Typical Culture Laboratory Bench

Transcription

1 Typical Culture Laboratory Bench

2 Microbial Classification and Identification 第十章 : 微生物的分类和鉴定

3 Content 1. Principle of microbial nomenclature 命名 Binomial Nomenclature 双名法 2. Microbial classification Bergey s Manual of Systematic Bacteriology 3. Steps of microbial Identification Classic Modern

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5 Seven Taxa 七个分类单位 Kingdom 界 ( 拉 : Regnum) Phylum (or Division) 门 ( 拉 : Phylum, Divisio) Class 纲 ( 拉 : Classis) Order 目 ( 拉 : Ordo) Family 科 ( 拉 : Familia) Genus 属 ( 拉 : Genus) Species 种 ( 拉 : Species)

6 The Concept of microbial SPECIES 微生物种的概念 Hard to define Sex 性 : not applicable due to the lack of sex Morphology 形态学 : too simple to be used for definition of species Type species 模式种 : type strain used to show species DNA identification 鉴定 : used in species definition

7 Binomial Nomenclature 双名法 Carl von Linne 林奈, 1753 Use only the Genus 属名 and species 种名 Genus & species either underlined or italicized Genus is always CAPITALIZED Species is never capitalized Homo sapien 人类,Felis domestica 家猫 Escherichia coli 埃希杆菌 Bacillus subtilis 枯草芽胞杆菌 Saccharomyces cerevisiae 酿酒酵母 Common name, vernacular name 俗名 Tubercle bacillu vs Mycobacterium tuberculosis 结核杆菌

8 Naming of Bacteria 细菌命名 Information usually given: 1. Describes an organism Bacillus 杆菌, Streptococcus 链球菌 2. Identifies a habitat 栖息地 E. Coli 3. Honors a scientist or researcher Shigella 志贺菌属 Genus 属 : Usually comes from Latin 拉丁语 Bac. or B. Bacillus 杆菌属 P. or Ps. Pseudomonas 假单胞菌 A. or Asp. Aspergillus 曲霉 Species 种 sp. or spp. (pl) species, Bacillus spp.

9 The Bible of microbiologists - Bergey s Manual of Systematic Bacteriology Bergey s Manual of Determinative Bacteriology - 1 st ed 1923 伯杰氏细菌学鉴定手册 Bergey s Manual of Systematic Bacteriology - 2 nd ed 2001 伯杰氏细菌学系统分类手册

10 1st Ed. Bergey s Manual of Systematic Bacteriology 4 volumes with 33 sections. Procaryotic groups are divided: Volume 1 - Gram-negative bacteria of general, medical, or industrial importance; 常见医学及工业重要的 G- 菌 Volume 2 - Gram-positive bacteria other than actinomycetes; 除放线菌外的 G+ 菌 Volume 3 - Gram-negative bacteria with distinctive properties, cyanobacteria, and archaea; 有特殊性状的 G- 菌, 蓝细菌及古生菌 Volume 4 - Actinomycetes (gram-positive filamentous bacteria) 放线菌 ( 丝状 G+ 菌 )

11 5 volumes: 2nd Ed. Bergey s Manual of Systematic Bacteriology Volume 1- The Archaea, and the Deeply Branching and Phototrophic Bacteria 古生菌, 深分支和光合细菌 Volume 2- The Proteobateria 变形杆菌 Volume 3- The Low G+C Gram-Positive Bacteria Volume 4- The High G+C Gram-Positive Bacteria Volume 5- The Planctomycetes 泛霉状菌, Spirochaetes 螺旋体, Fibrobacteres 丝杆菌, Bacteroidetes 拟杆菌, and Fusobacteria 梭形杆菌

12 Methodology of Microbial Classification and Identification 微生物分类和鉴定方法学

13 Microbial Classification 1. Cell 细胞 Morphology 形态学 Habit 习性 2. Cellular components 细胞组分 Cell wall, lipid, pigment Regular techniques, GC, HPLC, MS 3. Protein 蛋白质 AA sequence, gel electrophoresis, immunolabeling 4. Nucleic acid 核酸 G+C %, nucleic acid hybridization, gene sequencing, genome sequencing Classic Modern

14 Steps of Bacterial Identification 细菌鉴定步骤 1. Isolation (culture) 培养物分离 Agar plate/colonies Liquid media test tube 2. Identification & taxonomy 鉴定和分类 Family Genus Species Type Strain 3. Biochemical (physiological) tests 生理生化试验 Carbon sources, acid & gas Chemical profiling 化学组合 DNA-DNA homology 16S rrna sequencing 4. Non culture based detection 无培养物测定 Polymerase chain reaction- (PCR) Agglutination(antigen detection) 凝集 Stain 染色 Serology (antibody detection) 血清学

