1、第一篇 微生物学(wi shn w xu)基础,第8章微生物感染(gnrn)的病原学检查法Laboratory Diagnosis,第一页，共六十二页。,教学大纲(jio xu d n),掌握内容(nirng)细菌细菌感染的微生物学检查程序直接涂片镜检、分离培养和鉴定、生化试验、血清学试验及动物实验病原菌抗原成分、核酸及其他成分的检测方法血清学诊断原则及常用的方法病毒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数量与感染性的测定（TCID50、PFU的原理、应用）病毒成份的检测（抗原、核酸检测）病毒抗体检测常用方法的原理及应用、双份血清诊断意义熟悉内容标本的采集与送检原则革兰染色和抗酸染
2、色的原理细菌药敏试验真菌感染的微生物学检查法了解内容组织培养；聚合酶链反应、蛋白印迹技术的原理和应用,第二页，共六十二页。,Outline,Laboratory diagnosis for bacterial infectionsMorphological-microscopic MolecularSerologicalLaboratory diagnosis for viral infectionsMorphological-electron microscopic Isolation Molecular:nucleic acids and proteins SerologicalLabor
3、atory diagnosis for fungal infectionsMorphological-microscopicIsolation,第三页，共六十二页。,第四页，共六十二页。,Assays,Morphological assaysLight or electron microscopiesIsolation and differentiationSerological assaysAntigen-antibody assays Molecular assaysMicroorganisms gene(DNA&RNA),第五页，共六十二页。,Principles for Specime
4、n Collection,Sterile manipulation,avoid contaminationObtain a specimen from the infected siteaccording to the disease phasebefore administrating antibioticsDouble sera for antibody detections,acute and recovery phase eachPositive if the titer of the later specimen is above 4X to the first specimenTr
5、ansport,and store correctlyas soon as possibleStore at-4oC for most bacterial specimens,but keep warm for Neisseria meningitidis and Neisseria gonorrhoeaeStore at-4oC-70oC for viral specimens,第六页，共六十二页。,Laboratory Diagnosis for Bacterial Infections,Morphological Protein and Nucleic acidsAntibody,第七页
6、，共六十二页。,Bacteriological Diagnosis,Identifying the organismMorphological Isolation&cultureBiochemical reaction Serological assays,Pathogenesis&antibiotics susceptibilityAnimal experimentVirulence testantibiotics susceptibility,第八页，共六十二页。,Morphological,Non-stained microscopic observationDark-field mic
7、roscopyObserving the movement of live bacteriaStained microscopic observationsGram stainAcid-fast stainFluorescence stain,第九页，共六十二页。,第十页，共六十二页。,第十一页，共六十二页。,第十二页，共六十二页。,第十三页，共六十二页。,第十四页，共六十二页。,第十五页，共六十二页。,Isolation&Culture:Colony,SizeShapeColorSurface featuresSmooth-RoughTransparencyHemolysis,第十六页，共六
8、十二页。,Biochemical Reactions,Sugar Fermentation H2S Test Citrate utilization,第十七页，共六十二页。,Serological Assays,Detection antibody in the patients serumA current infection should beIgM positiveA 4-fold or greater rise on antibody titer between the acute serum sample and the convalescent serum sampleMajor
9、drawbacksA single IgG antibody titer is difficult to interpret because it is unclear whether it represents a current or a previous infectionthe convalescent sample is usually taken 10-14 days after the acute sample.By this time,the patient has often recovered and the diagnosis becomes a retrospectiv
10、e oneSome exceptionsIn certain diseases,a single titer of sufficient magnitude can be used as presumptive evidence of a current infection,第十八页，共六十二页。,Animal experiment,AnimalsMouseGuinea Pig Rabbit Dog MonkeyInoculation routesIntradermal SubcutaneousIntraperitoneal IntravenousIntracranial/intracereb
11、ral IntraspinalIntranasal Lavage,第十九页，共六十二页。,Virulence Test,Median lethal dose,LD50Median infective dose,ID50Elek PlateDiphtherotoxin Corynebacterium diphtheriaeEnterotoxinenterotoxigenic E.coli,第二十页，共六十二页。,Antibiotic Susceptibility Test,Method,第二十一页，共六十二页。,MIC&MBC,Minimum Inhibitory Concentration，M
12、ICMinimum Bactericidal Concentration，MBCBactericidal drugs usually have an MBC equal or very similar to the MICBacteriostatic drugs usually have an MBC significantly higher than the MIC,第二十二页，共六十二页。,第二十三页，共六十二页。,Bacterial Proteins,DNA&RNA,Antigens(Proteins)Known antibodiesAgglutination,coagulation,p
13、recipitation,ELISA,Immunofluorescence,radioimmunoassayDNA&RNAPCRNucleic acid hybridization,第二十四页，共六十二页。,Detection of Bacterial Antigens,Precipitation testCoagglutination testImmunoflorecence,IFMcAb technique,第二十五页，共六十二页。,第二十六页，共六十二页。,第二十七页，共六十二页。,第二十八页，共六十二页。,Bacterial DNA&RNA,PCRDNA probe&hybridiza
14、tionDNA hybridization(southern blot)Plasmid fingerprint analysisRestrict multimorphologic analysis,第二十九页，共六十二页。,第三十页，共六十二页。,第三十一页，共六十二页。,PCR,第三十二页，共六十二页。,第三十三页，共六十二页。,Gene Chip/microarray,第三十四页，共六十二页。,Diagnosis Strategy for Bacterial Infection,第三十五页，共六十二页。,Laboratory Diagnosis for Viral Infection,Mo
15、rphological Isolation&Culture Protein&Nucleic acids Serological,第三十六页，共六十二页。,Morphological,Electron microscopy,EM Immunoelectron microscopy,IEMLight MicroscopyInclusion bodyNegri body,第三十七页，共六十二页。,第三十八页，共六十二页。,Isolation and Culture,Animal inoculationChicken egg culture:914 daysTissue cultureOrgan cu
16、ltureTissue cultureCell culture,第三十九页，共六十二页。,Cell culture,第四十页，共六十二页。,第四十一页，共六十二页。,Type of Cultured Cells,Primary cell culturePassage 2-3 generationsDiploid cell culture2-fold chromosomes,Passage 50100 generations,safe,can be used for vaccine manufactureContinuous cell line culture Mutant diploid cells,mostly tumor cells,easy to passage and maintainNot sensitive as diploid cells,may have endogenous viruses,can not be used for vaccine manufacture,第四十二页，共六十二页。,Indications of Viral Propagation,Cyto