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PHT 313 Lab (1) Staphylococci
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Bacteria Gram’s Stain Gram’s +ve Cocci Bacilli Gram’s -ve
Corynbacterium Clostridum Bacillus Staphylococci Streptococci Micrococci Neisseria Enterobacteriaceae Pseudomonas.
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Staphylococci Three mainly species that are human pathogens:
Staph. aureus Staph. epidermidis Staph. saprophyticus
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Habitat: S. aureus: S. epidermidis: S. saprophyticus:
Nasal passages, skin, oral cavity and gastrointestinal tract. S. epidermidis: skin. S. saprophyticus: Rarely found in healthy humans.
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Microscopic Morphology:
Gram positive cocci, arranged in grape like clusters, non-motile, non-spore forming.
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Classification Coagulase positive e.g. S.aureus
According to Coagulase enzyme production Coagulase positive e.g. S.aureus Coagulase negative e.g. S. epidermidis & S. saprophyticus According to Pigment production (endopigment) Golden yellow colonies S .aureus White colonies S. epidermidis Yellow colonies S. saprophyticus
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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) .
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Staphylococci on Nutrient Agar
Organism S.aureus Media Nutrient agar Appearance Golden Yellow colonies
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Staphylococci on Nutrient Agar
Organism S.epidermidis Media Nutrient agar Appearance White colonies
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Staphylococci on Blood Agar
Organism S. aureus Media Blood agar Appearance Beta heamolysis (complete haemolysis)
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Staphylococci on Blood Agar
Organism S.epidermidis S saprophyticus Media Blood agar Appearance Non hemolytic
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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.
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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
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S. aureus S.epidermidis S saprophyticus
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Deoxyribonuclease (DNase) Test:
Principle: DNA DNase enzyme Nucleotides 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.
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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
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Biochemical Tests Catalase test:
Differentiative test to separate Staphylococci and Micrococci which are catalase +ve from Sterptococci which are catalase –ve. Principle: H2O2 Catalase enzyme H2o O2 Air bubbles Procedure:
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Results: Positive test: Rapid appearance of gas bubbles. Catalase +ve
Staphylococci or Micrococci Streptococci
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Coagulase Test: Definitive test to differentiate between S.aureus & other species of staphylococci (coagulase-negative staphylococci “CONS”) e.g. S.epidermidis Principle: Fibrinogen Plasma Coagulase enzyme Fibrin Visible Clot 1 Procedure: 2 3 Place at water bath at 37oC, observe for formation of visible clot for up to 4 hrs.
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Results: Positive test: Formation of visible clot. Coagulase -ve
S.epidermidis Coagulase +ve S.aureus
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Diseases Due to invasion: Toxin mediated
Local lesions of skin e.g. boils, carbauncles, abscesses. Systemic infections e.g. septicemia, meningitis. Toxin mediated Food poisoning Toxic shock syndrome Scalded skin syndrome
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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 .
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3.Culture : S. aureus: Nutrient agar: Golden yellow colonies
Blood agar: Beta hemolysis . MSA: Yellow colonies (mannitol fermentation)
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S. epidermidis: Nutrient agar: White colonies Blood agar: Non haemolytic MSA: Non fermentative S. saprophyticus: Nutrient agar: Yellow colonies
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4. Biochemical reactions:
Catalase test : positive Coagulase test: positive in case of s. aureus
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