethodforSalmonellainfoods.ThisstandardappliestotheexaminationofSalmonellainfoods.2. ApparatusandmaterialsIn addition to the conventional apparatus for sterilization and incubation in microbiological laboratory, otherapparatusandmaterialsareasfollows:2.1 Refrigerator:2℃~5℃.2.2 Constanttemperatureincubator:36℃±1℃,42℃±1℃.2.3 Homogenizer.2.4 Shaker.2.5 Electronicbalance:withsensitivityof0.1g.2.6 Sterileconicalflask:withnominalcapacitiesof500mLand250mL.2.7 Sterilepipette:withnominalcapacitiesof1mL(graduatedin0.01mLdivision)and10mL(graduatedin0.1mLdivision),ormicropipetteandpipettetips.2.8 Sterileculturedish:withdiameterof60mmand90mm.2.9 Steriletesttube:3mm×50mm,1 0 mm×75mm.2.10 pHmeterorpHcolorimetrictubeorprecisionpHtestpaper.2.11 Automaticmicroorganismbiochemicalidentificationsystem.2.12 Sterilecapillary.

3. Culturemediumsandreagents3.1 Bufferedpeptonewater(BPW):SeeSectionA.1inAnnexA.3.2 Tetrathionatebrilliantgreen(TTB)enrichmentbroth:SeeSectionA.2inAnnexA.3.3 Selenitecystinol(SC)enrichmentbroth:SeeSectionA.3inAnnexA.


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3.4 Bismuthsulfite(BS)agar:SeeSectionA.4inAnnexA.3.5 HEagar:SeeSectionA.5inAnnexA.3.6 Xyloselysinedeoxycholate(XLD)agar:SeeSectionA.6inAnnexA.3.7 Salmonellachromogenicmedium.3.8 Triplesugariron(TSI)agar:SeeSectionA.7inAnnexA.3.9 Peptonewater,Indolereagent:SeeSectionA.8inAnnexA.3.10 Ureaagar(pH7.2):SeeSectionA.9inAnnexA.3.11 Potassiumcyanide(KCN)medium:SeeSectionA.10inAnnexA.3.12 Lysinedecarboxylationtestbroth:SeeSectionA.11inAnnexA.3.13 Sugarfermentationtube:SeeSectionA.12inAnnexA.3.14 Ortho-nitrophenol-β-D-galactopyranoside(ONPG)medium:SeeSectionA.13inAnnexA.3.15 Semisolidmedium:SeeSectionA.14inAnnexA.3.16 Sodiummalonatemedium:SeeSectionA.15inAnnexA.3.17 SalmonellaO,HandViantisera.3.18 Biochemicalidentificationkit.4. ExaminationproceduresTheexaminationproceduresofSalmonellaareshowninFigure1.


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Figure1ExaminationproceduresofSalmonella

5. Operatingsteps5.1 Pre-enrichmentAseptictechnique:Weigh25g(mL)ofsampleandplaceintoasterilehomogenizingcupwhichcontains225mLofBPW,homogenizefor1min~2mininthespeedof8000r/min~10000r/min,orplaceintoasterilehomogenizingbag which contains 225mL of BPW, and homogenize it for 1 min~2min with a slapping homogenizer. If thesampleisliquid,shakeandmixwell,withnoneedforhomogenization.FordeterminationofpH,adjustthepHto6.8±0.2with1mol/mLsterileNaOHorHCl.Aseptically transfer thesample intoa500mLconical flaskorothervessels (if the homogenizing cup does not have hole cover, there is no need to transfer the sample). Directly


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incubateincaseofusingahomogenizingbag.Incubateat36℃±1℃for8h~18h.

Andthefrozenproductshouldbeunfrozenatbelow45℃forlessthan15min,orat2℃~5℃forlessthan18h.

5.2 EnrichmentLightlyshaketheculturedsamplemixture,take1mLofthecultureandtransferincubateinto10mLofTTB,and

thenincubateat42℃±1℃for18h~24h.Meanwhile,take1mLofthecultureandtransferincubateinto10mL

ofSC,andthenincubateat36℃±1℃for18h~24h.

5.3 IsolationTakeoneloopoftheenrichedculture,streakinoculateonaBSagarplateandaXLDagarplate(orHEagarplateor

Salmonellachromogenicmediumplate).Incubateat36℃±1℃for18h~24h(XLDagarplate,HEagarplateand

Salmonellachromogenicmediumplate)orfor40h~48h(BSagarplate).Observethecoloniesgrowingoneachplate,andthecolonycharacteristicsoneachplatecanbeseeninTable1.

Table1ColonycharacteristicsofSalmonellaondifferentselectivemediums

Selectiveagarplate Salmonella

BSagar

The colonies showblackwithmetallic luster, tanor grey, and themediumssurrounding thecolony showblackorbrown; thereare somegreyish-greencoloniessurroundbyanunchangedmedium.

HEagar

Thecoloniesshowblue-greenorgreen,withablackcenteroralmostablackwholecolony;somestrainsshowyellow,withablackcenteroralmostablackwholecolony.

XLDagar

Thecoloniesshowpink,withorwithoutablackcenter,somestrainshavealarge lustrous black center or a blackwhole colony; while some strainsshowyellow,withorwithoutablackcenter.

Salmonellachromogenicmedium

It should be judged in accordancewith the instruction of the chromogenicmedium.

5.4 Biochemicaltest5.4.1 Pick over 2 of the typical or suspected colonies from each selective agar plate, inoculate on TSI agar bystreakingontheslantfirstlyandthenstabbing inthebottom;withoutsterilizingthe incubationneedle,directly

inoculateonthelysinedecarboxylasetestmediumandnutrientagarplate,incubateat36℃±1℃for18h~24h,

upto48hifnecessary.Table2canbereferredtoforthereactionresultsofSalmonellaonTSIagarandlysineandlysinedecarboxylasetestmedium.

