EXERCISE 13

GRAM POSITIVE PYOGENIC COCCI

I. INTRODUCTION

The normal flora of the mucous membranes of man count among its members a variety of Gram-positive cocci. Two important genera, Staphylococcus and Streptococcus are presented in today's lab exercise.

A. STAPHYLOCOCCUS

The genus Staphylococcus is, for the most part composed of two noteworthy species: Staphylococcus aureus and Staphylococcus epidermidis. S. aureus is the major pathogen and can cause suppuration, abscess formation, a variety of pyogenic infections, and even fatal septicemia. S. epidermidis is normally non-pathogenic but can produce disease under certain conditions. The staphylococci produce the enzyme catalase that distinguishes them from the streptococci which do not. By definition, strains of staphylococci that produce the enzyme coagulase are S. aureus, thus differentiating them from all other staphylococci which do not. Most strains of S. aureus produce a non-diffusible golden yellow pigment and a zone of clear beta hemolysis on agar containing blood. Non-pathogenic strains of staphylococci usually produce white or grayish colonies and are coagulase negative. They may be hemolytic but usually are not.

B. STREPTOCOCCUS

The genus Streptococcus contains many species which are considered pathogens. Streptococci probably cause a greater variety of types of clinical diseases than any other genus. The most commonly isolated and/or the most virulent strains of streptococci are:

The ability to hemolyze red blood cells varies among the species in the Streptococcus genus, and it is the first feature observed in species classification. The hemolytic reactions of streptococci on sheep blood agar (BAP) are:

II. LAB WORK

Materials supplied: (work individually) Procedure:
  1. Each student has his/her own:
  2. Each student:

CATALASE TEST:

The Catalase test differentiates between groups of microorganisms on the basis of catalase production. This test is consistently positive for staphylococci and negative for streptococci. Catalase is an enzyme that splits hydrogen peroxide into water and oxygen. The oxygen released is seen as tiny bubbles.

  1. Pick up 1 or 2 isolated colonies with your sterile loop and spread on a slide. Do not add saline. Do not include any agar; blood cells have catalase in them.
  2. Add 1 drop of 3% hydrogen peroxide to the smear and look for vigorous release of bubbles. Bubbles indicate a positive reaction for catalase.

    GENUSCATALASE REACTION
    StreptococcusNo bubbles (no catalase)
    StaphylococcusBubbles (catalase present)

COAGULASE TEST:

The Coagulase test is the most reliable indicator of pathogenic, toxin-producing strains of Staphylococcus, i.e., for Staphylococcus aureus. Most pathogenic strains of Staphylococcus produce prostaphylocoagulase that reacts with an activator similar to prothrombin, to form the active clotting agent, coagulase. Coagulase in turn reacts with fibrinogen forming fibrin to produce the clotting of plasma.

  1. Use a sterile inoculating loop to remove a heavy inoculum from your presumptive Staphylococcus unknown (determined by the Gram stain and Catalase test).
  2. Emulsify this growth into 1 tube of Rabbit plasma, label with your name, and place in the 37C incubator until the next lab period.
  3. After incubation, look for cloting of the plasma by tilting the plasma tube and observe whether the liquid is solidified or not (do not spill).

ORGANISMCOAGULASE PRODUCTION
Staphylococcus aureusClot (Coagulase produced)
Staphylococcus epidermidisNo Clot (No Coagulase)

STREPTOCOCCUS GROUP IDENTIFICATION:

To speciate your Streptococcus unknown, it is first necessary to categorize it on the basis of its ability to lyse sheep red blood cells (hemolysis).
  1. Examine your unknown BAP carefully (look only at isolated colonies for this determination) and decide based upon the following descriptions, and the demonstration plates at the front table, which of the 3 types of hemolysis your unknown exhibits.
ALPHA HEMOLYSIS:
Incomplete lysis of RBC's with the formation of a green discoloration surrounding the colony.
BETA HEMOLYSIS:
Complete disruption of RBC's with release of hemoglobin. A clearing around the colony.
GAMMA HEMOLYSIS:
No change in the medium at all.
  1. If your strep unknown exhibits a-hemolysis, then it could belong to Group D or be Streptococcus pneumoniae or a Viridans streptococcus. Streak your unknown to one half of the BAP and place one Optochin disc in the area of densest inocululation. Next, use a sterile loop to inoculate the Bile-Esculin Agar plate and the 6.5% NaCl broth with your Strep unknown. (Do this only if your unknown exhibits a-hemolysis.)

  2. If your strep unknown exhibits b-hemolysis, then streak your unknown to one half of the BAP and place one Bacitracin disc in the area of densest inocululation. Use the other half of the BAP for the CAMP test (see diagram). Streak your unknown Strep. in a straight line across the curved side of the plate. Sterilize your needle and streak the Staphylococcus aureus (broth, not plate) perpendicular to your unknown streak, leaving a 1cm space between them.

  3. If your strep unknown exhibits no hemolysis (g), then ignore steps b and c. Instead, use a sterile loop to inoculate the Bile-Esculin Agar plate and the 6.5% NaCl broth with your Strep unknown.

  4. Incubate your BAP plates in a candle jar at 37C until the next lab period. Put the Bile-Esculin and 6.5% NaCl broth in the 37C incubator. Be sure to put your name on all of your material.

  5. Note that if the streptococcus culture is sensitive to Bacitracin or Optichin, you will see an area of no growth surrounding the disc.

  6. Discard your unknown BAP plates. Materials kept longer than 24 hrs. often give inconclusive or erroneous results.

CAMP TEST PROCEDURE:

CAMP test performed on a full size Blood Agar Plate. Note the enhanced hemolysis in the shape of an arrow.

NEXT LAB PERIOD

  1. Read and record the results of both your and your partner's unknown on the Report Sheet, using the table below:
    STREPTOCOCCUS IDENTIFICATION
    GroupHemolysisBacitracin
    Sensitivity
    CAMP
    Test
    Bile-
    Esculin
    Agar
    6.5%
    NaCl
    Broth
    Optochin
    Sensitivity
    ABeta+-


    BBeta-+


    CBeta--


    D
    Enterococcus
    Alpha or
    Gamma


    ++-
    D
    Non-Enterococcus
    Alpha or
    Gamma


    +--
    ViridansAlpha

    -+-
    Streptococcus
    pneumoniae
    Alpha

    +-+

  2. At the end of this lab period, turn in your Report Sheet, labeled with your name and unknown #s.



While every effort is made to ensure that this information is up-to-date and accurate, the statements found on this page are for informational purposes only.
LSUMC WWW Publication Policy, 9/23/96