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1、第一篇 微生物学基础第8章微生物感染的病原学检查法Laboratory Diagnosis教学大纲n n掌握内容掌握内容uu细菌细菌FF细菌感染的微生物学检查程序细菌感染的微生物学检查程序FF直接涂片镜检、分离培养和鉴定、生化试验、血清学试验及动物实验直接涂片镜检、分离培养和鉴定、生化试验、血清学试验及动物实验FF病原菌抗原成分、核酸及其他成分的检测方法病原菌抗原成分、核酸及其他成分的检测方法FF血清学诊断原则及常用的方法血清学诊断原则及常用的方法uu病毒病毒FF病毒分离鉴定方法及病毒在培养细胞中的增殖的指标病毒分离鉴定方法及病毒在培养细胞中的增殖的指标FF病毒数量与感染性的测定（病毒数量与感
2、染性的测定（TCID50TCID50、PFUPFU的原理、应用）的原理、应用）FF病毒成份的检测（抗原、核酸检测）病毒成份的检测（抗原、核酸检测）FF病毒抗体检测常用方法的原理及应用、双份血清诊断意义病毒抗体检测常用方法的原理及应用、双份血清诊断意义n n熟悉内容熟悉内容uu标本的采集与送检原则标本的采集与送检原则uu革兰染色和抗酸染色的原理革兰染色和抗酸染色的原理uu细菌药敏试验细菌药敏试验uu真菌感染的微生物学检查法真菌感染的微生物学检查法n n了解内容了解内容uu组织培养；聚合酶链反应、蛋白印迹技术的原理和应用组织培养；聚合酶链反应、蛋白印迹技术的原理和应用Outlinen nLabor
3、atory diagnosis for Laboratory diagnosis for bacterial infectionsbacterial infectionsuuMorphological - microscopic Morphological - microscopic uuMolecularMolecularuuSerologicalSerologicaln nLaboratory diagnosis for Laboratory diagnosis for viral infectionsviral infectionsuuMorphological - electron m
4、icroscopic Morphological - electron microscopic uuIsolation Isolation uuMolecular: nucleic acids and proteins Molecular: nucleic acids and proteins uuSerologicalSerologicaln nLaboratory diagnosis for Laboratory diagnosis for fungal infectionsfungal infectionsuuMorphological - microscopicMorphologica
5、l - microscopicuuIsolationIsolationAssaysn nMorphologicalMorphological assays assaysuuLight or electron microscopiesLight or electron microscopiesn nIsolation and differentiationIsolation and differentiationn nSerologicalSerological assays assaysuuAntigen-antibody assays Antigen-antibody assays n nM
6、olecularMolecular assays assaysuuMicroorganisms gene (DNA & RNA)Microorganisms gene (DNA & RNA)Principles for Specimen Collectionn nSterile manipulation, avoid contaminationSterile manipulation, avoid contaminationn nObtain a specimenObtain a specimen uufrom the infected sitefrom the infected siteuu
7、according to the disease phaseaccording to the disease phaseuubefore administrating antibioticsbefore administrating antibioticsn nDouble sera for antibody detections, acute and recovery Double sera for antibody detections, acute and recovery phase eachphase eachuuPositive if the titer of the later
8、specimen is above 4X to the first Positive if the titer of the later specimen is above 4X to the first specimenspecimenn nTransport, and store correctlyTransport, and store correctlyuuas soon as possibleas soon as possibleuuStore at -4Store at -4o oC for most bacterial specimens, but C for most bact
9、erial specimens, but keep warm for keep warm for Neisseria meningitidisNeisseria meningitidis and and Neisseria gonorrhoeaeNeisseria gonorrhoeaeuuStore at -4Store at -4o oC -70C -70o oC for viral specimensC for viral specimensLaboratory Diagnosis for Bacterial Infectionsn nMorphological n nProtein a
10、nd Nucleic acidsn nAntibodyBacteriological Diagnosisn nIdentifying the organismMorphological Morphological Isolation & cultureIsolation & cultureBiochemical reaction Biochemical reaction Serological assaysSerological assaysn nPathogenesis & antibiotics susceptibilityAnimal experimentAnimal experimen
11、tVirulence testVirulence testantibiotics susceptibility antibiotics susceptibility Morphologicaln nNon-stained microscopic observationuuDark-field microscopyDark-field microscopyuuObserving the movement of live bacteriaObserving the movement of live bacterian nStained microscopic observationsuuGram
