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A. GRAM'S POSITIVE COCCI

A. GRAM'S POSITIVE COCCI. 2. STREPTOCOCCUS SPP. Microscopic examination. Microscopic examination. Morphological Description Type of stain : gram stain Gram reaction : gm +ve Color : violet Shape : cocci Size : small

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A. GRAM'S POSITIVE COCCI

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  1. A. GRAM'S POSITIVE COCCI 2. STREPTOCOCCUS SPP.

  2. Microscopic examination

  3. Microscopic examination Morphological Description • Type of stain: gram stain • Gram reaction: gm +ve • Color: violet • Shape: cocci • Size: small • Arrangment: chains or pairs

  4. Classification of streptococci *S. pyogenes *S. pneumonia *S. faecalis *Viridans streptococci

  5. Cultivation Culture Media 1. Blood agar 2. Chocolate agar

  6. Blood agar Type: enriched, differential medium Constituents: nutrient agar +5- 10% defibrinated blood Principle: species of strepto- cocci give various types of hemolysis on blood agar Use: * for isolation of pathogenic bacteria * differentiate Strepto- coccusspp. according to their hemolytic activity Chocolate agar Type: enriched Constituents: blood agar heated at 56oC Principle: hemoglobin releases hemin (X-factor), required by fastidious organisms Use: permits growth of fastidious bacteria & to help in the recovery of Streptococcus spp Culture Media

  7. Tests • Blood hemolysis:α, β, γ • Biochemical tests:1.Bile solubility 2. Bacitracin sensitivity 3. Optochin sensitivity • Serological test: Antistreptolysin O test (ASO)

  8. Blood hemolysis

  9. Bile solubility • Medium: Nutrient broth + bile salt. • Principle: Lysis of m.o. sensitive to bile salts as Streptococcus pneumoniae • Use: To differentiate betweenα-haemolytic streptococci S. pneumoniae +ve &S. viridans -ve

  10. Bacitracin sensitivity • Medium: Blood agar • Reagent: 0.04 unit bacitracin disc is applied to the surface of the agar plat • Principle: S. pyogenes (β-hemolytic) is sensitive to bacitracin α & γ hemolytic streptococci are resistant to bacitracin • Use: To differentiate betweenStrept. pyogenes (S) from other α & γ hemolytic streptococci (R) Bacitracin Test+ve

  11. Optochin sensitivity • Medium: Blood agar • Reagent: Optochin (ethyl hydrocuprein HCl) • Principle: Optochin inhibits the growth of some m. o. • Use: To differentiate betweenα-haemolytic streptococciS. pneumoniae +ve &S. viridans -ve -ve S. viridans +ve S. pneumoniae Optochin Sensitivity Test +ve -ve

  12. Antistreptolysin O test (ASO)Serologic Agglutination Test • Medium: Polystyrene latex particles coated with Streptolysin O Antigen • Reagent: Streptolysin O Antigen • Principle: Reagent + Serum → agglutination (+ ve Ag-Ab reaction) • Use: Detection of anti-streptolysin O in patient’s serum is an indication for streptococcal infection.

  13. ASO test +ve -ve

  14. Mixed cultureGram stain and streak for isolation MAcConkey Day 1 Day 2 TSA Gram stain and select a Gram negative colony Oxidase (-) Oxidase (+) Lac negative on MAC Lac positive on MAC Lac negative on MAC Day 3 Urea (+) Green sheen on EMB Dark purple on EMB Glucose fermentation (-) H2S (+)on TSI Citrate (-) Citrate (+) Lipase (+) on tributyrin Day 4 Proteus vulgaris Escherichia coli E. aerogenes Ps. aeruginosa

  15. Mixed cultureGram stain and streak for isolation MSA Day 1 Day 2 TSA Gram stain and select a Gram positive colony Catalase (+) Negative for mannitol on MSA Positive for mannitol on MSA Day 3 Nitrate (+) Nitrate (-) Trehalose(-) Novobiocin sensitive Novobiocin sensitive Novobiocin resistant Day 4 Staphylococcus epidermidis Staphylococcus aureus Staphylococcus saprophyticus

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