Table2ReactionresultsofSalmonellaonTSIagarandlysineandlysinedecarboxylasetestmedium


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TSIagar Lysinedecarboxylasetestmedium Preliminaryjudgment

Slant Bottom Gasproduction H2S

K A +(-) +(-) + SuspectedSalmonella

K A +(-) +(-) - SuspectedSalmonella

A A +(-) +(-) + SuspectedSalmonella

A A +/- +/- - non-Salmonella

K K +/- +/- +/- non-Salmonella

Note:K:alkaliproduction,A:acidproduction;+:positive,-:negative;+(-):majorityispositive,minorityisnegative;+/-:positiveornegative.

5.4.2 Thecolony canbe inoculated inpeptonewater (for indole test),ureaagar (pH7.2) andKCNmediawhileinoculatingontheTSIagarandlysineandlysinedecarboxylasetestmedium.Alternatively,thesuspectedcolonies

onthenutrientagarplatecanbeusedforinoculationafterthepreliminaryjudgment.Incubateat36℃±1℃ for

18h~24h,upto48hifnecessary.JudgetheresultaccordingtoTable3.Thepickedplatesshouldbekeptat2℃

~5℃orroomtemperatureforatleast24h,forreexaminationwhennecessary.

Table3PreliminaryidentificationofthebiochemicalreactionofSalmonella

Reactionno. H2S Indole pH7.2Urea KCN Lysine

decarboxylaseA1 + - - - +

A2 + + - - +A3 - - - - +/-

Note:+:positive;-:negative;+/-:positiveornegative.

5.4.2.1 Reactionno.A1:ThestrainswithtypicalreactioncanbejudgedasSalmonella.ThestrainscanbejudgedasSalmonellaaccordingtotable4whenthereisanabnormalitemamongUrea,KCNandLysinedecarboxylase.Thestrainscanbejudgedasnon-Salmonellawhentherearetwoabnormalitems.

Table4PreliminaryidentificationofthebiochemicalreactionofSalmonella

pH7.2Urea KCN Lysinedecarboxylase

Judgmentresults

- - - SalmonellaparatyphiA(requireserologicalidentificationresults)

- + + SalmonellaIVorV(conform tothebiochemicalcharacteristicsofthegroupthereof)

+ - + Salmonellavariation(requireserologicalidentificationresults)Notes:+referstopositive;-referstonegative.


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5.4.2.2 Reactionno.A2:Performmannitolandsorbieritetestforsupplement,andtheabovetwo test resultsofSalmonella indolepositivevariationarebothpositive,buttheeventualjudgmentshouldbemadewiththehelpoftheserologicalidentificationresults.5.4.2.3 Reactionno.A3:PerformONPGtestforsupplement.WhilethestrainsoflysinedecarboxylasenegativeareSalmonellaparatyphiA,thestrainsofONPGnegativeandlysinedecarboxylasepositiveareSalmonella.5.4.2.4 IdentifySalmonellabiochemicalgroupsaccordingtoTable5ifnecessary.

Table5IdentificationofSalmonellabiochemicalgroups

Items I II III IV V VI

Galactitol + + - - + -

Sorbierite + + + + + -

Salicin - - - + - -

ONPG - - + - + -

Malonate - + + - - -

KCN - - - + + -Notes:+referstopositive;-referstonegative.

5.4.3 Ifbiochemical identificationkitorautomaticmicroorganismbiochemical identificationsystemisused.Pickthesuspectedcoloniesonthenutrientagarplate,andprepareabacterialsuspensionwithappropriateturbidity,thenidentifyitbythebiochemicalidentificationkitorautomaticmicroorganismbiochemicalidentificationsystemaccordingtothepreliminaryjudgmentresults.5.5 Serologicalidentification5.5.1 Identifytheself-agglutinationoftargetedtestingmaterialsGenerallyuse1.2%-1.5%of incubatedagar toslideagglutination.Firstly, self-agglutinationshouldbeexcluded.Addadropsalineonacleanslideandmixthetestingmaterialwiththesalinetoeventurbidsuspension.Shaketheslidelightlyfor30s-60s,andobservethereactionunderblackbackground(themagnifyingglasscomestohelpwhennecessary), ifthat isagglutinate,thenit isdeterminedasself-agglutination,otherwiseviewedasnon-self-agglutination.5.5.2 Identificationofmultivalentsomaticantigens(O)Respectiveplacehalfofacolonyontothetopofeachareaofslide,addonedropofmultivalentsomaticO-serumonthebottomofonearea,addotheronedropofsaline solutiononthebottomofotherareaascomparison.Dispersetwoareasastoobtainahomogeneousandturbidsuspension.Rocktheslidegentlyfor60s.Observetheresult against a dark background. All of agglutination phenomena are considered positive reaction. Place thecolonyonhighconcentrationagarculture(2%-3%)andobserveagainwhenthereisnoagglutinationphenomena.IfViantigenspreventtheagglutinationofOantigens,thenpickthecolonyandmixwith1mLsalineandheattoboilonthealcoholburnerandthencheckagain.5.5.3 Identificationofmultivalentflagellarantigens(H)ThesameasthatspecifiedinSection5.5.2.WhentheHantigensdoesnotgrowwell,placethecolonyontothecenter of 0.55%-0.65% semisolid agar culture, pick the colony around themarginwhen colony is growing and


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check;orplacethecolonyintothe0.3%-0.4%semisolidagarglasstubeforonceortwice,andpickthefar-endcolonyandcheck.5.6 Serologicaltyping(optional)5.6.1 IdentificationofO-antigensA~Fmultivalentanti-Oserumisusedforslideagglutinationtest,whilethenormalsalineisusedascontrol.Iftheauto-agglutinablestrainsinthenormalsalineareroughstrains,theycannotbeserotyped.ForthestrainsagglutinatedbyA~Fmultivalentanti-Oserum,performagglutinationtestusingO4;O3,O10;O7; O8; O9;O2 and O11 factor serum in turn. For the strains agglutinated by O3 and O10 serum, performagglutinationtestusingO10,O15,O34,O19single-factorserum,andjudgetheE1,E2,E3,E4subgroup.Thefinaljudgmentof eachO-antigencomponentshouldbe inaccordancewiththetestresultofOsingle-factorserum.ForthosehavenoOsingle-factorserum,theyshouldbeverifiedbytwoOcombined-factorserums.For the strains not agglutinated by A~Fmultivalent anti-O serum, firstly examine it with 9multivalent anti- Oserums. For the strains agglutinated by any serum, examine itwith theO-group serums included in the abovemultivalentserumtodeterminetheO-group.TheO-factorserumsincludedineachmultivalentanti-Oserumareasfollows:Omultivalent1A,B,C,D,EandFgroup(alsoinclude6and14group)Omultivalent213,16,17,18and21group