12、stainGram stainuuAcid-fast stainAcid-fast stainuuFluorescence stain Fluorescence stain Isolation & Culture: Colonyn nSizen nShapen nColorn nSurface featuresuuSmooth - RoughSmooth - Roughn nTransparencyn nHemolysisBiochemical Reactions Sugar Fermentation HSugar Fermentation H2 2S Test Citrate utiliza
13、tionS Test Citrate utilizationSerological Assaysn nDetection Detection antibodyantibody in the patients serum in the patients serumn nA A current infectioncurrent infection should be should beuuIgM positiveIgM positiveuuA 4-fold or greater rise on antibody titer between the acute serum A 4-fold or g
14、reater rise on antibody titer between the acute serum sample and the convalescent serum samplesample and the convalescent serum samplen nMajor Major drawbacksdrawbacksuuA single IgG antibody titer is difficult to interpret because it is unclear A single IgG antibody titer is difficult to interpret b
15、ecause it is unclear whether it represents a current or a previous infectionwhether it represents a current or a previous infectionuuthe convalescent sample is usually taken 10-14 days after the acute the convalescent sample is usually taken 10-14 days after the acute sample. By this time, the patie
16、nt has often recovered and the diagnosis sample. By this time, the patient has often recovered and the diagnosis becomes a retrospective onebecomes a retrospective onen nSome Some exceptionsexceptionsuuIn certain diseases, a single titer of sufficient magnitude can be used In certain diseases, a sin
17、gle titer of sufficient magnitude can be used as presumptive evidence of a current infectionas presumptive evidence of a current infectionAnimal experimentn nAnimalsAnimalsMouseMouseGuinea PigGuinea Pig Rabbit Dog MonkeyRabbit Dog Monkeyn nInoculation routesInoculation routesIntradermal Subcutaneous
18、Intradermal SubcutaneousIntraperitonealIntraperitoneal IntravenousIntravenousIntracranial / intracerebral IntraspinalIntracranial / intracerebral IntraspinalIntranasal LavageIntranasal LavageVirulence Testn nMedian lethal dose, Median lethal dose, LD50LD50n nMedian infective dose, Median infective d
19、ose, ID50ID50n nElek PlateElek PlateuuDiphtherotoxin Diphtherotoxin FFCorynebacterium diphtheriaeCorynebacterium diphtheriaeuuEnterotoxinEnterotoxinFFenterotoxigenic enterotoxigenic E. coliE. coliAntibiotic Susceptibility Testn nMethodMethodMIC & MBCn nMinimum Inhibitory ConcentrationMinimum Inhibit
20、ory Concentration，MICMICn nMinimum Bactericidal ConcentrationMinimum Bactericidal Concentration，MBCMBCn nBactericidal drugsBactericidal drugs usually have an MBC equal usually have an MBC equal or very similar to the MICor very similar to the MICn nBacteriostatic drugsBacteriostatic drugs usually ha
21、ve an MBC usually have an MBC significantly higher than the MIC significantly higher than the MIC Bacterial Proteins, DNA & RNAn nAntigens (Proteins)uuKnown antibodiesKnown antibodiesuuAgglutination, coagulation, precipitation, Agglutination, coagulation, precipitation, ELISA, Immunofluorescence, EL
22、ISA, Immunofluorescence, radioimmunoassayradioimmunoassayn nDNA & RNAuuPCRPCRuuNucleic acid hybridizationNucleic acid hybridizationDetection of Bacterial Antigensn nPrecipitation testn nCoagglutination testn nImmunoflorecence, IFn nMcAb techniqueBacterial DNA & RNA n nPCRn nDNA probe & hybridization
