微生物學(xué)課件：10 微生物鑒定

Chapter10

TheIdentificationofMicroorganisms1LearningobjectivesforthissectionFollowingcompletionofthissectionyoushouldbeableto;DeterminetheappropriatemethodsforidentificationofdifferentMOsDifferentiatebetweenculturemethodsandbiochemicalmethodsofMOidentificationUnderstandsomeoftherapidmethodsusedforMOdetectionIdentificationofMicrobesMaintainpurecultureAscertaincolonymorphologyStainingtechniques–cellshape,arrangementsandtypeofcellwallUseofSelectiveandDifferentialmediaBiochemicalandSerologicaltests-APIkits/EnterotubeandBiologRapidmethodsIdentificationofMicrobesInitialstepsinvolveestablishmentofpureculture(singlespecies):StreakplatePourplatePickisolates(individualcolonies)fromplatesandpreparepureculturebye.g.streakingontonewplateorinoculatingsterilebroth.ColonyMorphologyDifferenttypesofbacteriahavedifferentappearancesor“morphology”ondifferentagar.Candistinguishdifferentmorphologiesbasedonshape,size,elevation,colour,opacity,edgepattern,surfaceetcEg:Micrococcus

spponnutrientagarisyellow,circular,smoothedgewithconvexelevation.Eg:Bacillus

sppoftenhasirregularshaped,beige(淺褐色）,

lobate（葉狀）orcurled（卷發(fā)狀）,slimycoloniesonnutrientagar.MicrococcusleuteusBacilluscereusDifferentiationbetweentwobacteriaColonyMorphologyStainingTechniquesLivingbacteriaalmostcolourlessBacteriaarechemicallydifferenttotheirsurroundingsandthereforereactdifferentlytostainsthanbackground,makingthemdistinguishableandvisible.Commonlyusedstainsinclude:BasicstainGramstain,Acid-faststainStructuralstains:Endospore,capsulestainDirectstainingwithBasicdyesBacterialcellmemnormallynegativelychargedinneutralpHenvironmentThereforeattractpositivelychargedions,suchasthoseindyesE.g.methyleneblue-+ion.FixsmearofbacterialcultureonslideBasicdyes(wherecolourisin+veionofthestain)includecrystalviolet,safranin,malachitegreenandmethyleneblue.Othernegativelychargedstains,eosinandpicric,bindanyposchargedcellcomponentsDirectstainingSimplestain:SingledyeRevealsbasiccellshape,andarrangementExamples:Methyleneblue,safranin,carbolfuchsin,crystalvioletDifferentialstain:TwoormoredyesDistinguishbetweentwokindsoforganismortwopartoforganismExample:Gramstain,Ziehl-Neelsenacid-faststain,Schaeffer-FultonsporestainGramStainTypeofdifferentialstain–dividesbacteriainto2groups,GrampositiveorGramnegativeDevelopedin1884byDanishphysicianChristianGramBasedondifferencesincellwallHowdoGramnegativeandGrampositivebacterialcellwallsdiffer?Drawadiagramofboth.CulturemethodsforidentifyingmicroorganismsSelective&differentialmediaEMBagar–aselectiveanddifferentialagarSelective:methyleneblueinhibitsgrowthofGram+Differential:Lactosefermentersmetabolizethelactoseinthemediaandproducelacticacid(causingacolourchangeinthecolony).

Strongacidproduction(E.coli)resultsinametallicgreensheen.Weakerfermentationoflactoseresultsinpinkish-purplecolonies.

Coloniesofnonlactose

fermentersremaincolorless,oratleastarenodarkerthanthecolourofthemedia.McCA–aselectiveanddifferentialmediaSelective:BilesaltsandcrystalvioletpreventGram+growthDifferential:pHindicator(neutralreddye,bromocresolpurple)-byutilizingthelactoseavailableinthemedium,Lac+bacteriasuchasE.coliandKlebsiellawillproduceacid.OncethepHoftheagarlowersbelow6.8andresultsintheappearanceofred/pinkcolonies.Non-LactosefermentingbacteriasuchasSalmonellaandShigellacannotutilizelactose,andwillusepeptoneinstead.Thisformsammonia,whichraisesthepHoftheagar,andleadstotheformationofwhite/colorlesscolonies.Triple-sugarironagar(TSI)NotselectiveUsedinagarslantsDifferentiatesbetweenaerobic(slant)growthandanaerobic(butt)growthMediumcontains3keyingredientsinkeyconc’sGlucose(0.1%)Sucrose(1%)Lactose(1%)Sulphur&ironalsoMeasureofabacterium'sabilitytoutilizethesesugarsandferroussulfateFermisdetectedbypHindicatorAcidproduction=yellowcolourchangeinmediaAlkalinesurrounding=nocolorchange(red)InoculationofagarslantsStep1-SmearinocloopacrosssurfaceofagarStep2-StabinocloopdeeptobottomofagarIncubateagarslantsatappropriatetempSugarfermentationcanbedeterminedbyanalysingtheextentofacidproductionTube1-uninoculatedTSImediumTube2–obligateaerobeTube3–H2SformingbacteriaTube4–GlucoseonlyfermentorTube5-Lac+/Suc+ferm,H2STube6–Lac+/Suc+ferm,gasTube7–Lac+/Suc+ferm,nogas1345762InterpretationofresultsButtyellowonly-Ifonly