Omultivalent3 28,30,35,38and39groupOmultivalent4 40,41,42and43groupOmultivalent5 44,45,47and48groupOmultivalent6 50,51,52and53groupOmultivalent7 55,56,57and58groupOmultivalent8 59,60,61and62groupOmultivalent9 63,65,66and67group5.6.2 IdentificationofH-antigensForthecommonbacteriatypesinA~Fgroup,examinetheH-antigensinPhase1andPhase2withHfactorslistedinTable6.

Table6H-antigensforcommonbacteriatypesinA~FgroupOgroup Phase1 Phase2

A a No

B g,f,s No

B i,b,d 2

C1 k,v,r,c 5,z15

C2 b,d,r 2,5

D(Nongas-producing) d No

D(Gas-producing) g,m,p,q No

E1 h,v 6,w,x

E4 g,s,t No

E4 i

Fortheuncommonbacteriatypes,firstlyexamineitwith8multivalentanti-Hserums.Forthestrainsagglutinatedby one or two serum(s), examine it with the H-group serums contained in the above one or twomultivalentserum(s) to determine H-antigens in Phase 1 and Phase 2. The H-factor serums contained in the above 8


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multivalentanti-Oserumsareasfollows:

Hmultivalent1a,b,c,d,iHmultivalent2 eh,enx,enz15,fg,gms,gpu,gp,gq,mt,gz51Hmultivalent3k,r,y,z,z10,1v,1w,1z13,1z28,1z40Hmultivalent4 1,2;1,5;1,6;1,7;z6Hmultivalent5 z4z23,z4z24,z4z32,z29,z35,z36,z38Hmultivalent6z39,z41,z42,z44Hmultivalent7 z52,z53,z54,z55Hmultivalent8 z56,z57,z60,z61,z62ThefinaljudgmentofeachH-antigencomponentshouldbemadeinaccordancewiththetestresultofHsingle-factorserum.IftherearesomewithnoHsingle-factorserum,theyshouldbeverifiedbytwoHcombined-factorserums.AstothecasethatHantigens inPhase1weredetectedbutHantigens inPhase2werenotdetectedorthatHantigens inPhase2weredetectedbutHantigens inPhase1werenotdetected, retestcanbeperformedafterinoculatingontheagarslantfor1~2generations.IfstillonlyonephaseofHantigensisdetected,itisallowedtoemploy phase variationmethod to check the other phase. Single-phase bacteria do not need phase variationexamination.Phasevariationtestmethodsareasfollows:Easyplatemethod:Drythewateronthesurfaceof0.35%~0.4%semisolidagarplate, pickone loopof factorserumanddroponthesurfaceofflatplate,standforamoment.Inoculatethebacteriatobetestedonthecenterpointofserum,incubateitandthenpickthebacteriafromtheedgeofthespreadinglawntotestaftertheserumisabsorbedintotheagar.Smallglasstube:Meltthesemi-solidtube(eachtubeisabout1mL~2mL)onthealcohollampandcooldownto

50℃,add0.05mL~0.1mLoftheH-factorserumofknownphaseintothemeltedsemi-solid,mixwellandthen

pipetteitwithacapillaryintothesmallglasstubesforphasevariationtest.Pickthebacteriatobetestedwithaninoculationneedleand inoculateononeendaftersolidification.Placethesmallglasstube intoaflatplate,andplaceaballofwetcottonbesidethesmallglasstubetopreventwaterevaporationandagarshrinkage,checktheresults every day, and pick the culture on the other end for bacteriological examination after dissociation ofbacteria in theotherphase.Serumconcentrationof themediumshouldbe inappropriateproportion,becausethe bacteria cannot grow under a too high concentration, and themotility of the bacteria in the same phasecannotbeinhibited.Itshouldbeaddedintheproportionof1:200~1:800totheoriginalserumamount.Smallinvertedtubemethod:Placeasmallglasstubewithbothendsopened(leavingagapattheloweropenedend,anddonotmaketheendflat)inthesemi-solidtube,theupperendofthesmallglasstubeshouldbehigherthat theuppersurfaceof themedium,andstore foruseafter sterilization.Beforeuse,heatandmelt iton the

alcohollampandcooldownto50℃,pickoneloopoffactorserum,addintothesmallinvertedtubeinsidethe

semi-solid, slightly stir tomixwell. After solidification, inoculate the bacteria to be tested onto the semi-solidsurface inside the small inverted tube, check the results every day, and pick the culture for bacteriologicalexaminationthesemi-solidsurfaceinsidethesmallinvertedtubeafterdissociationofbacteriaintheotherphase,

ortransferandinoculateona1%softagarslant,incubateat36℃andthenperformagglutinationtest.

5.6.3 IdentificationofVi-antigensCheckthebacteriatypewithVifactorserum.TheknownbacteriatypeswithVi- antigensareSalmonellatyphi,SalmonellaparatyphiAandSalmonelladublin.5.6.4 JudgmentofbacterialtypeInaccordancewiththeresultsofserologicalconfirmation,thejudgementofthebacterialtypeshouldbemadebasedonAnnexBorSalmonellaantigentable.


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6. ResultsandreportsBased on the above results of biochemical tests and serological identification, report whether Salmonella isdetectedornotdetectedin25g(mL)ofthesample.