23、uuDNA hybridization (southern blot) DNA hybridization (southern blot) n nPlasmid fingerprint analysisuuRestrict multimorphologic analysisRestrict multimorphologic analysisPCRGene Chip / microarrayDiagnosis Strategy for Bacterial InfectionLaboratory Diagnosis for Viral Infectionn n Morphologicaln n I
24、solation & Culturen n Protein & Nucleic acidsn n SerologicalMorphological n nElectron microscopy, EM uuImmunoelectron microscopy, IEMImmunoelectron microscopy, IEMn nLight MicroscopyuuInclusion bodyInclusion bodyFFNegri bodyNegri bodyIsolation and Culturen nAnimal inoculationn nChicken egg culture:
25、914 days914 daysn nTissue cultureuuOrgan cultureOrgan cultureuuTissue cultureTissue cultureuuCell cultureCell cultureCell cultureType of Cultured Cellsn nPrimary cell culturePrimary cell cultureuuPassage 2-3 generationsPassage 2-3 generationsn nDiploid cell cultureDiploid cell cultureuu2-fold chromo
26、somes, Passage 50100 generations, safe, can 2-fold chromosomes, Passage 50100 generations, safe, can be used for vaccine manufacturebe used for vaccine manufacturen nContinuous cell line culture Continuous cell line culture uuMutant diploid cells, mostly tumor cells, easy to passage and Mutant diplo
27、id cells, mostly tumor cells, easy to passage and maintainmaintainuuNot sensitive as diploid cells, may have endogenous viruses, Not sensitive as diploid cells, may have endogenous viruses, can not be used for vaccine manufacture can not be used for vaccine manufacture Indications of Viral Propagati
28、onn nCytopathic effect, CPE Cytopathic effect, CPE n nHemadsorptionHemadsorptionn nHemagglutinationHemagglutinationn nViral interferenceViral interferencen nNeutralization test, NTNeutralization test, NTCytopathic Effect, CPEViral Quantity Determinationn nTCID50TCID50uu50% tissue culture infectious
29、dose50% tissue culture infectious doseuuDetermine the Determine the infectivityinfectivity of the examined viruses of the examined virusesn nPFUPFUuuPlaque formationPlaque formationuupfu/mlpfu/mluuPreciselyPrecisely quantitate the number of the living viral quantitate the number of the living viral
30、particlesparticlesPlaqueDifferences between viral particle (VP), plaque formation unit (PFU)n nViral particles (VPs)Viral particles (VPs) uurepresent the total number of viral particles (live and dead represent the total number of viral particles (live and dead combined). Due to variations in virus
31、preparations the ratio of combined). Due to variations in virus preparations the ratio of live/dead varies significantly and therefore, VP does not reflect live/dead varies significantly and therefore, VP does not reflect the amount of active virus in the preparation the amount of active virus in th
32、e preparation n nPFU (plaque formation unit)PFU (plaque formation unit) uurepresents the number of infectious or live viruses. It reflects the represents the number of infectious or live viruses. It reflects the amount of working viruses in the preparation amount of working viruses in the preparatio
33、n uuFor most virus preps, the VP/PFU ratio is 20:1 to 50:1 For most virus preps, the VP/PFU ratio is 20:1 to 50:1 n nWhich one better reflects the amount of active virus Which one better reflects the amount of active virus used?used?uuUsing VP (viral particles) as the unit will result in significant
