09 第8章 微生物感染的病原学检查法.ppt

第1页：第8章 微生物感染的病原学检查法封面

第8章 微生物感染的病原学检查法封面

本页以课件原图为主，源文件仅提取到页面标题，建议对照原图补充要点说明。

第2页：教学大纲与学习要求

第8章 微生物感染的病原学检查法教学大纲

掌握内容

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；

第3页：Outline

Outline

• Laboratory diagnosis forbacterial infections
  • Morphological--microscopic
  • Molecular
  • Serological
• Laboratory diagnosis forviral infections
  • Morphological--electron microscopic
  • Isolation
  • Molecular: nucleic acids and proteins
  • Serological
• Laboratory diagnosis forfungal infections
  • Morphological--microscopic
  • Isolation

第4页：

第5页：Assays

Assays

• Morphologicalassays
  • Light or electron microscopies
• Isolation and differentiation
• Serologicalassays
  • Antigen-antibody assays
• Molecularassays
  • Microorganism’s gene (DNA & RNA)

第6页：Principles for Specimen

Principles for Specimen

• Sterile manipulation, avoid contamination
• Obtain a specimen
  • from the infected site
  • according to the disease phase
  • before administrating antibiotics
• Double sera for antibody detections, acute and recoveryphase each
  • Positive if the titer of the later specimen is above 4X to the firstspecimen
• Transport, and store correctly
  • as soon as possible
  • Store at-4o

第7页：Laboratory Diagnosis for

Laboratory Diagnosis for

• Morphological
• Protein and Nucleic acids
• Antibody

第8页：Bacteriological Diagnosis

Bacteriological Diagnosis

• Identifying theorganismMorphologicalIsolation & cultureBiochemical reactionSerological assays
• Pathogenesis &antibioticssusceptibilityAnimal experimentVirulence testantibiotics susceptibility

第9页：Morphological

Morphological

• Non-stainedmicroscopic observation
  • Dark-field microscopy
  • Observing the movement of live bacteria
• Stainedmicroscopic observations
  • Gram stain
  • Acid-fast stain
  • Fluorescence stain

第10页：

第11页：

第12页：

第13页：

第14页：

第15页：

第16页：Isolation & Culture

Isolation & Culture

• Size
• Shape
• Color
• Surface features
  • Smooth-Rough
• Transparency
• Hemolysis

第17页：Biochemical Reactions Sugar

Biochemical Reactions Sugar

本页以课件原图为主，源文件仅提取到页面标题，建议对照原图补充要点说明。

第18页：Serological Assays

Serological Assays

• Detectionantibodyin the patient’s serum
• Acurrent infectionshould be
  • IgM positive
  • A 4-fold or greater rise on antibody titer between the acute serumsample and the convalescent serum sample
• Majordrawbacks
  • A single IgG antibody titer is difficult to interpret because it is unclearwhether it represents a current or a previous infection
  • the convalescent sample is usually taken 10-

第19页：Animal experiment

Animal experiment

• AnimalsMouseGuinea PigRabbit Dog Monkey
• Inoculation routesIntradermal SubcutaneousIntraperitonealIntravenousIntracranial / intracerebral IntraspinalIntranasal Lavage

第20页：Virulence Test

Virulence Test

• Median lethal dose,LD50
• Median infective dose,ID50
• Elek Plate
  • Diphtherotoxin
  • Corynebacterium diphtheriae
  • Enterotoxin
  • enterotoxigenicE. coli

第21页：

第22页：Minimum Inhibitory

Minimum Inhibitory

• Minimum Inhibitory Concentration，MIC
• Minimum Bactericidal Concentration，MBC
• Bactericidal drugsusually have an MBC equalor very similar to the MIC
• Bacteriostatic drugsusually have an MBCsignificantly higher than the MIC

第23页：

第24页：Bacterial Proteins, DNA &

Bacterial Proteins, DNA &amp

• Antigens (Proteins)
  • Known antibodies
  • Agglutination, coagulation, precipitation,ELISA, Immunofluorescence,radioimmunoassay
• DNA & RNA
  • PCR
  • Nucleic acid hybridization

第25页：Detection of Bacterial

Detection of Bacterial

• Precipitation test
• Coagglutination test
• Immunoflorecence, IF
• McAb technique

第26页：

第27页：

第28页：

第29页：Bacterial DNA & RNA

Bacterial DNA & RNA

• PCR
• DNA probe & hybridization
  • DNA hybridization (southern blot)
• Plasmid fingerprint analysis
  • Restrict multimorphologic analysis

第30页：

第31页：

第32页：

本页图示

本页以课件原图为主，源文件仅提取到页面标题，建议对照原图补充要点说明。

第33页：

第34页：Gene Chip / microarray

Gene Chip / microarray

本页以课件原图为主，源文件仅提取到页面标题，建议对照原图补充要点说明。

第35页：Diagnosis Strategy for

Diagnosis Strategy for

本页以课件原图为主，源文件仅提取到页面标题，建议对照原图补充要点说明。

第36页：Laboratory Diagnosis for Viral

Laboratory Diagnosis for Viral

• Morphological
• Isolation & Culture
• Protein & Nucleic acids
• Serological

第37页：Morphological

Morphological

• Electron microscopy, EM
  • Immunoelectron microscopy, IEM
• Light Microscopy
  • Inclusion body
  • Negri body

