PHT313 Lab. No. 3

Identification of Gram's +ve Cocci Microscopical Appearance:(Gram’s Stain) Gram’s +ve Cocci Irregular Clusters Chains or Pairs Tetrads Staphylococci Micrococci Streptococci

Catalase Test Catalase +ve Catalase -ve Staphylococci Micrococci Gram’s +ve Cocci Irregular Clusters Chains or Pairs Tetrads Staphylococci Micrococci Streptococci Catalase +ve Catalase -ve

Catalase Test Differentiative test to separate Staphylococci and Micrococci which are catalase +ve from Sterptococci which are catalase –ve. H2O2 Catalase enzyme H2o + O2 Air bubbles Principle: Procedure: 1 3 2

Catalase Test Results: Positive test: rapid appearance of gas bubbles. Staphylococci or Micrococci Catalase –ve Streptococci Catalase +ve

Identification of Sterptococci

Identification of Sterptococci 1- Microscopical Examination (Morphology): Gram’s +ve cocci arranged in: chains pairs (S. Pneumonia) Nonmotile, nonsporeforming.

2-macroscopical examination (cultural characteristics): Growth requires enriched medium containing blood or serum e.g., sheep blood agar →Transparent pin point colonies Catalase negative. 3-Biochemical reactions:

Classification of streptococci: Current classification of streptococci depends on 6 features; some of them are: Hemolytic reactions on sheep blood agar Lancefield serological groups (A-W with the exception of I and J). N.B: S.pyogenes, S.agalatiae & S.pneumoniae are the most important species.

Growth on Blood Agar Sterptococci are divided into three main groups accorging to its action on erythrocytes: β-hemolytic Sterptococci. α-hemolytic Sterptococci. Non-hemolytic streptococci

β-hemolytic Sterptococci. α-hemolytic Sterptococci. non-hemolytic Sterptococci. β-hemolytic Sterptococci. α-hemolytic Sterptococci.

α-hemolytic Sterptococci:

β-hemolytic Sterptococci: It causes complete hemolysis to RBCs (caused by hemolysins) leading to formation of clear zone around the colonies Example: S. pyogenes (group A β-hemolytic Strept.), S.agalactiae.

β-hemolytic Sterptococci Definitive test to differentiate between S.pyogenes & Non group A β-hemolytic streptococci Bacitracin Sensitivity Test: Principle: A low conc. of Bacitracin (0.04 units) will selectively inhibit the growth of S.pyogenes giving a zone of inhibition around the disc

Bacitracin Sensitivity Test: Procedure: 1. Inoculate blood agar plate with the test organism. 2. Aseptically apply Bacitracin disc onto the center of the streaked area. 3. Incubate the plate at 35oC for 18 hrs. B

Results: Positive test: any zone of inhibition around the disc. Bacitracin Sensitive Bacitracin Resistant Non group A β-hemolytic Streptococci S.pyogenes

Antibiotic sensitivity Penicillin: still uniformly effective in treatment of S.pyogenes disease.

α-hemolytic Sterptococci: It causes: 1. Zone of greenish discolouration around the colonies. 2. It is due to peroxidase release that changes haemoglobin to methemoglobin Example: S.pneumonia, viridans streptococci.

α-hemolytic Sterptococci Definitive test to differentiate between S.Pneumoniae & Viridans Streptococci 1- Optochin sensitivity. 2- Bile solubility test.

1. Optochin Sensitivity Test: Principle: S.pneumoniae is inhibited by less than 5µg/ml Optochin reagent giving a zone of inhibition more than 15 mm in diameter.

Procedure: 1. Inoculate blood agar plate with the test organism. 2. Aseptically apply Optochin disc onto the center of the streaked area. 3. Incubate the plate at 35oC for 18 hrs. 4. Accurately measure the diameter of the inhibition zone around the disc. O

Viridans Streptococci Results: Positive test: inhibition zone more than 15 mm in diameter. Optochin sensitive S.pneumoniae Optochin resistant Viridans Streptococci

2. Bile Solubility Test: Principle: S.pneumoniae produces a self-lysing enzyme to depress the growth of old colonies. The presence of bile salt accelerate this process.

10 ml broth culture of the test organism Procedure: Add 1 ml 10% bile salt solution 2 10 ml broth culture of the test organism 1 3. Incubate at 37oC for 15 min . 4. Observe for the visible clearing of the turbid culture.

Results: Positive test: Visible clearance of the turbid culture. Remain turbid S.Pneumoniae Viridans Streptococci

Antibiotic sensitivity 1-Penicillin-resistant pneumococci: reported all over the world. 2- Vancomycin + extended-spectrum cephalosporins e.g., ceftriaxone → used in serious infections; e.g; meningitis.

Effect on blood agar: Enterococci Gram-positive cocci arranged singly, in pairs, and in short chains. Catalase-negative Most strains react with Lancefield group D antisera. E.faecalis is the most important species. Effect on blood agar: It has no effect on RBCs (Non hemolytic)

Definitive test for Enterococcus faecalis Growth on MacConkey’s agar: Definitive test for Enterococcus faecalis Principle: MacConkey’s agar is a selective medium for Gram’s –ve bacteria. It contains bile salts and crystal violet to inhibit the growth of Gram’s +ve bacteria. Enterococcus faecalis is the only gram-positive cocci which can grow on MacConkey’s agar giving pink colonies.

Growth on MacConkey’s agar: Procedure: 1. Inoculate MacConkey’s agar plate with the test organism by streaking. Flam & Cool 2. Incubate the plate at 35oC for 24 hrs.

Results: Sterptococci Enterococcus faecalis No Growth Growth of pink colonies Sterptococci Enterococcus faecalis

Antibiotic sensitivity Enterococci can be very resistant to antibiotics. 1- Penicillin + aminoglycosides 2- Vancomycin → used for penicillin-resistant strains. 3- VRE (vancomycin-resistant enterococci)→ ttt: Linezolid.

Practical Work Gram’s Stain (spots) Catalase test Blood agar plate. Bacitracin & Optochin Sensitivity. MacConkey’s agar plate. B O Flam & Cool

Identification of Sterptococci β-hemolytic Sterptococci α-hemolytic Sterptococci Enterococci Gram’s Stain Catalase test Growth on blood agar Gram’s +ve Cocci arranged in chains - ve - ve - ve Complete hemolysis Non hemolytic Greenish discoloration

Identification of Sterptococci β-hemolytic Sterptococci α-hemolytic Sterptococci Enterococci Bacitracin sensitivity Inhibition zone No zone Bacitracin sensitive Bacitracin resistant - - S.pyogenes Non group A β-hemolytic Strept.

Identification of Sterptococci β-hemolytic Sterptococci α-hemolytic Sterptococci Enterococci Optochin sensitivity Inhibition zone >15mm No zone or <15mm - - Optochin sensitive Optochin resistant Bile Solubility - Visibile clearance Remain turbid - (+ve) (-ve) S.pneumoniae Viridans Streptococci

Identification of Sterptococci β-hemolytic Sterptococci α-hemolytic Sterptococci Enterococci Growth on MacConkey’s Agar No Growth No Growth Growth of pink colonies Enterococcus faecalis

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