Faculty:Dr.AlvinFoxSuggestedreading:Murray,Thirdedition Chapter2,16-18and21.TOPIC:CLASSIFICATION&IDENTIFICATIONOFINFECTIOUSAGENTS.KeyTermsIsolation(culture)Agarplate/coloniesLiquidmediatesttube-bulkIdentification&taxonomyFamilyGenusSpeciesTypeStrainBiochemical(physiological)testsMoleculartestsDNA-DNAhomology16SrRNAsequencingChemicalprofiling NonculturebaseddetectionPolymerasechainreaction-(PCR)Agglutination(antigendetection)StainSerology(antibodydetection).Bacterialidentificationinthediagnosticlaboratory1.Isolation&identificationaidstreatmentsinceinfectiousdiseases causedbydifferentbacteriahaveavarietyofclinicalcoursesandconsequences.Susceptibilitytestingofisolateshelpsinselectionofantibiotics fortherapy.Theidentificationisbasedontaxonomicprinciplesappliedto theclinicalmicrobiologicalsituation.4.Testsmustbeeasilylearned,lowincostandrapidlyperformed..ClassificationFamily:agroupofrelatedgenera.Genus:agroupofrelatedspecies.Species:agroupofrelatedstrains.Type:setsofstrainwithinaspecies(e.g.biotypes,serotypes).Strain:onelineorasingleisolateofaparticularspecies..StepsindiagnosticisolationandidentificationofbacteriaStep1.Samplesofbodyfluidsarestreakedoncultureplatesandisolatedcoloniesofbacteriaappearafterincubation.Observationofthesecoloniesforsize,texture,color,and(ifgrownonbloodagar)hemolysisreactions,ishighlyimportantasafirststepinbacterialidentification.Whethertheorganismrequiresoxygenforgrowthisanotherimportantdifferentiatingcharacteristic..BloodAgarPlate.Step2.ColoniesareGramstainedandindividualbacterialcellsobservedunderthemicroscope..Asinglecolonyisplacedonaslideanddried.Stage1.Stainingwithcrystalviolet.Stage2.Fixationwithiodinestabilizescrystalvioletstaining.Allbacteriaremainpurpleorblue.Stage3.Extractionwithalcohol.Decolorizessomebacteria (Gramnegative)andnotothers(Grampositive).Stage4.Counterstainingwithsafranin.Grampositive bacteriaarestainedwithcrystalvioletandremain purple.Gramnegativebacteriaarestainedpink.GramStain..GramstainmorphologyShapecocci(round)bacilli(rods)spiralorcurved(e.g.spirochetes)Singleormultiplecellsclusters(e.g.streptococci)chains(e.g.streptococci)Grampositiveornegative...Step3.Thebacteriaarespeciatedusingtheseisolatedcolonies.

.TypicalCultureLaboratoryBench.Step4.Antibioticsusceptibilitytestingisperformed..TaxonomiccharacterizationofbacteriaComparisonsofspeciesinvolvecomparisonsof multiplestrainsforeachspecies.Comparisonsareprimarilybasedonchemicalor molecularanalysis..MolecularanalysisItwouldbeidealtocomparesequencesofentirebacterialchromosomalDNA.Alternatively,genomicsimilarityhasbeenassessedbytheguanine(G)+cytosine(C)content(%GC).Thishasbeenreplacedbytwoalternatives:HybridizationSequencingspecificgenes.DNA-DNAhomology1.HowwelltwostrandsofDNAfromdifferent bacteriabind(hybridize)together. Thistechniqueisemployedtocomparethe geneticrelatednessofbacterialstrains/species.IftheDNAfromtwobacterialstrainsdisplaya highdegreeofhomology(i.e.theybindwell)the strainsareconsideredtobemembersofthesame species..“Goldstandard"inbacterialtaxonomySequencingof16SribosomalRNAmolecules(16SrRNA)The16SrRNAisapproximately1600nucleotidesinlength.The16SrRNAprovidesgenomicsimilarityabovethe specieslevelallowingcomparisonsofrelatednessacross theentirebacterialkingdom.CloselyrelatedbacterialspeciesoftenhaveidenticalrRNA

sequences.5. Thetechniqueprovidescomplementaryinformationto DNA-DNAhybridization.

Thedevelopmentofprobesbasedon16Sdatahaveimproved identificationofsomebacterialpathogens..ChemicalanalysisSophisticatedtoolsareavailableforstudyingthestructuralcompositionofbacteria(mostcommonlyfattyacid,carbohydrateorubiquinoneprofiling)ofbacteria.Characterizationofsecretedmetabolic

products(e.g.volatilealcoholsandshortchainfattyacids)isalsohelpful..Approachestorapiddiagnosiswithoutpriorculture

Certainhumanpathogenseithercannotbeisolatedinthelaboratorygrowextremelypoorly.Successfulisolationcanbeslowandinsome instancesimpossible.3.Directdetectionofbacteriawithoutcultureis possibleforsomeorganisms..AsimpleapproachtorapiddiagnosisAntigendetection Usedinmanydoctor'sofficesforthegroupAstreptococcus.Thepatient'sthroatisswabbedandstreptococcalantigenextracteddirectlyfromtheswab(withoutpriorbacteriologicalculture).Thebacterialantigenisdetectedbyaggregation(agglutination)ofantibodycoatedlatexbeads..BacterialDNAsequencesamplifieddirectlyfromhumanbodyfluidsThepolymerasechainreaction(PCR)canbeusedtoamplifylargeamountsofspecificgenesorportionsofgenes.Greatsuccesshasbeenachievedinrapiddiagnosisoftuberculosis..DirectmicroscopicobservationCertainclinicalsamplessuchassputumcanbedirectlyassessedfo