15 Example of methods for identification Microorganisms grow in pure culture first!

16 Use metabolic characteristics to identify enteric bacteria 乙偶姻

17 Identification in diagnostic laboratory 诊断化验室的鉴定 Aids treatment 酸处理 Susceptibility antibiotic selection 抗生素敏感性 Based on taxonomy 分类学 Simple, low cost, rapid

18 Serologic identification 血清学鉴定 antibody response to the infecting agent 对感染物的抗体反应 several weeks may needed 需数周时间

19 Antibiotic susceptibility testing 抗生素敏感性测试 Susceptible 易感的 Not susceptible Bacterial lawn 菌苔 No growth Antibiotic disk Growth

20 GC ratios Highly determines the Tm 解链温度 The same - maybe not related Different - sure unrelated Occasionally helpful but generally of minimal value

21 DNA base composition (GC%)

22 Modern microbial auto-identification 微生物的自动化鉴定 - Biolog 95 carbon sources Identify over 1900 species of bacteria, yeast & filamentous fungi

23 Modern microbial autoidentification 微生物的自动化鉴定 - API System culture no. O N P G D H L C O D C C I T 2 S U E T D A I N D V P G E L G L U M A N I O S O R R H A S A C M E L A M Y A R A identification Klebsiella pneumoniae Proteus vulgaris 变形菌 8P Salmonella sp.

24 Antibody Rapid Strep Test 快速链锁测试 Streptococcal antigenic extract Latex beads

25 Rapid diagnosis without culture 无培养物的迅速诊断 WHEN AND WHY? Grow poorly Isolation, slow, may not be clinically useful Can not be cultured Treponema pallidum 梅毒螺旋体

26 Molecular taxonomy (chemotaxonomy) 分子分类法 ( 化学分类法 ) DNA hybridization Ribotyping 核糖核酸图谱分型 FAME analysis 脂肪酸甲酯分析法

27 DNA-DNA hybridization Heat Strain 1 + Strain 2 0% Homology 100% Homology 同源

28 16S rrna Sequencing Relative constant (53%) during evolution Similarity above species level Closely related species: may be identical Allows relatedness comparisons of all bacteria Development of clinical tests based on sequence

29 FISH technology (Fluorescent in situ hybridization) Detection of microorganisms in environment with fluorescently labeled rrna probes 荧光原位杂交技术 : 在环境中用荧光标记的 rrna 探针来检测微生物 No probe Universal probe Eukaryal probe Bacillus megaterium 巨大芽胞杆菌 Saccharomyces cerevisiae

30 The probe that fluoresces red is specific for a signature sequence in the 16S rrna of ammonia-oxidizing Nitrite bacteria, oxidizing while the probe that fluoresces green is specific for a sequence present only in nitrite-oxidizing bacteria. 氨氧化细菌与亚硝酸氧化细菌的探针 In situ detection of nitrifying bacteria in activated sludge 活性污泥中硝化细菌的原位检测 Ammonia oxidizing nitrite-oxidizing bacteria

31 Metagenomics 元基因组 Genomic analysis of microbial DNA extracted directly from samples such as soil, water or faeces Unlock the massive uncultured microbial diversity

32 Evidence of cellulose metabolism by the giant panda gut microbiome The function of gut metagenomes from three wild giant pandas PNAS, 2011, Evidence of cellulose metabolism by the giant panda gut microbiome (A) Gene taxonomic assignment. NA, no matched data or no taxon information for matched genes. Others includes bacteria in other classes, such as Bacteroidetes and Actinobacteria. (B) Projection of the gut genome on the KEGG pathways using ipath (26). (C) Pathway of cellulose digestion. The box indicates that 1,4-β-cellobiosidase was partially found in the unassembly short-read sequences set. (D) Functional composition of the gut metagenome in the COG database.

33 Numerical Taxonomy 数值分类法 Taxonometrics: computer taxonomy 1957, P. H. Sneath Operational taxonomic units (OTU) 运算分类单位 : a variety of strains 1. Calculate correlation coefficient 相关系数 2. Give similarity matrices 相似度矩阵 3. Dendrogram 树状分枝图 Phylogenetics 系统发生学 - the study of phylogeny family history of microbes Genotype-based

34 不同年龄阶段牦牛肠道微生物聚类分析 0.5 岁牦牛 :1-3 号 ;1.5 岁牦牛 :4-6;2.5 岁牦牛 :7-9;3.5 岁牦牛 :10-12

35 Key Points 1. What are the five kingdoms 五界系统 2. The criterion of defining the three domains 三域划分的标准 3. Binomial Nomenclature 双名法 4. Eubacteria vs. Archaebacteria 5. The four major process of bacterial identification 细菌鉴定的四个主要步骤