医学微生物课件chapter4 Identify bacteria

Identify bacteria
1 Introduction
2 Bacterial classification
3 Identifying Bacteria
1 Introduction
Classification: The arrangement of organisms into taxonomic groups(taxa分类类群) on the basis of similarities or relationships.
Classification of bacteria requires a knowledge obtained by experimental as well as observational techniques, because biochemical, physiologic, genetic, and morphologic properties are often necessary for an adequate description of a taxon(分类单元). Nomenclature: Naming an organism by international rules according to its characteristics.
Binomial nomenclature is adopted in naming bacteria. The names of every bacteria are composed with two latin words. The first one is genus name, and the last one is species name. The genus name is capital. The species name is lower case. They must be in ital. Identification: Identification refers to the practical use of a classification scheme: (1) To isolate and distinguish desirable(想要的) organisms from undesirable（不合适的）
ones;
(2) To verify(核实) the authenticity(真实性) or special properties of a culture; or, in a
clinical setting;
(3) To isolate and identify the causative agent(病原体，病原物) of a disease.
An ideal identification system should contain the minimum number of features required for a correct diagnosis. Groups are split into smaller subgroups on the basis of the presence or absence of a diagnostic character.
numerical taxonomy
phylogenetic(系统发生的) classification
2 Bacterial classification
2.1 Bergey’s Manual of Systematic Bacteriology
The possibility that one might draw inferences about phylogenetic relationships among
bacteria is reflected in the organization of the latest edition of Brgey’s Manual of Systematic Bacteriology. The companion volume, published in 1994, serves as an aid in the identification of those bacteria that have been described and cultured.
2.2 Medically important bacteria
I.Gram-negative eubacteria(真细菌) that have cell walls
Group 1:The spirochetes(螺旋体，螺旋菌)
•Treponema(密螺旋体)
•Borrelia(包柔氏螺旋体)
•Leptospira(细螺旋体，钩端螺旋体)
Group 2:Aerobic/microaerophilic(需氧/微需氧的), motile helical/vibroid(螺旋形的/类病毒的) gram-negative bacteria
•Campylobacter(弯曲杆菌)
•Helicobacter(螺杆菌)
•Spirillum(螺旋菌)
Group 3:Nonmotile (or rarely motile) curved bacteria
none pathogens is found in this group
Group 4:Gram-negative aerobic/microaerophilic rods and cocci
•Alcaligenes(产碱杆菌属)
•Bordetella(博代氏杆菌属)
•Brucella(布鲁氏菌)
•Francisella(弗朗西斯氏菌属)
•Legionella(军团杆菌，军团菌属)
•Moraxella(莫拉克斯氏菌属)
•Neisseria(奈瑟氏菌属)
•Pseudomonas(假单胞菌属)
•Rochalimaea(罗沙利马体属)
•Bacteroides (拟杆菌属)(some species)
Group 5:Facultatively anaerobic(兼性厌氧的) gram-negative rods
•Escherichia(埃希氏杆菌属) (related coliform(大肠型) bacteria)
•Klebsiella(克雷白氏杆菌属)
•Proteus(变形杆菌属)
•Providencia(普罗维登斯菌属)
•Salmonella(沙门氏菌)
•Shigella(志贺氏杆菌)
•Yersinia(耶尔森氏鼠疫杆菌)
•Vibrio(弧菌)
•Haemophilus(嗜血杆菌)
•Pasteurella(巴斯德菌)
Group 6:Gram-negative, anaerobic,straight, curved,and helical rods
•Bacteroides(拟杆菌属)
•Fusobacterium(梭菌属)
•Prevotella(普雷沃菌属)
Group 7: Dissimilatory(异化的) sulfate- or sulfur-reducing bacteria
Group 8: Anaerobic gram-negative cocci
none pathogens is found in these two groups
Group 9:The rickettsiae(立克次氏体) and chlamydiae(衣原体)
•Rickettsia
•Coxiella(柯克斯氏体)
•chlamydia
Group 15:Nonphotosynthetic, nonfruiting gliding bacteria(滑行) •Capnocytophaga
II. Gram-positive bacteria that have cell walls
Group 17:Gram-positive cocci
•Enterococcus(肠球菌)
•Peptostreptococcus(消化链球菌属)
•Staphylococcus(葡萄球菌)
•Streptococcus(链球菌)
Group 18:Endospore-forming(形成内芽孢) gram-positive rods and cocci
