PHT 313 Lab (1) Staphylococci. Bacteria Gram’s Stain Gram’s +ve Cocci Bacilli Gram’s -ve Cocci...

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PHT 313 Lab (1) Staphylococci

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  • PHT 313Lab (1)Staphylococci

  • StaphylococciStreptococciMicrococci

    NeisseriaCorynbacteriumClostridumBacillusEnterobacteriaceae Pseudomonas.

  • StaphylococciThree mainly species that are human pathogens:Staph. aureusStaph. epidermidisStaph. saprophyticus

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

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

  • Culture Characteristics

    Environment: Facultative anaerobeTemp.: 37 CPH: 7.2Media:Nutrient agar (Simple medium).Blood agar (Enriched medium).Mannitol salt agar (Selective & diffrential medium) .

  • Staphylococci on Nutrient Agar

    OrganismS.aureus MediaNutrient agarAppearanceGolden Yellowcolonies

  • Staphylococci on Nutrient Agar

    OrganismS.epidermidisMediaNutrient agarAppearanceWhite colonies

  • Staphylococci on Blood Agar

    OrganismS. aureus MediaBlood agarAppearanceBeta heamolysis(completehaemolysis)

  • Staphylococci on Blood Agar

    OrganismS.epidermidisS saprophyticus MediaBlood agarAppearanceNon hemolytic

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

  • 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

  • S. aureus S.epidermidisS saprophyticus

  • Deoxyribonuclease (DNase) Test: Principle:Insoluble In acidsoluble 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.

  • 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 cloudyCloudiness in all the plateS.epidermidisS.aureus

  • Biochemical TestsCatalase test: Differentiative test to separate Staphylococci and Micrococci which are catalase +ve from Sterptococci which are catalase ve.

    Principle:Procedure:

  • Results:Positive test: Rapid appearance of gas bubbles.Staphylococci or Micrococci

    Catalase veStreptococci

    Catalase +ve

  • Coagulase Test:Definitive test to differentiate between S.aureus & other species of staphylococci (coagulase-negative staphylococci CONS) e.g. S.epidermidisPrinciple:Procedure:1

  • Results:Positive test: Formation of visible clot.Coagulase +veCoagulase -ve

  • DiseasesDue to invasion:Local lesions of skin e.g. boils, carbauncles, abscesses.Systemic infections e.g. septicemia, meningitis.

    Toxin mediatedFood poisoningToxic shock syndromeScalded skin syndrome

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

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

  • S. epidermidis:Nutrient agar: White coloniesBlood agar: Non haemolyticMSA: Non fermentative

    S. saprophyticus:Nutrient agar: Yellow coloniesBlood agar: Non haemolyticMSA: Non fermentative

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