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AnnexA

CultureMediumsandReagents

A.1 Bufferedpeptonewater(BPW)A.1.1 CompositionPeptone 10.0gSodiumchloride 5.0gSodiumhydrogenphosphate(containing12crystalwater) 9.0gPotassiumdihydrogenphosphate 1.5gDistilledWater 1000mLA.1.2 PreparationDissolvethecomponentsinthedistilledwater,stirwellandplacefor10min,byheatingtocompletelysolve,

adjustthePHto7.2±0.2,autoclave15minat121℃.

A.2 Tetrathionatebrilliantgreen(TTB)enrichmentbrothA.2.1 BasesolutionPeptone 10.0gBeefextract 5.0gSodiumchloride 3.0gCalciumcarbonate 45.0gDistilledwater 1000mLPlaceall theabovecomponentsexcludingcalciumcarbonate inthedistilledwater,boil todissolve,addcalcium

carbonate.AdjustthepHto7.0±0.2,andsterilizefor20minintheautoclavesetat121℃.

A.2.2 SodiumhyposulfitesolutionSodiumhyposulfite(containing5crystalwater) 50.0gDistilledwater addto100mLSterilizefor20minintheautoclavesetat121℃.A.2.3 IodinesolutionIodineplate 20.0gPotassiumiodide 25.0gDistilledwater addto100mLDissolvethepotassiumiodide ina littledistilledwatercompletely,add iodineplate,shaketheglassbottleuntilthe iodine plate is completely dissolved, and then add distilledwater to the specified volume, store in brownbottles,andplugthebottlestopper.Thentheobtainedsolutionisreadytouse.A.2.4 0.5%brilliantgreenaqueoussolution


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Brilliantgreen 0.5gDistilledwater 100mLAftercompletelydissolved,storeindarkplacefornotlessthan1d,andsterilizeitnaturally.A.2.5 OXbilesaltsolutionOXbilesalt 10.0gDistilledwater 100mLHeatandboiltocompletelydissolved,andsterilizefor20minintheautoclavesetat121℃.

A.2.6 PreparationBasesolution 900mLSodiumhyposulfitesolution 100mLIodinesolution 20.0mLBrilliantgreenaqueoussolution 2.0mLOXbilesaltsolution 50.0mLBeforeuse,asepticallyaddalltheabovecomponentsintothebasesolutionaccordingtotheaboveorder,shakewellaftereachadding.A.3 Selenitecystinol(SC)enrichmentbrothA.3.1 CompositionPeptone 5.0gLactose 4.0gSodiumhydrogenphosphate 10.0gSodiumhydrogenselenite 4.0gL-cystine 0.01gDistilledwater 1000mLA.3.2 PreparationPlace all the above components except sodium hydrogen selenite and L-cystine in the distilled water, boil to

dissolve, cool down to below 55℃, aseptically add sodium hydrogen selenite and 10 mL of 1 g/L L-cystine

solution(weigh0.1gofL-cystine,add15mLof1mol/LNaOHsolutiontodissolveit,andthenaddsteriledistilledwater to100mL.Theamountshouldbedoubled incaseofusingDL-cystine).Shakewellandadjust itspHto7.0±0.2.A.4 Bismuthsulfite(BS)agarA.4.1 CompositionPeptone 10.0gBeefextract 5.0gGlucose 5.0g


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Ironsulfate 0.3gSodiumhydrogenphosphate 4.0gBrilliantgreen 0.025gor5.0mLof5.0g/LaqueoussolutionAmmoniumbismuthcitrate 2.0g

Sodiumsulfite 6.0gAgar 18.0g~20.0gDistilledwater 1000mLA.4.2 PreparationPlacethe first threecomponents in300mLofdistilledwater (forbasesolution preparation).Placethe ferroussulfateanddisodiumhydrogenphosphate in20mLand30mLofdistilledwater,respectively.Placeammoniumbismuthcitrateandsodiumsulfiteinanother20mLand30mLofdistilledwater,respectively.Placetheagarin

600mLofdistilledwater.Thenshakewellandboiltodissolveit.Cooldownto80℃,firstlymixtheferroussulfate

and disodium hydrogen phosphate thoroughly, and pour into the base solution, and then mix well. Mix theammoniumbismuth citrateand sodiumsulfite thoroughly, andpour into thebase solution, and thenmixwell.

AdjustthepHto7.4±0.2,immediatelypourintotheagarsolution,mixwellagain,andthencooldownto50℃

~55℃.Addbrilliantgreensolution,mixwellandimmediatelypourplates.

Note: Pressure sterilization is not needed by the medium. Overheating is not advisable during preparation,becauseitcanleadtothereductionofitsselectivity.Storeitindarkplaceattheroomtemperature.Therewouldbeadeclineofitsselectivityifitexceeds48h,andthemediumshouldbepreparedonthatverydayandbeputintouseonthenextday.A.5HEagar(HektoenEntericAgar)A.5.1CompositionPeptone 12.0gBeefextract 3.0gLactose 12.0gSucrose 12.0gSalicin 2.0gBilesalt 20.0gSodiumchloride 5.0gAgar 18.0g~20.0gDistilledwater 1000mL0.4%bromothymolbluesolution 16.0mLAndradeindicator 20.0mLSolutionA 20.0mLSolutionB 20.0mLA.5.2PreparationDissolve the first sevencomponents in400mLofdistilledwater, asbase solution;place theagar in600mLofdistilledwater.Thenstirwellrespectivelyandboiltodissolvethem.AddsolutionAandBintothebasesolution,

andadjustthepHto7.4±0.2.Thenaddtheindicator,andmixwithagarsolution.Aftercoolingto50℃~55℃,

pourplates.


