Identification of Staphylococci by malj

VIEWS: 69 PAGES: 17

									Identification of Staphylococci
                &
       Oral Streptococci

        Exercise 19 & 22
                Gram Positives
• Staphylococci
  –   G+
  –   Spherical
  –   Grow in clusters
  –   Non-sporulating
• Streptococci
  –   G+
  –   Spherical
  –   Grow in chains
  –   Non-sporulating
                       Biochemical Tests
• Staphylococcus                               • Streptococcus
     – Catalase Positive                            – Catalase Negative
•   Staph sp.           •   Staph. Aureus      •   Streptococcus           •   Streptococcus
     –   Coagulase –         –   Coagulase +       Normal Flora                Pathogens
                                                    –   Mitis-Salivarius        –   Hemolysins
                                                        Agar                         •   No γ
                                                    –   Hemolysins                   •   Some α
                                                          •   Some γ                 •   Some β
                                                          •   Some α            –   Group D Strep
                                                          •   No β                   •   Positive for
                                                                                         Esculin Test
     Distinguishing between
Staphylococcus and Streptococcus
• Cell groupings can be visualized, but it is not always
  definitive.
• Biochemical Tests
   – Catalase Test

   – 2H2O2        catalase   H 2 O + O2

• Protects cell from damage due to hydrogen peroxide
• Streptococcus
   – Catalase -
• Staphylococcus
   – Catalase +
               Catalase Test
• Obtain a small square of parafilm
  – ~2x2 inches
• Make a dimple in it with your thumb
• Add a few drops of hydrogen peroxide to the
  dimple
• Get some bacteria on a sterile inoculating loop
  and place them in the hydrogen peroxide
• Watch for bubbles
  – Catalase positive
                       Biochemical Tests
• Staphylococcus                               • Streptococcus
     – Catalase Positive                            – Catalase Negative
•   Staph sp.           •   Staph. Aureus      •   Streptococcus           •   Streptococcus
     –   Coagulase –         –   Coagulase +       Normal Flora                Pathogens
                                                    –   Mitis-Salivarius        –   Hemolysins
                                                        Agar                         •   No γ
                                                    –   Hemolysins                   •   Some α
                                                          •   Some γ                 •   Some β
                                                          •   Some α            –   Group D Strep
                                                          •   No β                   •   Positive for
                                                                                         Esculin Test
What about potential pathogens
• If you know your organism is a Staphylococcus it is
  important to determine if it is Staphylococcus aureus
• Staphylococcus aureus - Important pathogen
   – Causes more diseases in the US than any other bacterium
      • Cause of many nosocomial infections
           – An secondary infection contracted in the hospital.
      • Staph infection (wound infection), conjunctivitis, toxic shock syndrome
      • Many are becoming antibiotic resistant
          – PRSA (penicillin), MRSA (methicillin), VRSA (vancomycin), LRSA
            (linezolid)
• Characteristics of Staph. aureus
   – Yellow pigmented colonies
      • Most other Staph. species are cream colored

   – Coagulase +
               Coagulase Test
• The coagulase enzyme causes plasma to
  coagulate (clot) at 37°C
  – Important characteristic of many pathogens
• The Test
  – Acquire a durham tube with rabbit plasma
  – Using a loop inoculate with Staph. aureus
  – Cover the top with parafilm and incubate at 37°C
     • Use an uninoculated tube as a negative control
  – May take between 30 min to 24 hrs for clotting to
    occur.
                       Biochemical Tests
• Staphylococcus                               • Streptococcus
     – Catalase Positive                            – Catalase Negative
•   Staph sp.           •   Staph. Aureus      •   Streptococcus           •   Streptococcus
     –   Coagulase –         –   Coagulase +       Normal Flora                Pathogens
                                                    –   Mitis-Salivarius        –   Hemolysins
                                                        Agar                         •   No γ
                                                    –   Hemolysins                   •   Some α
                                                          •   Some γ                 •   Some β
                                                          •   Some α            –   Group D Strep
                                                          •   No β                   •   Positive for
                                                                                         Esculin Test
    Normal Flora of the Mouth
• Many bacteria are present in the mouth
  – Streptococci of the viridians group (normal flora)
     • Streptococcus mitis
     • Streptococcus salivarius
     • Streptococcus mutans (dental caries)

