Identification of Gram Positive Cocci: Staphylococcus

Contains both pathogenic and non-pathogenic organisms Do not produce endospores, but are resistant to drying (desiccation) Found routinely on the surface of the skin May cause infection if the skin or mucous membranes are broken or damaged Three major species:
1. 2. 3. Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus

Staphylococcus aureus is considered the most pathogenic species, causing abscesses, boils, carbuncles, acne, impetego, and less commonly, pneumonia, osteomyelitis, endocarditis, cystitis, pyelonephritis, and food poisoning. The three species can be distinguished from each other by various biochemical tests. In this lab we will perform some of these tests and observe the results.

Chemical and Biochemical Tests

The identification of organisms is based on cellular, cultural, and biochemical characteristics All species of Staphylococcus are Gram Positive Cocci (GPC) On nutrient agar they tend to be white (or cream colored), circular, entire, convex colonies. On Sheep Blood Agar Staphylococcus aureus may exhibit hemolysis of the agar in the area around the colonies. Tests to be performed:
1. 2. 3. 4. Catalase test Coagulase test Growth and fermentation on Mannitol Salt Agar Susceptibility to the antibiotic Novobiocin

Catalase Test
The Catalase test determines if the organism produces the enzyme Catalase, which breaks down hydrogen peroxide (H2O2) to water and oxygen (O2).
Catalase

2 H2O2

2 H2O + O2 (g)

Catalase allows organisms to break down harmful metabolites of aerobic respiration and may be seen in aerobic and facultatively anaerobic organisms. There are other enzymes produced by some organisms to handle other toxic endproducts of metabolism, such as superoxide dismutase. Not all organisms produce catalase.

Coagulase Test
Pathogenic organisms require mechanisms to help them overcome host defense systems. One mechanism involves coating the bacterial cells in a body substance, such as fibrin, to hide the bacterial cells from the immune system. This coating will not trigger an immune response by the host cells. The enzyme coagulase causes fibrin to be deposited on bacterial cells helping them to become invisible to the host immune system.

High Salt Tolerance

Some organisms cannot tolerate a high salt concentrations. Media containing higher than normal salt concentrations will inhibit the growth of these non-salt tolerant organisms. Mannitol salt agar contains a high salt concentration so only salt tolerant organisms will grow on it. Also, Mannitol salt agar contains the sugar Mannitol. Some organisms can utilize this sugar as a food source and will produce acidic by-products from this metabolism. The addition of acid to the medium by the fermentation of Mannitol changes the pH. If a pH indicator is present in the medium (such as Phenol red) a color change will occur dependant upon the pH of the medium (agar or broth). Mannitol Salt Agar contains the pH indicator Phenol Red This pH indicator is red at neutral pH (around 7.0), but turns yellow under acidic conditions. Antibiotic susceptibility is another test that can be used to identify bacteria. A paper disc impregnated with the antibiotic, in this case Novobiocin, is placed on a lawn of bacteria following inoculation. The antibiotic in the disc diffuses into the surrounding agar. If the bacterial species is susceptible to the antibiotic there is a circle of no -growth around the disc where bacterial growth is inhibited by the antibiotic. If the bacteria is resistant to the antibiotic the cells grow right up the the antibiotic disc. The bacterial species or strain is reported as being resistant to the antibiotic (R) or susceptible to the antibiotic (S) depending on the observations made. The diameter of the area of no-growth around the disc may determine the susceptibility or resistance of the organism to the antibiotic.

Antibiotic Susceptibility/Resistance

Interpretation of Results
Catalase
Bubbling indicates a positive test for the presence of the catalase enzyme.

Coagulase
Agglutination of the Test latex with no agglutination of the Control latex is considered a positive (+) test for the presence of this enzyme. All reactions occurring after 20 seconds should be ignored. Agglutination of the Test latex with no agglutination of the Control latex is considered a positive (+) test for the presence of this enzyme.

Mannitol Salt Agar

Two different characteristics of the organism are determined with this agar. The first is the organisms ability to tolerate a high salt environment. Evidence of growth on the slant indicates the organism can grow in a high salt environment. Organisms that can ferment the sugar Mannitol produce an acid end-product that changes the red pH indicator (Phenol red) in the media to yellow. Any yellow in the media is considered a positive test for Mannitol fermentation. It is possible to have growth, but no Mannitol fermentation.

Novobiocin Susceptibility
A zone of growth inhibition 17 mm or less in diameter indicates resistance (R) to Novobiocin. If the zone is greater than 11 mm the organism is susceptible (S) to Novobiocin.