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PHT 313 Lab (1)

PHT 313 Lab (1). Staphylococci. Corynbacterium Clostridum Bacillus. Enterobacteriaceae Pseudomonas. Neisseria. Staphylococci Streptococci Micrococci. Staphylococci. Three mainly species that are human pathogens: Staph. aureus Staph. epidermidis Staph. saprophyticus. Habitat:.

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PHT 313 Lab (1)

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  1. PHT 313Lab (1) Staphylococci

  2. Corynbacterium Clostridum Bacillus Enterobacteriaceae Pseudomonas. Neisseria Staphylococci Streptococci Micrococci

  3. Staphylococci • Three mainly species that are human pathogens: • Staph. aureus • Staph. epidermidis • Staph. saprophyticus

  4. Habitat: • S. aureus: • Nasal passages, skin, oral cavity and gastrointestinal tract. • S. epidermidis: • skin. • S. saprophyticus: • Rarely found in healthy humans.

  5. Microscopic Morphology: • Gram positive cocci, arranged in grape like clusters, non-motile, non-spore forming.

  6. Culture Characteristics • Environment: Facultative anaerobe • Temp.:37 ° C • PH: 7.2 • Media: • Nutrient agar (Simple medium). • Blood agar (Enriched medium). • Mannitol salt agar (Selective & diffrential medium) .

  7. Staphylococci on Nutrient Agar

  8. Staphylococci on Nutrient Agar

  9. Staphylococci on Blood Agar

  10. Staphylococci on Blood Agar

  11. Mannitol Salt Agar (MSA) • The name: Mannitol salt agar • Appearance: Solid, opaque, pink. • Type: Selective & diffrential medium • Composition: Contains high conc. of salt (about 7.5%). • Test sugar: mannitol. • pH indicator: phenol red (red in alkaline, yellow in acidic pH). • Sterilization: autoclave.

  12. S. aureus gives yellow colonies on MSA because of mannitol fermintation which leads to decrease in PH this turns the color of phenol red to yellow. • Note: MSA is differential for S.aureus

  13. S. aureus S.epidermidis S saprophyticus

  14. DNase enzyme DNA Nucleotides Deoxyribonuclease (DNase) Test: Principle: Insoluble In acid soluble In acid Procedure: 1. Inoculate DNase agar plate with the test organism. 2. Incubate the plate at 35oC for 24 hrs. 3. Flood the plate with 1M HCl.

  15. Results: • DNase activity is indicated by a clear zone around the growth after addition of Hcl Clear zone around the growth while the rest of the plate appears cloudy Cloudiness in all the plate S.epidermidis S.aureus

  16. Catalase enzyme H2O2 H2o + O2 Air bubbles Biochemical Tests Catalase test: Differentiative test to separate Staphylococci and Micrococci which are catalase +ve fromSterptococciwhich are catalase –ve. Principle: Procedure:

  17. Results: Positive test: Rapid appearance of gas bubbles. Catalase +ve Catalase –ve Staphylococci or Micrococci Streptococci

  18. Coagulase enzyme Fibrinogen Plasma Fibrin Visible Clot 2 3 Place at water bath at 37oC, observe for formation of visible clot for up to 4 hrs. Coagulase Test: • Definitive test to differentiate between S.aureus & other species of staphylococci (coagulase-negative staphylococci “CONS”) e.g. S.epidermidis Principle: 1 Procedure:

  19. S.epidermidis S.aureus Results: Positive test:Formation of visible clot. Coagulase -ve Coagulase +ve

  20. Diseases • Toxin mediated • Food poisoning • Toxic shock syndrome • Scalded skin syndrome • Due to invasion: • Local lesions of skin e.g. boils, carbauncles, abscesses. • Systemic infections e.g. septicemia, meningitis.

  21. Diagnosis 1.The specimen: • Swab from wounds, abscesses • Blood in case of septicemia • Urine in case of UTI • CSF in case of meningitis. 2. Direct examination : • Film is prepared and examined by gram stain .

  22. 3.Culture : S. aureus: • Nutrient agar: Golden yellow colonies • Blood agar: Beta hemolysis . • MSA: Yellow colonies (mannitol fermentation)

  23. S. epidermidis: • Nutrient agar: White colonies • Blood agar: Non haemolytic • MSA: Non fermentative S. saprophyticus: • Nutrient agar: Yellow colonies • Blood agar: Non haemolytic • MSA: Non fermentative

  24. 4. Biochemical reactions: • Catalase test : positive • Coagulase test: positive in case of s. aureus

  25. 5-Typing • Used for epidemiological purposes. • Phage typing. • Antibiogram. • Molecular typing (ribotyping or PCR).

  26. Phage Typing Staphylococci isolated from wounds, from nose and nail bed of attending personnel (doctors, nurses, ….) and from fomites are identified by phage typing to reveal the source of infection. Also it is used to trace the source of food poisoning.

  27. Antibiogram • Helpful in tracing sources of staphylcoccal infections

  28. Molecular typing (ribotyping or PCR) • Used to document the spread of epidemic disease-producing clones of S.aureus

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