Classify and identifi

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  • Laboratory procedures employed in the identification of bacteria

    Isolation of organism in pure culture Bacterial colony morphologyMicroscopic morphology and Staining reaction4. 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:

    WHOLE SHAPE OF COLONY EDGE/MARGIN OF COLONY

  • 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 lightOPACITY OF COLONY: transparent (clear), opaque, translucent (almost clear, but distorted visionlike looking through frosted glass iridescent (changing colors in reflected light) CONSISTENCY: butyrous (buttery), viscid (sticks to loop, hard to get off)brittle/friable (dry, breaks apart)EMULSIFIABILITY OF COLONY: Is it easy or difficult to emulsify? Does it form a uniform suspension, a granular suspension, or does not emulsify at all?

  • SURFACE OF COLONY: 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 substrateCatalase, oxidase, indole, ureaseReactions in sugar fermentation broth Nitrate Broth reactions

    60% of common pathogens can be identified by metabolic test

  • Serological procedure Antigen and antibody determinationSerological TestsUse group specific antiserum isolated from the plasma of animals that have been sensitized to the organismThe antiserum contains antibody proteins that react with antigens on the unknown organism.Procedures: agglutination, precipitation test, hemagglutination inhibition, complement fixation, ELISA, RIA, Western blot assayAdvantages:Highly specificDoes not usually require the organism to be isolated into pure cultureCan be used to identify organisms that cant 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 vivoMethods of testing:Broth dilutionThe lower the dilution, the greater the antibiotic contentAgar dilutionDisk diffusionthe 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

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