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Notes:1. The medium does not need pressure sterilization, do not heat it excessively during preparation to avoidreducingitsselectivity.2. PreparationofSolutionASodiumhyposulfite 34.0gAmmoniumironcitrate 4.0gDistilledwater 100mL3. PreparationofSolutionBSodiumdeoxycholate 10.0gDistilledwater 100mL4. AndradeindicatorAcidfuchsin 0.5g1mol/Lsodiumhydroxidesolution 16.0mLDistilledwater 100mLDissolve the acid fuchsin in distilledwater, add sodiumhydroxide solution.Add another 1mL~2mLof sodiumhydroxidesolutionifthecolorfadingoftheacidfuchsinisnotcompleteafterseveralhours.A.6 Xyloselysinedesoxycholate(XLD)agarA.6.1 CompositionYeastextract 3.0gL-lysine 5.0gXylose 3.75gLactose 7.5gSucrose 7.5gSodiumdeoxycholate 2.5gAmmoniumironcitrate 0.8gSodiumthiosulfate 6.8gSodiumchloride 5.0gAgar 15.0gPhenolred 0.08gDistilledwater 1000mLA.6.2 PreparationPlacealltheabovecomponentsexceptphenolredandagarin400mLofdistilledwater,boiltodissolvethem,andadjustthepHto7.4±0.2.Placetheagarintoanother600mLofdistilledwaterandboiltodissolveit.

Mixtheabovetwosolutionsthoroughly,addtheindicator,cooldownto50℃~55℃,andpourplates.

Notes: Pressure sterilization is not needed by the medium. Overheating is not advisable during preparation,because it can lead to the reductionof its selectivity. Store it in darkplace at the room temperature.And themediumshouldbepreparedonthatverydayandbeputintouseonthenextday.


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A.7 Triplesugariron(TSI)agarA.7.1 CompositionPeptone 20.0gBeefextract 5.0gSucrose 10.0gLactose 10.0gGlucose 1.0gAmmoniumferroussulfate(containing6crystalwater) 0.2gPhenolred 0.025gor5.0mLof5.0g/LsolutionSodiumchloride 5.0gSodiumthiosulfate 0.2gAgar 12.0gDistilledwater 1000mLA.7.2 PreparationPlacealltheabovecomponentsexceptphenolredandagarin400mLofdistilledwater,boiltodissolvethem,andadjustthepHto7.4±0.2.Placetheagarinanother600mLofdistilledwater,andboiltodissolveit.Mixtheabovetwosolutionsfully,addtheindicator,mixwellanddispenseintotesttubesinquantitiesof2mL~4

mL,sterilizefor10minintheautoclavesetat121℃or15minat115℃.Settinginaslopingpositionshowing

orangeredcolorisallowable.A.8 Peptonewater,indolereagentA.8.1 PeptonewaterPeptone(ortryptone) 20.0gSodiumchloride 5.0gDistilledwater 1000mLAddalltheabovecomponentsintodistilledwater,boiltodissolvethem,andadjustthepHto7.4±0.2.Dispense

intosmalltesttubes,andsterilizefor15minintheautoclavesetat121℃.

A.8.2 IndolereagentA.8.2.1 Kovacsreagent:Dissolve5gofp-Dimethylaminobenzaldehydein75mLofpentanol,andthenslowlyadd25mLofconcentratedhydrochloricacid.A.8.2.2 O-Bohrreagent:Dissolve1gofp-Dimethylaminobenzaldehydein95mLof95%ethanol,andthenslowlyadd20mLofhydrochloricacid.A.8.3 TestmethodPickasmallamountofthecultureandinoculate,andthenincubateat36℃±1℃for1d~2d,up to4d~5d if

necessary. Add approximately 0.5mL of the Kovacs reagent and lightlyshakethetube,andthe formationofacrimsoncolorindicatesapositivereaction.Oradd0.5mLofO-Bohrreagent,flowdownalongthetubewalland


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covertheculturesurface,andtheformationofarosecolorattheconnectingsurfaceindicatesapositivereaction.A.9Ureaagar(pH7.2)A.9.1CompositionPeptone 1.0gSodiumchloride 5.0gGlucose 1.0gPotassiumdihydrogenphosphate 2.0g0.4%phenolred 3.0mLAgar 20.0gDistilledwater 1000mL20%ureasolution 100mLA.9.2 PreparationPlacealltheabovecomponentsexcludingurea,agarandphenolredinto400mLofdistilledwater,boiltodissolvethemandadjustthepHto7.2±0.2.Placetheagarintoanother600mLofdistilledwater,andboiltodissolveit.Mixtheabovetwosolutionsfully,addtheindicator,mixwell,dispenseandsterilizefor15minintheautoclave

setat121℃.Cooldownto50℃~55℃andaddthesterilefilteredureasolution.Thefinalconcentrationofurea

is2%.Dispensethemediumintosteriletesttubes,andallowittosetinaslopingposition.Theobtainedmediumisreadytouse.A.9.3 TestmethodPick theagarcultureand inoculate,andthen incubateat36℃±1℃ for24h,andobserve theresults.For the

ureasepositivestrains,themediumturnsintoredbecauseofthealkali-production.A.10 Potassiumcyanide(KCN)mediumA.10.1 Composition

Peptone 10.0gSodiumchloride 5.0gPotassiumdihydrogenphosphate 0.225gSodiumhydrogenphosphate 5.64gDistilledwater 1000mL0.5%Potassiumcyanide 20.0mLA.10.2 PreparationPlace all the above components excluding potassium cyanide into distilled water and boil to dissolve them,

dispense and sterilize for 15 min in the autoclave set at 121 ℃. Cool themedium fully in the refrigerator.