glucose

isfermented(0.1%)Insufficientacidproductsareformedtoaffectthemethylredintheslant.Buttandslantyellow-eithersucroseorlactose(both1%)arefermentedIfgasisformedduringthefermentation,itwillshowinthebutteitherasbubblesorascrackingoftheagar.Buttandslantred-Nofermentation(e.g.obligateaerobes).Blackprecipitateinthebutt-bacteriumformsHydrogensulphate(H2S),thischemicalwillreactwithsodiumthiosulphateinmediumtoformferroussulfide(black).InterpretationofresultsRedslant+Redbutt

(tube2)=Pseudomonas,Acinetobacter（不動菌屬）,

AlcaligenesYellowslant+Yellowbutt

(tube7)=Escherichia,Enterobacter,KlebsiellaYellowslant+Yellowbutt

+H2S(tube5)=Citrobacter,Arizona,someProteussp’sRedslant

+Yellowbutt(tube4)=ShigellaandsomeProteussp’sRedslant

+Yellowbutt

+H2S(glucoseonly)=mostSalmonella,citrobacter,ArizonaCROMagar–IdentificationofyeastDifferentiationofseveralpathogensbyamulticolormethodCHROMagarOrientation-allowsdetectionanddifferentiationofurinarytractpathogensCHROMagar

Candida-allowsthedifferentiationofvariousCandidaspecies.ColoniesofspecificmicroorganismarerecognizablebytheircolorCHROMagarMRSA,CHROMagar

SalmonellaCROMagarIdentificationofbacteriausingbiochemicaltestsBiochemicaltestsCatalasetestCoagulasetestOxidasetestIMViCtestAPIkits/Enterotube/BiologTheCatalaseTestTestsforpresenceofenzyme‘catalase’.Whatdoescatalasedo?Add3%hydrogenperoxide(H2O2)toculture(oncleanglassslide)orglassslidewithculturepresent.Catalasepositive-E.g.Staphylococcus(+),Streptococcus(–)E.g.Bacillus(+),Clostridium(–).CatalasetestCoagulasetestDifferentiatesbetweenpathogenicandnon-pathogenicstrainsofStaphylococcus.Coagulaseconvertsfibrinogen（纖維蛋白原）

tofibrin（纖維蛋白）–asinhostclottingmethodsIfaStaphsamplehasnocoagulaseactivity–excludesS.aureus++--CoagulasetestBacteriathatproducecoagulaseuseitasadefensemechanismClotbloodplasmasurroundingorganismHidingfromphagocycticcellsofhost'simmunesystem.Inoculateculturesampleinto0.5mlofrabbitplasmaandincubateat37oCfor1-4hours.Apositivetestisdenotedbyaclotformationinthetesttubeaftertheallottedtime.S.aureus(+),S.epidermidis(-)OxidasetestSomebacteriacontainanenzymecalledcytochromeoxidasethathastheabilitytotransferelectronstooxygen–finaleacceptorInthistestareagentactsastheeacceptorinsteadofO2.Dropofoxidasereagentaddedtothesuspectcolonyonapieceoffilterpaper.Ifpositivethecytochrome

oxidasewillcatalysetransferofetoreagent.Thisresultsinacolourchange-blueOxidasetestNocolourchangeindicatesanegativetest–reagentremainsclear.Enterobacteriaceaeareoxidaseneg.Pseudomonas

sppandNeisseriaareoxidasepos.IMViCtestsIMViCisshortforIndole,MethylRed,Voges-ProskauerandCitrate.IMViCtestisusefulfordifferentiatingEscherichiacoli,Enterobacter

aerogenes,Enterobactercloacae,andKlebsiella

pneumoniae

E.coliandE.aerogenesgivethefollowingresultstotheIMViCtests:E.coli

++--,E.aerogenes--++Plus’sandminus’sareaccumulatedfromthe4testsIMViCtestsIndoletest:

IndoleisacomponentoftheaatryptophanTryptophanisbrokendownreleasingindolebytheenzymetryptophanase–enzproducedbysomebacteria.Kovac’sreagentaddedtocultureIfindoleispresent-brightpinkcolourwillbeformedIfnoindolepresent–Kovac’sreagentremainsyellowIMViCtestsMethylredtest:

DemonstratesthemetabolismofglucosewithsubsequentacidproductionusingapHindicatorcalledmethylred,whichisyellowatpH6andredatpH4.Apositivetest:redcolourNegativetestresultsinyellowcolour.IMViCtestsVoges-Proskauertest:

Alsodemonstratesthemetabolismofglucose,butwiththeproductionofaneutralendproductinsteadofanacidendproduct.AdditionofVPreagents(alpha-naphtholfollowedbypotassiumhydroxide)willresultinaredcolourformationifthetestispositive.IMViCtestsCitratetest:

Simmon'scitrateagarcontainssodiumcitrateasthesolecarbonsource.Ifthecitrateisutilized,alkalineproductsareproducedpHindicatorinmediumturnsblueifpositive.Nogrowthifnegative–mediumremainsgreen+-AnalyticalProfileIndex(API)testkitsSeriesofbiochemicaltestsConsistsofmicrotubulescontainingdehydratedsubstratesPatternofpositiveandnegativeresultsusedtobuildprofilewhichcanthenbeenteredintosoftwaresystemtogiveidentification.DifferenttypesofAPIkits,API20EforEnterobacteriaAPI20NEforPseudomonadsAPIstaphforStaphylococcusspp.APU50CHforBacillusspp.MusthavepurecultureandbeID’dtothegenuslevelAPItest20separatetestcompartmentsareonthestripBacterialsuspensiontobetestedisusedtore-hydrateeachofthewellsSomeofthewellswillhavecolorchangesduetopHdifferencesOthersproduceendproductsthathavetobeidentifiedwithreagents.Aprofilenumberisdeterminedfromthesequenceof+and-testresultsExplainhowtheAPInumberprofileisgeneratedfromtheresultsbelowMicrotubulesinincubationtrayEnterotubesPlastictubewith12compartmentseachwithadifferentmedium.InoculatingwirepassesthroughmediaandprotrudesatbothendsOneendofwiretouchedagainstcolonyandisthendrawnthroughcompartmentsTherebyinoculatingallcompartmentsPatternsoffermentationandbiochemicalreactionsusedtoproduceprofile,whichcanbeusedtomakeanIDEnterotubesEnterotubesEnterotubesBiologBasedonCarbon-sourceutilisationConsistsofmicroplateof95wellscontainingvariouscarbonsourcesWhenorganismisintroducedtowells,producescharacteristicpatternor‘fingerprint’,whichiscomparedtoextensivedatabase.CanIDupto1,900microbesVariousBiologsystemsavailableFullyautomated–e.g.“Omnilog?”

automaticallyincubates,readsandinterpretstheBiolog

microplatesGivesresultsinaslittleas4hoursQuicker,lesslabourintensive,accurateSemiautomatic–e.g.“MicrologTM”Manuallyreadsystems–e.g.“Microlog2TM”RapidmethodsofmicrobialdetectionWhatdowemeanbyrapidmethods?RapidmethodsgiverapidresultsAlsogiveextremelyreliableresultsUsuallymoretechnicallycomplicatedExamples:PCR,DNAhybridisation,westernblotting,phenotypicmicroarraysDNABASEDMETHODSSpeciesidentificationusingrRNAgeneprobes:DNAhybridisation(spotordotblots)PCRamplificationandsequencing(useantibodiesinsteadofDNAhybridisation)Methods:Alternatively,canisolatedwholeDNA.AgarosegelelectrophoresisRestrictionenzymesREscutDNAsequenceatveryspecificsites.ManydifferentREsthatcutattheirownspecificsequencesE.g.EcoR1cutsatfollowingsite.Animation

DNAhybridizationTechnique:DNAisolatedfromsampleEndonucleases(restrictionenzymes)usedtobreakupDNAstrandintofragments(RFLP)-animationThismixtureofDNAfragsisthenrunonanagarosegelDNAfragmentsongelaretransferredtomembranesupportMembranesupportisthenprobedwithspecies-specificDNAprobe

ProbeislabelledandlabeldetectedbyexposingmembranetophotographicpaperPCRamplificationPolymerasechainreaction(PCR)TechniquebywhichwecanmanipulatereplicationofonespecificregionofDNAoverandoveruntiltherearemillionsofcopiesi.e.amplifyingspecificgenes.Basictechniques:DNAisisolatedfromsampleAmplifyspecies-specificgenesRunampliconsonagarosegelPresenceofampliconisapositiveresult.AnimationPCRMolecularbiologytechniqueforenzymatically

replicatingspecificgeneswithintheDNAwithoutusingalivingorganismDetailedtechnique:IsolatemicrobialDNAAddprimerstomix–smallRNAsequencescomplimentarytostartofsequencetobeamplifiedAddDNApolymerase