•Bacillus(芽孢杆菌)
•Clostridium(梭菌)
Group 19: Regular, nonsporing gram-positive rods
•Erysipelothrix(丹毒丝菌)
•Listeria (李斯特菌属)
Group 20: Irregular, nonsporing gram-positive rods
•Actinomyces(放线菌)
•Corynebacterium(棒状杆菌)
•Mobiluncus(动弯杆菌属)
Group 21: The mycobacteria
•Mycobacterium(分支杆菌属)
Group 22-29: Actinomycetes
•Nocardia(诺卡氏菌属)
•Streptomyces (链霉菌属)
•Rhodococcus (红球菌属)
III. Cell wall-less eubacteria(真细菌): The mycoplasmas(支原体) or mollicutes(柔膜
细菌)
Group 30: Mycoplasmas
•Mycoplasma
•Ureaplasma(尿原体)
IV. Archaeobacteria(古细菌)
none pathogens is found in this catagory
3 Identifying Bacteria
3.1 Steps of identifying bacteria
•Isolation
•Purification
•Preliminary identification by observing
•Examination with microscope
•Biochemical test
•Serologic test
•Other identification
3.2 Biochemical test of bacteria
Mechanism
Different pathogens has different enzyme system, they can only use some certain kind carbon resource and nitrogen resource. And, their metabolin(代谢产物) such as degradation products of saccharide or protein are different, though the substrate(底物) are the same. According to the different substance, the bacteria can be identified. Metabolin related to bacteria identification
•Pigment(色素)
•Bacteriocin(细菌素)
•Degradation products of saccharide: including acid, aldehyde, alcohol, ketone and CO2
•Degradation products of protein
Application of biochemical test
➢Fermentation(发酵) test of saccharide
The saccharides used in this test commonly are glucose(葡萄糖), sugar and lactose(乳糖). After fermentation, organic acid and/or gas be generated. The result of fermentation can be used to help identifying bacteria.
➢Judgement of organic acid
To judge whether some certain organic acid is generated during the fermentation of saccharide, bromcresol purple(溴甲酚紫)is added into the media as an indicator. If some organic acid is generated, the color of the media will change from purple to yellow. This is named a positive result(+). If there is not any organic acid generated, we get a negative result(-).
➢Judgement of gas
To judge whether some gases would be generated during the fermentation of saccharide, we could use an inverted tiny tube in the fermentation tube. For the positive result (+), the gas generated would form bubble in the tiny tube. When a negative result (-) come out, no bubble can be seen.
➢Some examples of fermentation test
Escherichia coli (大肠杆菌)can utilize glucose and lactose as carbon resource. Salmonella typhi (伤寒杆菌)can utilize glucose only. When they ferment glucose, Escherichia coli could produce methanoic acid(甲酸) first, and then decompose it into CO2 and H2 further. Salmonella typhi could produce methanoic acid only.
➢VP (Voges-Proskauer伏-普) test
This test is designed to distinguish Escherichia coli and Enterobacter aerogenes(产气肠杆菌). They can utilize glucose and get the same result in fermentation test. But some of products of fermentation of them is different. In VP test, trace creatin(肌氨酸) is added to the basic liquid media.
Result of VP test
The color of the solution in the tube inoculated with Enterobacter aerogenes would change into red(+). Because acetymethyl carbinol(甲酰甲基甲醇) would be generated in this tube, it could be oxidized to form diacetyl(二乙酰) under basic condition. Biochemical test of bacteria
➢Methyl red test
This test is also designed to distinguish Escherichia coli and Enterobacter aerogenes. In this test, methyl red is added to the liquid media as an indicator. Its color is orange(-) when pH>4.5 and red(+) when pH ≤4.5.
