Experiment Three：Isolation and identification of pyogenic cocci（《医学微生物学》课件）.ppt

Experiment Three：Isolation and identification of pyogenic cocci（《医学微生物学》课件）.ppt

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IsolationandidentificationofpyogeniccocciExperimentThreeObjectiveofExperimentExperimentnnTomasterbasicprincipleandmethod,whichusetoisolateandidentifypyogeniccoccifromclinicalspecimens.nnTodiagnoseclinicaldiseaseandguidedoctorstoselectpropermedicines.Specimens（Gram-stain）Morphologicalcharacteristics（Microscopicexamination）IsolationcultureSmears（bloodagarplate）ColonialcharacteristicsHemolyticreactionPigmentationPROCEDUREPROCEDUREmorphologyobservationcolonialtypicalcoloniesstainedandobservePurecultureAntibioticsusceptibilitytestDirectidentificationIdentifiedmethodforStaphylococcinnGram-stainGram-stainnnIsolationIsolationandcultureandculturennPureculturePureculturennDirectDirectidentifidentificationicationStaphylococciareGram-positivecocci,typicallyarrangedinclumpsorGrape-likeclustersDirectidentificationnnThemannitolfermentationtest.nnTheCoagulaseTestnnTheDnaseTestnnThephagetypingtestnnAnimalExperimentThemannitolfermentationtest.nnInoculatethebacteriaintoamannitolmicro-tube,incubateat370Cfor18h.S.aureuswillfermentmannitoltoproduceacid,whichcausesthemediumtoturnyellow.TestofMicrobiologicalexperimentnnWhileapatientisinfectedbypathogenicenterobacteria（Salmonella）,howtodiagnosewithmicrobiologicalmethodsinlab?

（simpleprocedure）nnWhileapatientisinfectedbypyogeniccocci,howtodiagnosewithmicrobiologicalmethodsinlab?

（simpleprocedure）nnWhileapatientisinfectedbyinfluenzavirus,howtodiagnosewithmicrobiologicalmethodsinlab?

（simpleprocedure）TheCoagulaseTestnnCoagulaseCoagulaseisanenzymeconvertingfibrinogenisanenzymeconvertingfibrinogenintofibrinpromotingbloodclotting.intofibrinpromotingbloodclotting.nnItmightbeavirulencefactorwiththeItmightbeavirulencefactorwiththecoagulatedbloodaroundthebacteriaprotectingcoagulatedbloodaroundthebacteriaprotectingthemfromtheimmunesystem.themfromtheimmunesystem.nnCoagulaseCoagulase-negativestrainsareoftenas-negativestrainsareoftenaspathogenicaspathogenicascoagulasecoagulase-positivestrains.-positivestrains.TheSlideCoagulaseTestTheTubecoagulaseTestLeftLefttubetubecoagulasecoagulasepositivepositiveRighttubeRighttubecoagulasecoagulasenegativenegativeTheDnaseTestnnInoculateInoculateDnaseDnaseagarplateswithaloopsoagarplateswithaloopsothatthegrowthisinplaquesabout1cminthatthegrowthisinplaquesabout1cmindiameter.Incubateat37diameter.Incubateat3700Covernight.FloodCovernight.Floodtheplatewith1Nhydrochlorictheplatewith1Nhydrochloricacid.Clearingaroundthecoloniesacid.ClearingaroundthecoloniesindicatesindicatesDnaseDnaseactivity.Thehydrochloricactivity.Thehydrochloricacidreactswithunchangedacidreactswithunchangeddeoxyribonucleicacidtogiveacloudydeoxyribonucleicacidtogiveacloudyprecipitate.Afewotherbacteria,e.g.precipitate.Afewotherbacteria,e.g.SerratiaSerratia,maygiveapositivereaction.,maygiveapositivereaction.ThephagetypingtestnnThistestisusedtotracetheinfectiveagentinepidemiologyifnecessary.nnItisusuallynotdoneforroutineclinicalpurpose.AnimalExperimentvomitexcrementremainderedfoodIsolationandidentificationofbacteriaMeatsoupmediafilterinjection68wcatobservationFoodpoisoningTheantibioticsusceptibilitytestnnThistestishelpfulforthetreatmentofS.aureusinfection.nnmaterialsuuStaphaureusStaphaureus（isolatedfromthepusofisolatedfromthepusofapatientapatient）.uuSeveralkindsoffilterpaperSeveralkindsoffilterpaper（eacheachcontainsdifferentkindsofantibioticscontainsdifferentkindsofantibiotics）uuNutrientagarplateNutrientagarplateTheantibioticsusceptibilitytestStreakingthestaphaureusonagarplate（thoroughlycoveredtheplate）1.Put4kindsofpapercontaineddifferentantibioticsontheplate（eachpaperarefarawayabout2cm）Incubateat370Cfor18-24hours.ObservetheresultstheplatesThesensitivityoftheorganismisindicatedbythediameterofthezoneofgrowthinhibition.LaboratoryDiagnosisofPathogenicEnterobacterialInfectionDimidiationoftheenterobacteriaaccordingtothefermentationoflactosennLactaseLactasefermentersfermenters:

saprophyticandsaprophyticandcommensalcommensalEscherichiaKlebsiellaCitrobaterEscherichiaKlebsiellaCitrobaterEnterobacterEnterobacternnNonlactaseNonlactasefermentersfermenters:

pathogenspathogensSalmonellaSalmonellaShigellaShigellasomesomeCitrobacterCitrobacterProteusProteusSerratiaSerratiaColonialcharacteristicobservationSpecimensisolationGramStaining（SS/EMBplate）SerologicalidentificationTSIBiochemicalreactionProcedureSpecimensnnDifferentspecimensshouldbetakenDifferentspecimensshouldbetakendependingonthekindandtheprocessofdependingonthekindandtheprocessofthedisease.thedisease.bloodbloodbonemarrowbonemarrowUrineUrinestoolstoolIsolationnnCulturemedium:

S.SagarnnMethod:

streakplatennResult:

Pathogeneticcolonies:

middlesize,redSuspectcolonies:

colorless,small,opaqueBiochemicalreactionso