Classify and identifi


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Classify and identifi

  1. 1. Classification and Identification of Microorganisms
  2. 2. <ul><li>Laboratory procedures employed in the </li></ul><ul><li>identification of bacteria </li></ul><ul><li>Isolation of organism in pure culture </li></ul><ul><li>Bacterial colony morphology </li></ul><ul><li>Microscopic morphology and Staining reaction </li></ul><ul><li>4. Biochemical test </li></ul><ul><li>5. Serological procedure </li></ul><ul><li>6. Antibiotic sensitivity </li></ul>
  3. 3. <ul><li>Isolation of organism in Pure Culture </li></ul><ul><li>Pure culture (axenic culture) </li></ul><ul><li>– Population of cells arising from a single cell </li></ul><ul><li>- the approach used for the isolation of organism depends </li></ul><ul><li> upon the source of clinical specimen </li></ul><ul><li> Blood, spinal fluid and closed abscesses may yield almost pure bacterial culture </li></ul><ul><li> </li></ul><ul><li> specimen of sputum, stool, materials from the skin and body orifices usually contains mixture of organism </li></ul>
  4. 4. <ul><li>Bacterial colony morphology </li></ul><ul><li>Bacteria  grow on solid media as colonies </li></ul><ul><li>colony is defined as a visible mass of microorganisms all originating from a single mother cell. </li></ul><ul><li>therefore a colony constitutes a clone of bacteria all genetically alike </li></ul><ul><li>• Useful in bacterial identification </li></ul><ul><li>• Colonies - size, shape, texture, elevation, pigmentation, effect on growth medium </li></ul>
  5. 5. <ul><li>To identify the following colonial characteristics/culture characteristics: </li></ul><ul><li>WHOLE SHAPE OF COLONY EDGE/MARGIN OF COLONY </li></ul>
  6. 6. <ul><li>ELEVATION OF COLONY (turn the place on end to determine height) </li></ul><ul><li>CHROMOGENESIS (pigmentation) </li></ul><ul><li>- Some bacterial species form an array of pigments: white, red, purple, etc. </li></ul><ul><ul><li>• Some pigments are contained within the cell (i.e., probably not water soluble) </li></ul></ul><ul><ul><li>• Some pigments readily diffuse throughout the medium (i.e, water soluble) </li></ul></ul><ul><ul><li>• Some pigments fluoresce in UV light </li></ul></ul><ul><li>OPACITY OF COLONY: </li></ul><ul><li>transparent (clear), opaque, </li></ul><ul><li>translucent (almost clear, but distorted vision–like looking through frosted glass </li></ul><ul><li>iridescent (changing colors in reflected light) </li></ul><ul><li>CONSISTENCY: </li></ul><ul><ul><li>butyrous (buttery), viscid (sticks to loop, hard to get off) </li></ul></ul><ul><ul><li>brittle/friable (dry, breaks apart) </li></ul></ul><ul><li>EMULSIFIABILITY OF COLONY: </li></ul><ul><ul><li>Is it easy or difficult to emulsify?  Does it form a uniform suspension, a granular suspension, or does not emulsify at all? </li></ul></ul>
  7. 7. <ul><li>SURFACE OF COLONY: </li></ul><ul><li>smooth, mucoid/glistening, rough, dull (opposite of glistening), rugose (wrinkled) </li></ul><ul><li>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 </li></ul><ul><li>Mucoid - colonies exhibits a water-like glistening confluent appearance commonly seem among organism which from slime layer or capsule. Ex. Kleb. pneumoniae, S. pneumoniae </li></ul><ul><li>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 </li></ul>
  8. 8. <ul><li>Microscopic morphology </li></ul><ul><li>Provide presumptive identification of an organism </li></ul><ul><li>Bacterial Morphology </li></ul><ul><li>Shape </li></ul><ul><li>Arrangement </li></ul><ul><li>Staining reaction </li></ul>
  9. 9. Biochemical Test <ul><li>Various species of organism exhibits characteristic pattern of substrate utilization, metabolic product formation and sugar fermentation </li></ul><ul><ul><li>Enzyme based test – based on its reaction with a substrate </li></ul></ul><ul><ul><ul><li>Catalase, oxidase, indole, urease </li></ul></ul></ul><ul><ul><li>Reactions in sugar fermentation broth </li></ul></ul><ul><ul><li>Nitrate Broth reactions </li></ul></ul><ul><li>60% of common pathogens can be identified by metabolic test </li></ul>
  10. 10. <ul><li>Serological procedure </li></ul><ul><li>Antigen and antibody determination </li></ul><ul><li>Serological Tests </li></ul><ul><ul><li>Use group specific antiserum isolated from the plasma of animals that have been sensitized to the organism </li></ul></ul><ul><ul><ul><li>The antiserum contains antibody proteins that react with antigens on the unknown organism. </li></ul></ul></ul><ul><li>Procedures: agglutination, precipitation test, hemagglutination inhibition, complement fixation, ELISA, RIA, Western blot assay </li></ul><ul><li>Advantages: </li></ul><ul><ul><ul><li>Highly specific </li></ul></ul></ul><ul><ul><ul><li>Does not usually require the organism to be isolated into pure culture </li></ul></ul></ul><ul><ul><ul><li>Can be used to identify organisms that can’t be grown on medium </li></ul></ul></ul>
  11. 11. <ul><li>Antibiotic sensitivity </li></ul><ul><li>antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics </li></ul><ul><li>Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo </li></ul><ul><li>Methods of testing: </li></ul><ul><ul><li>Broth dilution </li></ul></ul><ul><ul><ul><li>The lower the dilution, the greater the antibiotic content </li></ul></ul></ul><ul><ul><li>Agar dilution </li></ul></ul><ul><ul><li>Disk diffusion </li></ul></ul><ul><ul><ul><li>the Kirby-Bauer test for antibiotic susceptibility, called the disc diffusion test, is a standard that has been used for years </li></ul></ul></ul><ul><li>  </li></ul>
  12. 12. <ul><li>The bacterium is swabbed on the agar </li></ul><ul><li> and the antibiotic discs are placed on top </li></ul><ul><li>The antibiotic diffuses from the disc into </li></ul><ul><li>the agar in decreasing amounts the further </li></ul><ul><li>it is away from the disc </li></ul><ul><li>Bacteria are not able to grow around antibiotics </li></ul><ul><li>to which they are sensitive </li></ul><ul><li>If the organism is killed or inhibited by the </li></ul><ul><li>concentration of the antibiotic, there will be </li></ul><ul><li>NO growth in the immediate area around the disc: </li></ul><ul><li>called the zone of inhibition </li></ul><ul><li>The zone sizes are looked up on a standardized chart to </li></ul><ul><li>give a result of sensititive, resistant, or intermediate </li></ul><ul><li>Many charts have a corresponding column that also gives the MIC (minimal inhibitory concentration) for that drug </li></ul>
  13. 13. Conventional diagnosis methods