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Classify and identifi

Classify and identifi






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    Classify and identifi Classify and identifi Presentation Transcript

    • Classification and Identification of Microorganisms
      • Laboratory procedures employed in the
      • identification of bacteria
      • Isolation of organism in pure culture
      • Bacterial colony morphology
      • Microscopic morphology and Staining reaction
      • 4. Biochemical test
      • 5. Serological procedure
      • 6. Antibiotic sensitivity
      • Isolation of organism in Pure Culture
      • Pure culture (axenic culture)
      • – Population of cells arising from a single cell
      • - the approach used for the isolation of organism depends
      • upon the source of clinical specimen
      • Blood, spinal fluid and closed abscesses may yield almost pure bacterial culture
      • specimen of sputum, stool, materials from the skin and body orifices usually contains mixture of organism
      • Bacterial colony morphology
      • Bacteria  grow on solid media as colonies
      • colony is defined as a visible mass of microorganisms all originating from a single mother cell.
      • therefore a colony constitutes a clone of bacteria all genetically alike
      • • Useful in bacterial identification
      • • Colonies - size, shape, texture, elevation, pigmentation, effect on growth medium
      • To identify the following colonial characteristics/culture characteristics:
      • ELEVATION OF COLONY (turn the place on end to determine height)
      • CHROMOGENESIS (pigmentation)
      • - Some bacterial species form an array of pigments: white, red, purple, etc.
        • • Some pigments are contained within the cell (i.e., probably not water soluble)
        • • Some pigments readily diffuse throughout the medium (i.e, water soluble)
        • • Some pigments fluoresce in UV light
      • transparent (clear), opaque,
      • translucent (almost clear, but distorted vision–like looking through frosted glass
      • iridescent (changing colors in reflected light)
        • butyrous (buttery), viscid (sticks to loop, hard to get off)
        • brittle/friable (dry, breaks apart)
        • Is it easy or difficult to emulsify?  Does it form a uniform suspension, a granular suspension, or does not emulsify at all?
      • smooth, mucoid/glistening, rough, dull (opposite of glistening), rugose (wrinkled)
      • Smooth - colonies gives the appearance of homogeneity and uniform texture without appearing as liquid or as mucoid colonies such as gram- negative enterobacteria Ex. Salmonella, Shigella
      • Mucoid - colonies exhibits a water-like glistening confluent appearance commonly seem among organism which from slime layer or capsule. Ex. Kleb. pneumoniae, S. pneumoniae
      • Rough – colonies are granulated and rough in appearance, usually produced by mutant strain that lacks surface protein and polysaccharide of freshly isolated wild-type parent organism
      • Microscopic morphology
      • Provide presumptive identification of an organism
      • Bacterial Morphology
      • Shape
      • Arrangement
      • Staining reaction
    • Biochemical Test
      • Various species of organism exhibits characteristic pattern of substrate utilization, metabolic product formation and sugar fermentation
        • Enzyme based test – based on its reaction with a substrate
          • Catalase, oxidase, indole, urease
        • Reactions in sugar fermentation broth
        • Nitrate Broth reactions
      • 60% of common pathogens can be identified by metabolic test
      • Serological procedure
      • Antigen and antibody determination
      • Serological Tests
        • Use group specific antiserum isolated from the plasma of animals that have been sensitized to the organism
          • The antiserum contains antibody proteins that react with antigens on the unknown organism.
      • Procedures: agglutination, precipitation test, hemagglutination inhibition, complement fixation, ELISA, RIA, Western blot assay
      • Advantages:
          • Highly specific
          • Does not usually require the organism to be isolated into pure culture
          • Can be used to identify organisms that can’t be grown on medium
      • Antibiotic sensitivity
      • antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics
      • Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo
      • Methods of testing:
        • Broth dilution
          • The lower the dilution, the greater the antibiotic content
        • Agar dilution
        • Disk diffusion
          • the Kirby-Bauer test for antibiotic susceptibility, called the disc diffusion test, is a standard that has been used for years
      • The bacterium is swabbed on the agar
      • and the antibiotic discs are placed on top
      • The antibiotic diffuses from the disc into
      • the agar in decreasing amounts the further
      • it is away from the disc
      • Bacteria are not able to grow around antibiotics
      • to which they are sensitive
      • If the organism is killed or inhibited by the
      • concentration of the antibiotic, there will be
      • NO growth in the immediate area around the disc:
      • called the zone of inhibition
      • The zone sizes are looked up on a standardized chart to
      • give a result of sensititive, resistant, or intermediate
      • Many charts have a corresponding column that also gives the MIC (minimal inhibitory concentration) for that drug
    • Conventional diagnosis methods