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A. GRAM'S POSITIVE COCCI 2. STREPTOCOCCUS SPP. Microscopic examination Microscopic examination Morphological Description Type of stain: Gram reaction: Color: Shape: Size: Arrangment: gram stain gm +ve violet cocci small chains or pairs Classification of streptococci 1. hemolysis on blood agar α hemolysis (Partial hemolysis ) β hemolysis (Complete hemolysis) γ hemolysis (non hemolysis *S. pyogenes *S. pneumonia *Viridans streptococci *S. faecalis Cultivation Culture Media 1. Blood agar 2. Chocolate agar Culture Media Blood agar Type: enriched, differential medium Constituents: nutrient agar +510% defibrinated blood Principle: species of streptococci give various types of hemolysis on blood agar Use: * for isolation of pathogenic bacteria * differentiate Streptococcus spp. 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 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 (α) Alpha haemolysis (β) Beta-haemolysis (γ) Gamma-haemolysis Principle Use Incomplete blood haemolysis green zone around the growth due to H2O2 production, hemoglobin Met hemoglobin Complete blood haemolysis producing clear zone around the growth To differentiate m.o. according to their haemolytic activity S. pneumoniae(α) S. viridans (α) Absence of blood haemolysis around the growth Streptococcus faecalis (γ) Staphylococcus epidermidis (γ) To differentiate m.o. according to their haemolytic activity e.g. Staph. aureus (β) Strept. pyogenes(β) 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 Principle: S. pyogenes (β-hemolytic) is sensitive to bacitracin Bacitracin Test +ve 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 -ve -ve S. viridans +ve S. pneumoniae 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 Day 1 Mixed culture Gram stain and streak for isolation MAcConkey Day 2 TSA Gram stain and select a Gram negative colony Oxidase (-) Lac negative on MAC Oxidase (+) Lac positive on MAC Lac negative on MAC Day 3 Day 4 Urea (+) Green sheen on EMB Dark purple on EMB Glucose fermentation (-) H2S (+) on TSI Citrate (-) Citrate (+) Lipase (+) on tributyrin Proteus vulgaris Escherichia coli E. aerogenes Ps. aeruginosa Day 1 Mixed culture Gram stain and streak for isolation MSA Day 2 TSA Gram stain and select a Gram positive colony Catalase (+) Day 3 Day 4 Negative for mannitol on MSA Positive for mannitol on MSA Trehalose (-) Nitrate Nitrate (+) (-) Novobiocin sensitive Novobiocin sensitive Novobiocin resistant Staphylococcus epidermidis Staphylococcus aureus Staphylococcus saprophyticus