34、 Using VP (viral particles) as the unit will result in significant variations in the amount of actual live viruses used, and using variations in the amount of actual live viruses used, and using IFU or PFU as the viral unit will give more consistent outcomes. IFU or PFU as the viral unit will give m
35、ore consistent outcomes. 病毒的数量与感染性测定n n蚀斑测定：准确滴定病毒感染性的方法uu蚀斑（蚀斑（PlaquePlaque）uu每个蚀斑是由一个有感染性病毒颗粒形成，每个蚀斑是由一个有感染性病毒颗粒形成，称为称为蚀斑形成单位蚀斑形成单位（Plaque forming unitPlaque forming unit，PFUPFU）。病毒的感染量用）。病毒的感染量用PFU/mlPFU/ml表示表示n n50%感染量（ID50）或50%组织细胞感染量（TCID50）uu不能准确地测定感染性病毒颗粒的数量不能准确地测定感染性病毒颗粒的数量Proteins and Nucl
36、eic Acidsn nProteins (antigens)uuFluoresece-labeled, enzyme-labeled Fluoresece-labeled, enzyme-labeled (enzyme linked immunosorbent assay, (enzyme linked immunosorbent assay, ELISA) and radio-labeled anitbodiesELISA) and radio-labeled anitbodiesn nViral DNA or RNAn nGene chipuuDNA chip / cDNA Microa
37、rrayDNA chip / cDNA MicroarrayVirus-specific Antibodiesn nNeutralization test, NTn nHemagglutination inhibition test, HIn nComplement fixation, CFn nELISAn nWestern blotELISA for HIV antibodyMicroplate ELISA for HIV antibody: coloured wells indicate reactivityMicroplate ELISA for HIV antibody: colou
38、red wells indicate reactivityDiagnosis Strategy for Viral Infection Rapid Diagnosis for Viral Infectionn nMorphologicalMorphologicaluuEM & IEM, occasionallyEM & IEM, occasionallyuuLight Microscopy: inclusion bodyLight Microscopy: inclusion bodyn nViral antigensViral antigensuuImmunofluorescenceImmun
39、ofluorescenceuuRadioimmunoassaysRadioimmunoassaysuuELISA ELISA n nSpecific antibodiesSpecific antibodies: : IgMIgMn nNucleic acids: PCRNucleic acids: PCRLaboratory Diagnosis for Fungal infection Direct microscopic Direct microscopic CultureCultureSerological Serological Nucleic acidsNucleic acidsMic
40、roscopic Observationn nSamplesSamplesuuSputum, skin scrapings, nail, hair, lung biopsy, etc.Sputum, skin scrapings, nail, hair, lung biopsy, etc.n nMethodMethoduuTreated with Treated with 10% KOH10% KOH to dissolve tissue material, to dissolve tissue material, leaving the alkali-resistant fungi inta
41、ct, or stained with leaving the alkali-resistant fungi intact, or stained with special fungal stains, then observe by microscopyspecial fungal stains, then observe by microscopyn nObservationObservationuuCharacteristic Characteristic spores and hyphaespores and hyphaeuuThe The wide capsule of wide c
42、apsule of Cryptococcus neoformansCryptococcus neoformans in in India ink preparation of spinal fluidIndia ink preparation of spinal fluidCulturen nSabourauds agaruupH5.6pH5.6, not suitable for bacterial growth, not suitable for bacterial growthuuWith With additions of antibioticsadditions of antibio
43、tics, eg, penicillin, , eg, penicillin, stretomycin, etcstretomycin, etcuu2525o oC 28C 28o oC for days and weeksC for days and weeksn nSerological n nNucleic acidsuuPCRPCRuuProbe and hybridizationProbe and hybridization小结n n细菌感染的微生物学检查法细菌感染的微生物学检查法病原体检查病原体检查 核酸的检测核酸的检测 抗原、抗体的检测抗原、抗体的检测n n病毒感染的微生物学检查法病毒感染的微生物学检查法电镜及免疫电镜电镜及免疫电镜 病毒的分离培养和鉴定病毒的分离培养和鉴定 核酸的检测核酸的检测 抗原、抗体的检测抗原、抗体的检测n n真菌感染的微生物学检查法真菌感染的微生物学检查法直接镜检直接镜检 真菌的分离培养真菌的分离培养