  – Opportunistic Pathogens (not normal flora)
     • Enterococcus faecalis (tooth infections, urinary tract
       infections)
     • Streptococcus pneumoniae (pnuemonia)
     • Streptococcus pyogenes (strep throat)
     • Neisseria meningitis (meningitis)
     • Haemophilus influenzae (meningitis)
     Identifying Oral Bacteria
• Mitis-Salivarius Agar
  – differential medium
     • What is a differential medium?
  – Distinguishes between different oral strep.
     • Streptococcus mutans
        – Light blue colonies, 3-4 mm in diameter, frosted
          appearance
     • Streptococcus salivarius
        – Blue gum drop colonies, 1-5 mm in diameter
     • Streptococcus mitis
        – Small navy blue to black colonies
                       Biochemical Tests
• Staphylococcus                               • Streptococcus
     – Catalase Positive                            – Catalase Negative
•   Staph sp.           •   Staph. Aureus      •   Streptococcus           •   Streptococcus
     –   Coagulase –         –   Coagulase +       Normal Flora                Pathogens
                                                    –   Mitis-Salivarius        –   Hemolysins
                                                        Agar                         •   No γ
                                                    –   Hemolysins                   •   Some α
                                                          •   Some γ                 •   Some β
                                                          •   Some α            –   Group D Strep
                                                          •   No β                   •   Positive for
                                                                                         Esculin Test
        Hemolysin Production
• Many pathogens found in the throat and mouth
  produce hemolysins
  – Hemolysins lyse red blood cells (RBCs)
• Three Classes of Hemolysin which can be
  visualized on Blood Agar (differential medium)
  – β hemolysin – completely lyse RBCs
     • Clear zone on blood agar
     • Characteristic of Group A Streptococci
  – α hemolysin – partially lyse RBCs
     • Green zone on blood agar
  – γ hemolysis – have no effect on RBCs
     • No alteration to blood agar (red)
         Testing for hemolysins
• Each table choose one patient. Preferably someone who
  has a sore throat.
   – Aim for tonsils (on the sides).
   – Please be careful not to gag them!
• Swab their throat.
• Use the swab to swab a blood agar plate.
                       Biochemical Tests
• Staphylococcus                               • Streptococcus
     – Catalase Positive                            – Catalase Negative
•   Staph sp.           •   Staph. Aureus      •   Streptococcus           •   Streptococcus
     –   Coagulase –         –   Coagulase +       Normal Flora                Pathogens
                                                    –   Mitis-Salivarius        –   Hemolysins
                                                        Agar                         •   No γ
                                                    –   Hemolysins                   •   Some α
                                                          •   Some γ                 •   Some β
                                                          •   Some α            –   Group D Strep
                                                          •   No β                   •   Positive for
                                                                                         Esculin Test
                   Group D strep.
• Group D Strep contain many pathogens
   – Enterococcus faecalis (life threatening nosocomial infections like
     endocarditis, bladder, prostate and epididymal infections)
   – Streptococcus faecium
   – Streptococcus avium
   – Streptococcus bovis (human endocardiatis)
   – Streptococcus faecium
• Bile Esculin Test
   – Differential Medium
       • Identifies a bacterium as Group D Strep (pathogen)
   – Tests ability to:
       • Grow in 4% bile (very harsh compound found in stomach)
       • hydrolize esculin (break down to use as a food source)
           – Turns medium black
                        Activities
• Each table test Staphylococcus epidermidis and
  Enterococcus faecalis for Catalase
• Observe the prepared Coagulase test
• Each table streak Enterococcus faecalis on a Bile
  Esculin Agar Slant
• Each student
   – pair up and swab each others throat
   – Smear the swab across the surface of a blood agar plate
• Each student will divide a Mitis-Salivarius Plate in half.
   – Swab your gum line and inoculate one side
   – Swab your tongue and inoculate the other side

								
To top