Add 2.0 mL of 0.5% potassium cyanide solution into each 100 mL of the medium (the final concentration is1:10000),dispenseintosteriletesttubesinthequantitiesof4mL.Stuffthetubeswiththesterilerubberstoppers

immediately, and then store in a 4℃ refrigerator, at least stable for 2months.Meanwhile, take themedium

withoutpotassiumcyanideascontrolmedium,dispenseintotesttubesforlateruse.A.10.3 Testmethod


18

Inoculatetheagarmediumtothepeptonewatertogetadilutedbacteriasolution,andpickaloopofthecultureand inoculate on the potassium cyanide (KCN) medium, and take another loop to inoculate on the control

medium.Incubateat36℃±1℃for1d~2d,andobservetheresults.Thegrowthofbacteriaindicatepositive

(notinhibit),andnogrowthofbacteriaafter2dindicatenegative(inhibit).Note:Becausepotassiumcyanideishighlytoxic,itshouldbeusedwithcaution.Toavoidpoisoning,donottouchit. The medium should be dispensed in a refrigerator in summer. The main cause of the failure to finish theexperimentisuntightseal.Thusthepotassiumcyanidedecomposesgraduallyandproduceshydrocyanicacidgas.Then the gas escapes, the reagent concentration decreases and bacteria grows. Finally, it causes false positivereactions.A.11LysinedecarboxylasemediumA.11.1 Composition

Peptone 5.0gYeastExtract 3.0gGlucose 1.0gDistilledwater 1000mL1.6%Bromocresolpurple-ethanolsolution 1.0mLL-lysineorDL-lysine 0.5g/100mLor1.0g/100mLA.11.2 PreparationHeattodissolvealltheabovecomponentsexceptlysine,dispensethemediumintobottlesinthequantities100mL,andthenrespectivelyaddlysine(L-lysine:0.5%,DL- lysine:1%).AdjustthepHto6.8±0.2.Donotaddlysineintothecontrolmedium.Dispensethemediumintosterilesmalltesttubesinquantitiesof5mL,dropwiseadda

layerofliquidparaffin,andsterilizefor10minintheautoclavesetat115℃.

A.11.3 TestmethodPicktheculturefromtheagarslantandinoculate,thenincubateat36℃±1℃for18h~24h,andobservethe

results. Because of alkali production, themedium should be in purple color for the amino acid decarboxylasepositive strains.While themedium turns to yellow because of the acid production of glucose, the amino aciddecarboxylasepositivestrainshavenobasicproduct.Thecontroltubeshouldbeinyellowcolor.A.12SugarfermentationtubeA.12.1CompositionBeefextract 5.0gPeptone 10.0gSodiumchloride 3.0gSodiumhydrogenphosphate(containing12crystalwater) 2.0g0.2%Bromthymolphenolbluesolution 12.0mLDistilledwater 1000mLA.12.2 Preparation


19

A.12.2.1 Preparetheglucose fermentationtubewith theabovecomponents,andadjust thepHto7.4±0.2.Add0.5%glucose,dispensethemediumintosmalltesttubescontaininginversetubes,andsterilizefor15mininthe

autoclavesetat121℃.

A.12.2.2 Prepareotherglucosefermentationtubeswiththeabovecomponents,dispensethemediumintobottles

inthequantitiesof100mL,andsterilizefor15min intheautoclavesetat121℃.Preparethe10%solutionof

eachglucose,andsterilizeintheautoclave.Dispense5mLofglucosesolutioninto100mLofmedium,andthenasepticallydispenseintosmalltesttubes.Notes: If sucrose is not pure, it can hydrolyze itself after heating. Filtration method should be employed toremovebacteria.A.12.3 Testmethod:Pickasmallamountofthecultureandinoculate,andthenincubateat36℃±1℃for2d~3d,

andexamine.Delayedreactionshouldbeobservedfor14d~30d.A.13 ONPGmediumA.13.1 CompositionO-Nitrophenyl-β-D-galactopyranoside(ONPG) 60.0mg0.01mol/Lsodiumphosphatebuffersolution(pH7.5) 10.0mL1%peptonewater(pH7.5) 30.0mLA.13.2 PreparationDissolveOPNGinthebuffersolution,addpeptonewater,sterilizebyfiltration,dispensethemediumintosterilesmalltesttubesinthequantitiesof0.5mL,andstuffthetubeswithrubberstoppers.A.13.3 TestmethodPickafullloopoftheculturefromtheagarslantandinoculate,incubateat36℃±1℃for1h~3hand24h,and

observetheresults.Thecolorwillturntoyellowwithin1h~3hifitproducesβ-galactosidase;otherwise,therewillbenochangeinthecolor.A.14 SemisolidmediumA.14.1 CompositionBeefextract 0.3gPeptone 1.0gSodiumchloride 0.5gAgar 0.35g~0.4gDistilledwater 100mLA.14.2 PreparationPreparetheabovecomponents,heattodissolvethem,andadjustthepHto7.4±0.2.Dispenseintosmalltest


20

tubes.Sterilizefor15minintheautoclavesetat121℃.Allowthemediumtosolidifyverticallyforlateruse.

Notes:Itisusedformotilityobservation,strainstorage,andphasevariationof H-antigens.A.15SodiummalonatemediumA.15.1CompositionYeastextract 1.0gAmmoniumsulfate 2.0gPotassiumhydrogenphosphate 0.6gPotassiumdihydrogenphosphate 0.4gSodiumchloride 2.0gSodiummalonate 3.0g0.2%Bromthymolphenolbluesolution 12.0mLDistilledwater 1000mLA.15.2PreparationDissolvealltheabovecomponentsexcludeyeastextractinwater,adjustthepH to6.8±0.2,addtheindicator,dispenseintotesttubes,andsterilizefor15minintheautoclavesetat121℃.A.15.3TestmethodInoculatethefreshagarculture,incubateat36℃±1℃for48h,andobservetheresults.Ifthecolorturnstoblue

fromgreen,itcanbeviewedasanindicatorofapositivereaction.


21

AnnexB

CommonSalmonellaAntigen

RefertoTableB.1forCommonSalmonellaAntigen.

