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Laboratory diagnosis of bacteria

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steps of bacterial diagnosis in lab

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Laboratory diagnosis of bacteria

  1. 1. Laboratory diagnosis of bacteria Prepared by Samira Fattah Assis. Lec. College of health sciences-HMU Lab 1
  2. 2. Methods of bacterial identification • Microscopic examination. • Cultural appearance. • Biochemical reactions. • Serological identification. • Animal inoculation. • Bacteriophage typing. • Molecular methods.
  3. 3. Microscopic examination • Gram stain: pneumococci, gonococci, staphylococci , meningococci • Acid fast stain: mycobacterium tuberculosis, leprae • Fluorescence : -direct -immunoflorescence
  4. 4. Fluorescent viability stain for showing live (green) and dead (red) bacteria
  5. 5. Cultural characters • Pigment production: o Endopigment (restricted to the colonies): • Golden yellow with Staphylococcus aureus. • White with Staph. epidermidis. o Exopigment (the color diffuses in the surrounding medium): • Green exopigment with Pseudomonas aeruginosa.
  6. 6. Hemolysis on blood agar: o Complete (beta) hemolysis: • Staphylococcus aureus and Streptococcus pyogenes. o Partial (alpha) hemolysis: • Streptococcus viridans and pneumococci. o No (gamma) hemolysis: • Enterococci.
  7. 7. Effect on lactose of MacConkey’s agar: o Lactose fermenters: • Appear as rose pink colonies. • Example: E. coli & klebsiella. o Non Lactose fermenters: • Appear as pale colonies. • Example: salmonella & shigella.
  8. 8. Biochemical reactions 1) Sugar fermentation 2) Indol test 3) Urease test 4) Oxidase test 5) Catalase test 6) Coagulase test 7) DNAse test 8) Gelatinase test
  9. 9. - Catalase test: • Is used to differentiate between staphylococci(catalase +ve) and streptococci(catalase –ve). • Principle: Catalase enzyme 2 H2O2 2 H2O + O2 • Procedure – Smear a colony of the organism to a slide – Drop H2O2 onto smear – Observe
  10. 10. Coagulase test is used to differentiate Staphylococcus aureus from coagulase-negative staphylococci. fibrinogen fibrin (clot formation) coagulase
  11. 11. Oxidase Test • All Enterobacteriaceae are oxidase-negative. • This test is used to differentiate enterobacteriaceae from Pseudomonas which is oxidase positive.
  12. 12. Motility test • to determine whether a bacterium is motile. • Non-motile organisms which lack flagella, are usually going to form a single line of growth that does not spread into the surrounding area. While a motile bacterium will grow and make a hazy zone around the stab line.
  13. 13. Analytical profile index (API):
  14. 14. Automated bacterial identification systems: o Principle: - Examples: Vitek system - These systems identify the organism and its antibiotic sensitivity by detecting color changes or turbidity in special plastic cards inoculated with the organism. - Such cards are composed of tiny wells that contain substrates for detection of biochemical reactions and antibiotic sensitivity. - Once the card has been inoculated and placed in the instrument, it will automatically perform all readings. - Results are available within 4-6 hours.
  15. 15. Vitek system Vitek card
  16. 16. Serological identification • Antigen detection – e.g. latex agglutination • Antibody detection – e. g. agglutination tests, complement fixation tests, indirect immunofluorescence
  17. 17. Animal inoculation • The use of laboratory animals (mice, guinea pigs, rabbits) is now limited due to the advancement in medical microbiological techniques. Guinea pig Mouse
  18. 18. But they could be used : 1. For growing the organisms that do not grow on culture such as lepra bacilli. 2. To determine the virulence factor of an organism. For example if injection of diphtheria in a guinea pig caused its death, this means that the organism is toxigenic.
  19. 19. Bacteriophage typing • Bacteriophages are viruses which infect the bacterial cells and cause their lysis. • Different types of a certain bacteria are lysed by different phage groups. • If a phage is added to a plate inoculated with susceptible bacteria, a zone of lysis will appear around the phage drop. •
  20. 20. Molecular methods Nucleic acid probes: o Principle The probe consists of sequence of single stranded DNA or RNA that forms a covalently bonded hybrid with the specific complementary strand of the nucleic acid of the organism. The probe is labeled either to radioactive substance as iodine 131 or enzyme.