第38页：

第39页：Isolation and Culture

Isolation and Culture

• Animal inoculation
• Chicken egg culture:9~14 days
• Tissue culture
  • Organ culture
  • Tissue culture
  • Cell culture

第40页：Cell culture

Cell culture

本页以课件原图为主，源文件仅提取到页面标题，建议对照原图补充要点说明。

第41页：

第42页：Type of Cultured Cells

Type of Cultured Cells

• Primary cell culture
  • Passage 2-3 generations
• Diploid cell culture
  • 2-fold chromosomes, Passage 50~100 generations, safe, canbe used for vaccine manufacture
• Continuous cell line culture
  • Mutant diploid cells, mostly tumor cells, easy to passage andmaintain
  • Not sensitive as diploid cells, may have endogenous viruses,can not be used for vaccine manufacture

第43页：Indications of Viral

Indications of Viral

• Cytopathic effect, CPE
• Hemadsorption
• Hemagglutination
• Viral interference
• Neutralization test, NT

第44页：Cytopathic Effect, CPE

Cytopathic Effect, CPE

本页以课件原图为主，源文件仅提取到页面标题，建议对照原图补充要点说明。

第45页：Viral Quantity Determination

Viral Quantity Determination

• TCID50
  • 50% tissue culture infectious dose
  • Determine theinfectivityof the examined viruses
• PFU
  • Plaque formation
  • pfu/ml
  • Preciselyquantitate the number of the living viralparticles

第46页：

第47页：Plaque

Plaque

本页以课件原图为主，源文件仅提取到页面标题，建议对照原图补充要点说明。

第48页：Differences between viral

Differences between viral

• Viral particles (VPs)
  • represent the total number of viral particles (live and deadcombined). Due to variations in virus preparations the ratio oflive/dead varies significantly and therefore, VP does not reflectthe amount of active virus in the preparation
• PFU (plaque formation unit)
  • represents the number of infectious or live viruses. It reflects theamount of working viruses in the preparation

第49页：病毒的数量与感染性测定

病毒的数量与感染性测定

• 蚀斑测定：准确滴定病毒感染性的方法
  • 蚀斑
  • 每个蚀斑是由一个有感染性病毒颗粒形成，称为蚀斑形成单位（ ，PFU）。病毒的感染量用 表示
• 50%感染量（ID50）或50%组织细胞感染量（TCID50）
  • 不能准确地测定感染性病毒颗粒的数量

第50页：Proteins and Nucleic Acids

Proteins and Nucleic Acids

• Proteins (antigens)
  • Fluoresece-labeled, enzyme-labeled (enzymelinked immunosorbent assay, ELISA) andradio-labeled anitbodies
• Viral DNA or RNA
• Gene chip
  • DNA chip / cDNA Microarray

第51页：Virus-specific Antibodies

Virus-specific Antibodies

• Neutralization test, NT
• Hemagglutination inhibition test, HI
• Complement fixation, CF
• ELISA
• Western blot

第52页：

第53页：ELISA for HIV antibody

ELISA for HIV antibody

本页以课件原图为主，源文件仅提取到页面标题，建议对照原图补充要点说明。

第54页：

第55页：Diagnosis Strategy for Viral

Diagnosis Strategy for Viral

本页以课件原图为主，源文件仅提取到页面标题，建议对照原图补充要点说明。

第56页：Rapid Diagnosis for Viral

Rapid Diagnosis for Viral

• Morphological
  • EM & IEM, occasionally
  • Light Microscopy: inclusion body
• Viral antigens
  • Immunofluorescence
  • Radioimmunoassays
  • ELISA
• Specific antibodies:IgM
• Nucleic acids: PCR

第57页：Laboratory Diagnosis for

Laboratory Diagnosis for

本页以课件原图为主，源文件仅提取到页面标题，建议对照原图补充要点说明。

第58页：Microscopic Observation

Microscopic Observation

• Samples
  • Sputum, skin scrapings, nail, hair, lung biopsy, etc.
• Method
  • Treated with10% KOHto dissolve tissue material,leaving the alkali-resistant fungi intact, or stained withspecial fungal stains, then observe by microscopy
• Observation
  • Characteristicspores and hyphae
  • Thewide capsule ofCryptococcus neoformansinIndia ink preparation of spinal fluid

第59页：Culture

Culture

• Sabouraud’s agar
  • pH5.6, not suitable for bacterial growth
  • Withadditions of antibiotics, eg, penicillin,stretomycin, etc
  • 25oC–28oC for days and weeks

第60页：Serological

Serological

• Serological
• Nucleic acids
  • PCR
  • Probe and hybridization

第61页：本章小结

本章小结

• 细菌感染的微生物学检查法病原体检查核酸的检测抗原、抗体的检测
• 病毒感染的微生物学检查法电镜及免疫电镜病毒的分离培养和鉴定核酸的检测抗原、抗体的检测
• 真菌感染的微生物学检查法直接镜检真菌的分离培养