TableB.1CommonSalmonellaAntigen

Bacterianame Latinbacterianame OantigenH antigen

Phase1 Phase2GroupA

甲型副伤寒沙门氏菌 S.paratyphiA 1,2,12 a [1,5]GroupB

基桑加尼沙门氏菌 S.kisangani 1,4,[5],12 a 1,2阿雷查瓦莱塔沙门氏菌 S.arechavaleta 4,[5],12 a 1,7马流产沙门氏菌 S.abortusequi 4,12 - e,n,x

乙型副伤寒沙门氏菌 S.paratyphiB 1,4,[5],12 b 1,2利密特沙门氏菌 S.limete 1,4,12,[27] b 1,5阿邦尼沙门氏菌 S.abony 1,4,[5],12,27 b e,n,x

维也纳沙门氏菌 S.wien 1,4,12,[27] b l,w伯里沙门氏菌 S.bury 4,12,[27] c z6斯坦利沙门氏菌 S.stanley 1,4,[5],12,[27] d 1,2圣保罗沙门氏菌 S.saintpaul 1,4,[5],12 e,h 1,2里定沙门氏菌 S.reading 1,4,[5],12 e,h 1,5彻斯特沙门氏菌 S.chester 1,4,[5],12 e,h e,n,x德尔卑沙门氏菌 S.derby 1,4,[5],12 f,g [1,2]阿贡纳沙门氏菌 S.agona 1,4,[5],12 f,g,s [1,2]埃森沙门氏菌 S.essen 4,12 g,m -

加利福尼亚沙门氏菌 S.california 4,12 g,m,t [z67]金斯敦沙门氏菌 S.kingston 1,4,[5],12,[27] g,s,t [1,2]布达佩斯沙门氏菌 S.budapest 1,4,12,[27] g,t -鼠伤寒沙门氏菌 S.typhimurium 1,4,[5],12 i 1,2拉古什沙门氏菌 S.Lago 1,4,[5],12 i 1,5布雷登尼沙门氏菌 S.Lago 1,4,12,[27] l,v 1,7基尔瓦沙门氏菌Ⅱ S.kilwaII 4,12 l,w e,n,x海德尔堡沙门氏菌 S.heidelberg 1,4,[15],12 r 1,2印地安纳沙门氏菌 S.indiana 1,4,12 z 1,7斯坦利维尔沙门氏菌 S.stanleyville 1,4,[5],12,[27] z4,z23 [1,2]伊图里沙门氏菌 S.ituri 1,4,12 z10 1,5

C1group

奥斯陆沙门氏菌 S.oslo 6,7,14 a e,n,x

爱丁保沙门氏菌 S.edinburg 6,7,14 b 1,5

布隆方丹沙门氏菌Ⅱ S.bloemfonteinII 6,7 b [e,n,x]:z42丙型副伤寒沙门氏菌 S.paratyphiC 6,7,[Vi] c 1,5猪霍乱沙门氏菌 S.choleraesuis 6,7 c 1,5猪伤寒沙门氏菌 S.typhisuis 6,7 c 1,5


22

罗米他沙门氏菌 S.lomita 6,7 e,h 1,5布伦登卢普沙门氏菌 S.braenderup 6,7,14 e,h e,n,z15里森沙门氏菌 S.rissen 6,7,14 f,g -

蒙得维的亚沙门氏菌 S.montevideo 6,7,14 g,m,[p],s [1,2,7]里吉尔沙门氏菌 S.riggil 6,7 g,[t] -奥雷宁堡沙门氏菌 S.oranieburg 6,7,14 m,t [2,5,7]奥里塔蔓林沙门氏菌 S.oritamerin 6,7 i 1,5汤卜逊沙门氏菌 S.thompson 6,7,14 k 1,5康科德沙门氏菌 S.concord 6,7 l,v 1,2伊鲁木沙门氏菌 S.irumu 6,7 l,v 1,5姆卡巴沙门氏菌 S.mkamba 6,7 l,v 1,6波恩沙门氏菌 S.bonn 6,7 l,v e,n,x波茨坦沙门氏菌 S.potsdam 6,7,14 l,v e,n,z15格但斯克沙门氏菌 S.gdansk 6,7,14 l,v z6维尔肖沙门氏菌 S.virchow 6,7,14 r 1,2婴儿沙门氏菌 S.infantis 6,7,14 r 1,5巴布亚沙门氏菌 S.papuana 6,7 r e,n,z15巴累利沙门氏菌 S.bareilly 6,7,14 y 1,5哈特福德沙门氏菌 S.hartford 6,7 y e,n,x三河岛沙门氏菌 S.mikawasima 6,7,14 y e,n,z15姆班达卡沙门氏菌 S.mbandaka 6,7,14 z10 e,n,z15田纳西沙门氏菌 S.tennessee 6,7,14 z29 [1,2,7]

布伦登卢普沙门氏菌 S.braenderup 6,7,14 e,h e,n,z15耶路撒冷沙门氏菌 S.jerusalem 6,7,14 z10 l,w

C2group习志野沙门氏菌 S.narashino 6.8 a e,n,x名古屋沙门氏菌 S.nagoya 6,8 b 1,5加瓦尼沙门氏菌 S.gatuni 6,8 b e,n,x慕尼黑沙门氏菌 S.muenchen 6,8 d 1,2曼哈顿沙门氏菌 S.manhattan 6,8 d 1,5纽波特沙门氏菌 S.newport 6,8,20 e,h 1,2科特布斯沙门氏菌 S.kottbus 6,8 e,h 1,5茨昂威沙门氏菌 S.tshiongwe 6,8 e,h e,n,z15林登堡沙门氏菌 S.lindenburg 6,8 i 1,2塔科拉迪沙门氏菌 S.takoradi 6,8 i 1,5波那雷恩沙门氏菌 S.bonariensis 6,8 i e,n,x利齐菲尔德沙门氏菌 S.litchfield 6,8 l,v 1,2病牛沙门氏菌 S.bovismorbificans 6,8,20 r,[i] 1,5查理沙门氏菌 S.chailey 6,8 z4,z23 e,n,z15

C3group巴尔多沙门氏菌 S.bardo 8 e,h 1,2依麦克沙门氏菌 S.emek 8,20 g,m,s -肯塔基沙门氏菌 S.kentucky 8,20 i z6

