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1、EntericbacterianDemonstrationEntericbacteria(E.coli,Shigellae, Salmonellae, Proteus)nMorphology&stainingnGrowthcharacteristicsonSSSS agar plate and EMBagar plate and EMB agar agar plate)plate)nBiochemical reaction: kligerBiochemical reaction: kligers iron slant, s iron slant, Urease testUrease testn
2、OperationWidaltestLaboratorydiagnosisofentericbacteriamannitolfermentationtest(Continued)AntimicrobialSusceptibilitytest(Continued)n VideoAnaerobicbacteriaClassificationEnterobacteriaceaeShigellaEscherichiaSalmonellaProteusKlebsiellaETEC, EIEC, EPEC, EHEC, EAECS. dysenteriae,S.flexneri,S. boydii,S.
3、sonneiS. typhi, S.entericaBasiccharacteristicsnGram-negative rodnEasy to culture, form circular,convex, smooth colonies with distinct edges on basic medianBiochemical reaction: activenComplx antigenic structurenResistance: not strongnEasy to mutate Widaltestbeusedtohelpmakeapresumptivediagnosisofent
4、ericfevernPrinciple:S.typhi OantigenS.typhiHantigenS.paratyphiAHantigenS.schottmuelleriHantigenKnown antigensUnknown antibody in patient serumagglutination12345678S.typhi O0.2ml0.2mlS.typhi H 0.2ml0.2mlS.paratyphi A H0.2ml0.2mlS.Schottmuelleri H0.2ml0.2mlInitial dilution1:201:401:801:1601:3201:6401:
5、1280ControlSalineFinal dilution1:401:801:1601:3201:6401:12801:2560 Ag test Abknown unknownSerum 1:10 1:20 0.2ml4 0.8 ml 1.6mlSaline 0.8 ml mixing 0.8ml mixing 1:40 0.2ml4 Saline 0.8 ml 1.6ml 0.8ml mixing Saline 0.8 ml 1:80 Adding antigen1. Diluting Antibody(serum)2. Adding antigen3. Incubating at 37
6、, 60(at 45, 30)4. Reading the resultsLaboratory diagnosis of pathogenic Enteric bacteria (stool)2 suspected colonies2 suspected colonies(Small,colorless,transparent)InoculatedinKliglersironslantstoolSpecimen:SS agar platedifferential and selective medium Kliglers iron slantBiochemical reactionSlide
7、agglutinationserologictestCultivatedSS agar plate(Shigella & Salmonella) adifferentialandselectivemediumusedforinitialisolationofentericbacteriaContains:basicmedium,lactose,sodiumcitrate,sodiumthiosulfate,ironcitrate,bilesalts,brilliantgreen,neutralred(pH6.8-8.0, redpH6.8-8.0, redyellowyellow)Usage:
8、lactose,neutralred:nLactose fermented:fermentlactosewithsufficientacidproductiontochangetheneutralredformredcoloniesnLactose not fermented: colorless coloniesSodiumcitrate,brilliantgreen,bilesalts:inhibitE.coliResult:Result:E.coliShigellae & SalmonellaeRed,large,opaquecoloniesSmall,colorless,transpa
9、rentcoloniesEMB agar plate Contains:basicmedium,lactose,eosin,methyleneblueUsage:Lactose fermented:fermentlactosetoproduceacid,thenmakeeosinandmethylenebluetobeaBLACKcompound.Lactose not fermented: colorless coloniesResult:Result:E.coliShigellae & Salmonellaelarge,black,opaquecoloniesSmall,colorless
10、,transparentcolonies双糖管n接种方法n观察上层含乳糖部分n发酵乳糖致病菌红色,非致病菌黄色;nH2S试验大肠杆菌无黑色肖氏沙门菌出现黑色然后观察下层n发酵葡萄糖(产酸产气)及动力乳糖乳糖,Fe2+固体固体葡萄糖葡萄糖半固体半固体指示剂指示剂:酚红(酚红(pH6.8-8.4, 黄黄 红)红)Biochemicalreaction(Kliglersironslant改良克氏双糖铁琼脂斜面)nOberservationLactose(10)Glucose(1)Ferrous sulfateindicator:phenolredpH6.8-8.4pH6.8-8.4YellowYel
11、lowredredGlucoselactoseH H2 2S S+-+SlantYellowRedblackBottomYellowandbubbleYellowblackInocuatingKliglersironagarslantnInoculatingneedlenRemovingcellsfromssagarplatewithaneedlenStabneedletobottomofthetubenAstheneedleiswithdrawnfromtheagar,streakitacrossthefaceoftheslantfrombottomtothetopnIncubateat37
12、 Lactose Glucose H2S Urease E.coli +S. dysenterias + -S. typhi + +/-S. paratyphi A + -Proteus + + Laboratory diagnosis of pathogenic Enteric bacteria (stool)stoolSpecimen:SS agar platedifferential and selective medium Kliglers iron slantBiochemical reactionSlide agglutinationserologictestReadingther
13、esultsofWidaltestnExaminingthecontroltubesfirst:negativenTheagglutinationofOantigenasa“matt”or“carpet”atthebottom.nTheagglutinationofHantigensloose,woolyorcottonyn()()liquid clear, all antigen are agglutinate at the bottom.()() liquid clear, most of antigen(75%) are agglutinate at the bottom()() liq
14、uid semitransparent , part of antigen are agglutinate at the bottom obviously.()() liquid turbid, a little antigen are agglutinate at the bottom.()()negative, same as controlAgglutination titer: the highest dilution of “+”Interpretationofwidaltestnbaseline:S.typhi Oantibody 1:80, S.typhi Hantibody 1:160, S.paratyphiAHantibody 1:80nA rise in titre between two sera specimens is more meaningful than a single test.0 antibodyH antibodyInterpretationHighHighentericfeverNormalNormalNormalNormalHighsufferedinthepast,vaccinatedHighNormalintheearlystages,Cross-reactionTheEndThanks