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Transcript
Survey of Microbial Diseases •How to identify bacteria in patient specimens or in samples from nature? Or the MM project;) - phenotypic: considers macroscopic and microscopic morphology, physiology, and biochemistry - immunologic: serological analysis - genotypic: genetic techniques increasingly being used as a sole resource for identifying bacteria •Data from these methods can provide a unique profile for any bacterium Survey of Microbial Diseases: Phenotypic Methods Physiological/Biochemical Characteristics •Traditional mainstay of bacterial identification •Enzyme production and other biochemical properties are reliable ways to ID microbes •Dozens of diagnostic tests exist for determining the presence of specific enzymes and to assess nutritional and metabolic activities: - fermentation of sugars - capacity to digest complex polymers - production of gas - sensitivity to antibiotics - nutrient sources Beta-hemolysis Blood agar as a differential medium Alpha-hemolysis No hemolysis (gamma-hemolysis) Survey of Microbial Diseases: Phenotypic Methods Tests for fermentation and gas production Durham tube (inverted tube to trap gas) No fermentation Acid fermentation with gas Phenotypic Methods: Direct Examination of Specimen •Direct observation of fresh or stained specimen •Stains most often used - Gram stain - acid-fast stain Survey of Microbial Diseases: Phenotypic Methods • Isolation Media and Morphological Testing - Selective media: encourage the growth of only the suspected pathogen - Differential media: used to identify definitive characteristics and fermentation patterns MacConkey Agar: Selective and Differential Selects for Gram-negative and tells you if the bacterium ferments lactose Phenotypic Methods: Biochemical Testing • Physiological reactions: indirect evidence of enzymes present in a species. If bacteria tests + for cytochrome c oxidase what does that tell you? Phenotypic Methods: Biochemical Testing Unknown microbe + different substrates Results (+/–) DNPG ADH LDC – – + ODC |CIT | H2S + – – URE TDA IND – – + |VP| |GEL | GLU – – + MAN INO SOR – – – RHA SAC MEL – – – AMY ARA – • Enzyme-mediated metabolic reactions often visualized by a color change - microbe is cultured in a medium with a special substrate, then tested for a particular end product - microbial expression of the enzyme is made visible by a colored dye – Flowchart: We will use this to ID our MM! Cocci Gram (+) Gram (–) Catalase (–), pairs, chain arrangement Catalase (+), irregular clusters, tetrads Aerobic, oxidase (+), catalase (+) Anaerobic, oxidase (–), catalase (–) Streptococcus Strictly aerobic Micrococcus Facultative anaerobic Staphylococcus Planococcus Neisseria Branhamella Moraxella Veillonella Phenotypic Methods: Phage Typing - Testing for sensitivity to various phage groups - a lawn of bacterial cells is inoculated onto agar, mapped off into blocks, and phage are exposed to each block - cleared areas corresponding to lysed cells indicate sensitivity to that phage - Ex. S. aureus Phage Group I vs. Group II Determining Clinical Significance of Cultures •Important to rapidly determine if an isolate from a specimen is clinically important or if it is merely a contaminant or normal biota - a few colonies of E. coli in a urine sample can indicate normal biota, but several hundred can mean an active infection - a single colony of a true pathogen such as Mycobacterium tuberculosis in a sputum culture, or an opportunist in a sterile site, is highly suggestive of disease - repeated isolation of a relatively pure culture of any microorganism can mean it is an agent of disease