➢Citrate(柠檬酸盐) utilization test
Some bacteria can utilize ammonium salt as unique nitrogen resource and citrate as unique carbon resource. Its metabolins include carbonate and ammonia(氨). They could make the indicator Bromothymol Blue(溴百里酚蓝) change color from green to deep blue. When the media turn deep blue after incubating, a positive result is obtained. Biochemical test of bacteria
Result of citrate utilization test
➢Indol(吲哚) test
Some bacterium could degrade tryptophan(色氨酸) into indol. Indol could react with 1,4-dimethyl carbamate benzaldehyde(1,4-二甲基氨基苯甲醛) and produce rose indoles which is red(+).
Biochemical test of bacteria
Result of indol test
➢Hydrogen sulfide(H2S) test
Amino acids containing S could be degraded into H2S by Salmonella sp.(沙门氏菌)and P. species(变形杆菌属). H2S then could react with Pb2+ or Fe3+ and form sulfide that is black(+).
➢Urease(脲酶) test
This test is designed to identify Bacillus proteus(变形杆菌). In this test urea is added to the media. The indicator is phenol red. For a positive result, the media would turn red.
➢Other biochemical test
•Gelatin(明胶) liquidation test
•Starch utilize test
➢API 20E
API 20E is the standard identification system of Enterobacteriaceae(肠杆菌科) and other gram negative bacterium. It includes 23 standardized mini biochemical tests and an identification informations bank.
Mechanism of API 20E
Every API 20E kit contains 20 mini tubes in which dry substrate is filled. Then bacterium suspension solutions would be inoculated in these tubes. After being cultured for a certain time, the color changes due to the metabolism or due the adding of reagents can be observed.
According to the related table contained in this system and other references or tools, the identification result is obtained.
Reagent in a kit of ref.20100
•25 API 20 E test bar
•25 cultivation boxes
•25 report sheets
• 1 sealing clip
• 1 operation instruction
Reagent in a kit of ref.20160
•100 API 20 E test bar
•100 cultivation boxes
•100 report sheets
• 1 sealing clip
• 1 operation instruction
Extra reagents
•5ml 0.85% suspension of NaCl for ref. 20230
•5ml suspension medium for ref. 20150
•TDA, IND, VP 1, VP 2, NIT 1, NIT 2 and OX or single extra reagent
•Zn reagent
•paraffin oil
•Specified one-off adding tube for API
•API LAB PLUS analysis software
If it is necessary, the following extra reagents are needed
•API of Medium(ref. 50110): determine the type of fermentation or oxidation
•API of Medium(ref. 51020):obverse the motility of facultative anaerobic bacterium Common equipments needed for API
•Incubator (35~37℃)
•Refrigerator
•Alcohol lamp
•Marking pen
3.3 Serological tests of bacteria
Mechanism
Antigen(抗原) could react with responding antibody under some certain condition out of being body, and then deposit and agglutination(凝集) can be seen.
Using a known antibody, we can know the species and types of the unknown bacteria in the specimens or isolation culture. We also can know what kind antigen the bacteria has.
➢OXOID Strep
OXOID is a famous trade brand. It supply many kits for identification of bacterium. And OXOID Strep is a kind kit for the identification of Strep.
➢Agglutination test on glass slide
Mechanism
Granular(颗粒) antigen could combine with responding antibody which contained in immune serum(免疫血清) and form visible agglutination gradually when proper electrolyte(电解质) is present.
Agents and apparatus
•Typhoid bacillus(伤寒杆菌) diagnosis serum
•Slant or plate culture of target bacterium
•Glass slides
•Capillary pipets(毛细移液管)
•physiological saline(生理盐水)
Steps
•Line on a clean glass slide and get two parts using a wax pencil and mark the number.
•Using a inoculating loop, add 1~2 drops Typhoid bacillus diagnosis serum diluted by the ratio of 1:10 on part 1, while 1~2 drops physiological saline on part 2. •Under aseptic condition, pick up some culture with an inoculating loop and add to part 2 and part 1 orderly.
•Mix the bacterium with the serum or physiological saline to form suspension. •Shake the slide lightly and observe in 1~2 minutes.
•If a milky agglutination is seen, it means a positive result. If it is necessary, a microscope could be used.
3.4 Other tests
➢Other serological tests
•Widal test
•Felix test
•Latex agglutination test
•Sedimentation test •Complement fixation reaction •Neutralization test
•ELISA
➢DNA-based test •Sampling
•DNA extraction
•PCR
•Sequencing of DNA •Comparison with gene bank •Get identification result。