Dgroup仙台沙门氏菌 S.sendai 1,9,12 a 1,5伤寒沙门氏菌 S.typhi 9,12,[Vi] d -


23

塔西沙门氏菌 S.tarshyne 9,12 d 1,6伊斯特本沙门氏菌 S.eastbourne 1,9,12 e,h 1,5以色列沙门氏菌 S.israel 9,12 e,h e,n,z15肠炎沙门氏菌 S.enteritidis 1,9,12 g,m [1,7]布利丹沙门氏菌 S.blegdam 9,12 g,m,q -沙门氏菌Ⅱ SalmonellaII 1,9,12 g,m,[s],t [1,5,7]都柏林沙门氏菌 S.dublin 1,9,12,[Vi] g,p -芙蓉沙门氏菌 S.seremban 9,12 i 1,5巴拿马沙门氏菌 S.panama 1,9,12 l,v 1,5戈丁根沙门氏菌 S.goettingen 9,12 l,v e,n,z15爪哇安纳沙门氏菌 S.javiana 1,9,12 L,z28 1,5鸡-雏沙门氏菌 S.gallinarum-pullorum 1,9,12 - -

E1group奥凯福科沙门氏菌 S.okefoko 3,10 c z6瓦伊勒沙门氏菌 S.vejle 3,{10},{15} e,h 1,2明斯特沙门氏菌 S.muenster 3,{10}{15}{15,34} e,h 1,5鸭沙门氏菌 S.anatum 3,{10}{15}{15,34} e,h 1,6纽兰沙门氏菌 S.newlands 3,{10},{15,34} e,h e,n,x火鸡沙门氏菌 S.meleagridis 3,{10}{15}{15,34} e,h l,w雷根特沙门氏菌 S.regent 3,10 f,g,[s] [1,6]西翰普顿沙门氏菌 S.westhampton 3,{10}{15}{15,34} g,s,t -

阿姆德尔尼斯沙门氏菌 S.amounderness 3,10 i 1,5新罗歇尔沙门氏菌 S.new-rochelle 3,10 k l,w恩昌加沙门氏菌 S.nchanga 3,{10}{15} l,v l,2新斯托夫沙门氏菌 S.sinstorf 3,10 l,v 1,5伦敦沙门氏菌 S.london 3,{10}{15} l,v 1,6吉韦沙门氏菌 S.give 3,{10}{15}{15,34} l,v 1,7鲁齐齐沙门氏菌 S.ruzizi 3,10 l,v e,n,z15乌干达沙门氏菌 S.uganda 3,{10}{15} l,z13 1,5乌盖利沙门氏菌 S.ughelli 3,10 r 1,5

韦太夫雷登沙门氏菌 S.weltevreden 3,{10}{15} r z6克勒肯威尔沙门氏菌 S.clerkenwell 3,10 z l,w列克星敦沙门氏菌 S.lexington 3,{10}{15}{15,34} z10 1,5

E4group萨奥沙门氏菌 S.sao 1,3,19 e,h e,n,z15

卡拉巴尔沙门氏菌 S.calabar 1,3,19 e,h l,w山夫登堡沙门氏菌 S.senftenberg 1,3,19 g,[s],t -斯特拉特福沙门氏菌 S.stratford 1,3,19 i 1,2塔克松尼沙门氏菌 S.taksony 1,3,19 i z6索恩保沙门氏菌 S.schoeneberg 1,3,19 z e,n,z15

Fgroup昌丹斯沙门氏菌 S.chandans 11 d [e,n,x]阿柏丁沙门氏菌 S.aberdeen 11 i 1,2布里赫姆沙门氏菌 S.brijbhumi 11 i 1,5威尼斯沙门氏菌 S.veneziana 11 i e,n,x

阿巴特图巴沙门氏菌 S.abaetetuba 11 k 1,5


24

鲁比斯劳沙门氏菌 S.rubislaw 11 r e,n,xOthergroups

浦那沙门氏菌 S.poona 1,13,22 z 1,6里特沙门氏菌 S.ried 1,13,22 z4,z23 [e,n,z15]

密西西比沙门氏菌 S.mississippi 1,13,23 b 1,5古巴沙门氏菌 S.cubana 1,13,23 z29 -苏拉特沙门氏菌 S.surat [1],6,14,[25] r,[i] e,n,z15松兹瓦尔沙门氏菌 S.sundsvall [1],6,14,[25] z e,n,x非丁伏斯沙门氏菌 S.hvittingfoss 16 b e,n,x威斯敦沙门氏菌 S.weston 16 e,h z6上海沙门氏菌 S.shanghai 16 l,v 1,6自贡沙门氏菌 S.zigong 16 l,w 1,5巴圭达沙门氏菌 S.baguida 21 z4,z23 -迪尤波尔沙门氏菌 S.dieuoppeul 28 i 1,7卢肯瓦尔德沙门氏菌 S.luckenwalde 28 z10 e,n,z15拉马特根沙门氏菌 S.ramatgan 30 k 1,5阿德莱沙门氏菌 S.adelaide 35 f,g -旺兹沃思沙门氏菌 S.wandsworth 39 b 1,2雷俄格伦德沙门氏菌 S.riogrande 40 b 1,5莱瑟沙门氏菌 S.letheII 41 g,t -达莱姆沙门氏菌 S.dahlem 48 k e,n,z15沙门氏菌IIIb SalmonellaIIIb 61 l,v 1,5,7

Note:explanationonmarksinthetable{}={}Oelementmeansexclusive.Theserotypeelementinthe{}cannotcoexistwithotherelementsinthe{}, take O:3 as an example, when 0:3, 10 colony produces O:15 or O15,34, then it will replace O:10elements.[]=O(withoutunderline)orthereisnorelationshipbetweentheexistingofHelementandtransformationofphage,suchas[5]elementintheO:4colony.Helementinthe[]meansraretoseeinthewildcolony,forexample,mostS.ParatyphiAhasa(a)phase,butrarelyhas2phase(1,5)colony.Soitcanbeillustratedas1,2,12:a:[15]._=underlinemeanstheOelementiscreatedbylysogenizationofphage.

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