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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 Arrangment: chains or pairs Classification of streptococci 1. hemolysis on blood agar α hemolysis β hemolysis γ hemolysis (Partial hemolysis ) (Complete hemolysis) (non hemolysis *S. pyogenes *S. pneumonia *S. faecalis *Viridans streptococci Cultivation Culture Media 1. Blood agar 2. Chocolate agar Culture Media Blood agar Chocolate agar Type: enriched, differential medium Type: enriched Constituents: nutrient agar +5- Constituents: blood agar heated at 10% defibrinated blood 56oC Principle: species of strepto- Principle: hemoglobin releases cocci give various types of hemin (X-factor), required hemolysis on blood agar by fastidious organisms Use: * for isolation of Use : permits growth of fastidious pathogenic bacteria bacteria & to help in the * differentiate Strepto- recovery of Streptococcus spp coccus spp. according to their hemolytic activity Tests • Blood hemolysis: α, β, γ • Biochemical tests: 1. Bile solubility 2. Bacitracin sensitivity 3. Optochin sensitivity • Serological test: Antistreptolysin O test (ASO) Blood hemolysis Hemolysis on blood agar Principle Use (α) Alpha haemolysis Incomplete blood To differentiate m.o. haemolysis according to their green zone around the haemolytic activity growth due to H2O2 S. pneumoniae(α) production, hemoglobin Met hemoglobin S. viridans (α) (β) Beta-haemolysis Complete blood To differentiate m.o. haemolysis according to their producing clear zone haemolytic activity around the growth e.g. Staph. aureus (β) Strept. pyogenes(β) (γ) Gamma-haemolysis Absence of blood Streptococcus faecalis haemolysis around (γ) the growth Staphylococcus epidermidis (γ) Bile solubility Medium: Nutrient broth + bile salt. Principle: Lysis of m.o. sensitive to bile salts as Streptococcus pneumoniae Use: To differentiate between α-haemolytic streptococci S. pneumoniae +ve & S. viridans -ve Bacitracin sensitivity Medium: Blood agar Reagent: 0.04 unit bacitracin disc is applied to the surface of the agar plat Bacitracin Test +ve Principle: S. pyogenes (β-hemolytic) is sensitive to bacitracin α & γ hemolytic streptococci are resistant to bacitracin Use: To differentiate between Strept. pyogenes (S) from other α & γ hemolytic streptococci (R) Optochin sensitivity Medium: Blood agar Reagent: Optochin (ethyl hydrocuprein HCl) Principle: Optochin inhibits the growth of some m. o. Use: To differentiate between α-haemolytic streptococci S. pneumoniae +ve & S. viridans -ve -ve S. viridans +ve S. pneumoniae +ve -ve Optochin Sensitivity Test 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. ASO test +ve -ve Mixed culture Day Gram stain and streak for isolation 1 MAcConkey Day TSA 2 Gram stain and select a Gram negative colony Oxidase (-) Oxidase (+) Lac negative on Lac positive on Lac negative MAC MAC on MAC Day 3 Urea (+) Green sheen Dark purple Glucose on EMB on EMB fermentation (-) H2S (+) Citrate (-) Citrate (+) Lipase (+) on on TSI tributyrin Day Proteus vulgaris Escherichia coli E. aerogenes Ps. aeruginosa 4 Mixed culture Day Gram stain and streak for isolation 1 MSA Day TSA 2 Gram stain and select a Gram positive colony Catalase (+) Negative for Positive for mannitol on MSA mannitol on MSA Day 3 Trehalose Nitrate Nitrate (-) (+) (-) Novobiocin Novobiocin Novobiocin sensitive sensitive resistant Day Staphylococcus Staphylococcus Staphylococcus 4 epidermidis aureus saprophyticus
"A. GRAMS POSITIVE COCCI"