微生物实验：实验5 肠道杆菌English

1、Enteric bacterianDemonstrationEnteric bacteria(E.coli, Shigellae, Salmonellae, Proteus)nMorphology & stainingnGrowth characteristics on SSSS agar plate and EMBagar plate and EMB agar plate)agar plate)nBiochemical reaction: kligers iron slant, Biochemical reaction: kligers iron slant, Urease test

2、Urease testnOperationWidal testLaboratory diagnosis of enteric bacteriamannitol fermentation test （Continued）Antimicrobial Susceptibility test （Continued）n VideoAnaerobic bacteriaClassificationEnterobacteriaceaeShigellaEscherichiaSalmonellaProteusKlebsiellaETEC, EIEC, EPEC, EHEC, EAECS. dysenteriae,

3、S.flexneri,S. boydii,S. sonneiS. typhi, S.entericaBasic characteristicsnGram-negative rodnEasy to culture, form circular,convex, smooth colonies with distinct edges on basic medianBiochemical reaction: activenComplx antigenic structurenResistance: not strongnEasy to mutate Widal test be used to help

4、 make a presumptive diagnosis of enteric fevernPrinciple ：S.typhi O antigenS.typhi H antigenS.paratyphi A H antigen S.schottmuelleri H antigen Known antigensUnknown antibody in patient serumagglutination12345678S.typhi O0.2ml0.2mlS.typhi H 0.2ml0.2mlS.paratyphi A H0.2ml0.2mlS.Schottmuelleri H0.2ml0.

5、2mlInitial dilution1:201:401:801:1601:3201:6401:1280ControlSalineFinal 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 Ant

6、ibody(serum)2. Adding antigen3. Incubating at 37, 60(at 45, 30)4. Reading the resultsLaboratory diagnosis of pathogenic Enteric bacteria (stool)2 suspected colonies2 suspected colonies(Small, colorless, transparent )Inoculated in Kliglers iron slantSpecimen: stooldifferential and selective medium SS

7、 agar plateBiochemical reaction Kliglers iron slantserologic test Slide agglutinationCultivated SS agar plate(Shigella & Salmonella) a differential and selective medium used for initial isolation of enteric bacteriaContains：basic medium, lactose, sodium citrate, sodium thiosulfate, iron citrate,

8、 bile salts, brilliant green, neutral red (pH6.8-8.0, redpH6.8-8.0, redyellowyellow）Usage：lactose , neutral red ：nLactose fermented: ferment lactose with sufficient acid production to change the neutral red form red coloniesnLactose not fermented: colorless coloniesSodium citrate, brilliant green, b

9、ile salts: inhibit E.coli Result:Result:E.coliShigellae & SalmonellaeRed, large, opaque coloniesSmall, colorless, transparent coloniesEMB agar plate Contains：basic medium, lactose, eosin , methylene blueUsage：Lactose fermented: ferment lactose to produce acid, then make eosin and methylene blue

10、to be a BLACK compound. Lactose not fermented: colorless coloniesResult:Result:E.coliShigellae & Salmonellaelarge, black，opaque coloniesSmall, colorless, transparent colonies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

11、-8.4, 黄黄 红）红）Biochemical reaction（ Kliglers iron slant 改良克氏双糖铁琼脂斜面）nOberservation Lactose（10）Glucose（1）Ferrous sulfateindicator: phenol redpH6.8-8.4pH6.8-8.4YellowYellowredredGlucoselactoseH H2 2S S+-+SlantYellow Red blackBottomYellow and bubbleYellow blackInocuating Kliglers iron agar slant nInocul

12、ating needlenRemoving cells from ss agar plate with a needlenStab needle to bottom of the tube nAs the needle is withdrawn from the agar, streak it across the face of the slant from bottom to the topnIncubate at 37 Lactose Glucose H2S Urease E.coli +S. dysenterias + -S. typhi + +/-S. paratyphi A + -

13、Proteus + + Laboratory diagnosis of pathogenic Enteric bacteria (stool)Specimen: stooldifferential and selective medium SS agar plateBiochemical reaction Kliglers iron slantserologic test Slide agglutinationReading the results of Widal testnExamining the control tubes first: negativenThe agglutinati

14、on of O antigenas a “matt” or “carpet” at the bottom.nThe agglutination of H antigens loose, wooly or cottonyn（）（）liquid clear, all antigen are agglutinate at the bottom.（）（） liquid clear, most of antigen(75%) are agglutinate at the bottom（）（） liquid semitransparent , part of antigen are agglutinate

15、 at the bottom obviously.（）（） liquid turbid, a little antigen are agglutinate at the bottom.（）（）negative, same as controlAgglutination titer: the highest dilution of “+”Interpretation of widal testnbaseline：S.typhi O antibody 1：80， S.typhi H antibody 1：160, S.paratyphi A H antibody 1：80nA rise in titre between