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A. GRAMS POSITIVE COCCI

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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

								
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