Add“spare”nucleotides(dNTPs)forbuildingofnewDNAmoleculesThisisalladdedtoaPCRtubeandtubesareplacedintoPCRmachine(thermalcycler)Thermalcyclergoesthrough3mainsteps:Denaturation–92-95oCAnnealing–55oCExtension–72oCDNAmoleculesofspecificregionofDNAwillbeamplifiedRunthesesamplesongelCanperformsouthernblottoIDspecificmicrobialspeciesWesternblottingTheuseofproteinandantibodiestospecificproteinsProteinsfromsuspectmicrobialspecimenextractedRunonSDSgelTransferofproteinstonitrocellulosemembraneMembranetreatedwithantibodiesforaparticularproteinthatisspeciesspecificPrimaryantibody–bindstoantigenSecondaryantibody–bindstoprimaryantibodyandislabeledfordetection.AnimationPlatingTechniques(culturemethods)Advantages:Simple,Sensitive,WidelyusedforcountingbacteriaandothermicroorganismsDisadvantages:LowcountswillresultifclumpsnotbrokenupResultsexpressedinCFU’sratherthannumberoforganismsIfwrongagarused,willgetinaccurateresultsHotagarinpourplatemethodorhotspreaderinspreadplatemethodmaydamagesomecells.SerialdilutionsUnderproperconditions,onlyalivingbacteriumwilldivideandformavisiblecolonyonaagarplate-i.e.measuresCFU’sratherthannumberofmicrobes.ThisisaviablecellcountItisdifficulttocountmorethan300coloniesononeplate,sowehavetodilutetheoriginalbacterialsolnbeforeyouplateit.Serialdilutionsaccomplishthispurpose.Somesamplesmayneedtobecrushedandshakenfirsttoreleasemicrobes–e.g.–tablets.SerialdilutionsSerialdilutionsStartwithorganisminliquidmedia(stock)Add1mlofthisto9mlofwater,mix(1/10diln)–wehavedilutedtheculturebyafactorof10Add1mlofthe1/10dilnto9mloffreshwater,mix(1/100diln)–wehavefurtherdilutedtheculturebyanotherfactorof10,bothdilutionsaccountforafactorof100Add1mlof1/100dilnto9mlfreshwater(1/1000diln)–wehavenowdilutedtheculturebyafurtherfactorof10.Threefactorsof10=1/1000diln.Repeatthisprocessuntilthedesireddilutionhasbeenreached100μlofeachdilutionistheneitherspreadontoplateoraddedtoagarinthepourplatemethodSerialdilutionsThePourplatemethod1mlofadilutionfromserialdilutionseriesisaddedto9mlofmeltednutrientagar.MediaismixedwellandpouredintoPetridish.MediaisallowedtosolidifyandincubatedatappropriatetempColonieswillformbothwithintheagarandonthesurfaceoftheagari.ecanestimateaerobesandmicroaerophilesTheSpreadplatemethodUsually100μlofadilutionfromthedilutionseriesistransferredtothesurfaceofasolidifiedagarplate.Sampleisspreadoversurfaceevenlyusingasterilespreader.Followingincubationattheappropriatetemp,viablecolonieswillformontheagarsurfaceonly(aerobic).CountingofcoloniesEachbacteriumrepresentsaCFU.ShouldplateeachdilutionintriplicateDonotincubatelongerthan24/48h,ascoloniesmaygrowintoeachotherandbeundistinguishable.TodeterminethenumberofCFU’sintheoriginalculture(stock):multiplycountbythedilutionfactor(ifitisafractionusethedenominator)thenbythereciprocalofsamplevolume(fractionof1mlvolspread).Countxdilutionfactorxreciprocalofsamplevol.ThisgivesyoutheCFU/mlCountingofcoloniesExample:Youcounted183,156and175coloniesontriplicateplatesspreadwith0.1mlofa1/100dilutionofyouroriginalculture.Howmanybacteriaaretherein1mloftheoriginalstock?SampleexamquestionAsampleofwellwaterwastestedforthepresenceofbacteria.Aseriesof10-folddilutionswerepreparedand0.2mlofeachdilutionwasplatedontoagarplatesinduplicate.Afterincubation,theplatesrepresentingthe10-1dilutionwerefoundtohave120and114coloniesrespectively.Calculatethelevelofmicrobialcontaminationpe