Lab. No. 3

Microscopical Appearance:(Gram’s Stain) Microscopical Appearance:(Gram’s Stain) Gram’s +ve Cocci Irregular ClustersChains or PairsTetrads Staphylococci MicrococciStreptococci

Gram’s +ve Cocci Irregular ClustersChains or PairsTetrads Staphylococci MicrococciStreptococci Catalase Test Catalase +veCatalase -ve

Catalase Test Differentiative test to separate Staphylococci and Micrococci which are catalase +ve from Sterptococci which are catalase –ve. Differentiative test to separate Staphylococci and Micrococci which are catalase +ve from Sterptococci which are catalase –ve. H2O2H2O2 Catalase enzyme H 2 o + O 2 Air bubbles Principle: Procedure: 1 3 H2O2H2O2 2

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

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

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

Current classification of streptococci depends on 6 features; some of them are: Hemolytic reactions on sheep blood agar Hemolytic reactions on sheep blood agar Lancefield serological groups (A-W with the exception of I and J). 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: 1.β-hemolytic Sterptococci. 2.α-hemolytic Sterptococci. 3.Non-hemolytic streptococci

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

β -hemolytic Sterptococci: α -hemolytic Sterptococci:

 It causes complete hemolysis to RBCs (caused by hemolysins) leading to formation of clear zone around the colonies β -hemolytic Sterptococci:  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 S.pyogenes & Non group A β-hemolytic streptococci A low conc. of Bacitracin (0.04 units) will selectively inhibit the growth of S.pyogenes giving a zone of inhibition around the disc Principle: Bacitracin Sensitivity Test:

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

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

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

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

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

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

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

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

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.

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

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

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

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

Definitive test for Enterococcus faecalis Growth on MacConkey’s agar: 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. 2. Incubate the plate at 35 o C for 24 hrs. Flam & Cool

Results: Enterococcus faecalis Growth of pink colonies No Growth Sterptococci

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

Practical Work 1. Gram’s Stain (spots) 2. Catalase test 3. Blood agar plate. 4. Bacitracin & Optochin Sensitivity. 5. MacConkey’s agar plate. O B 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 Complete hemolysis Greenish discoloration Non hemolytic

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

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

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