Identification of Gram Positive Cocci: Staphylococcus

1
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
• 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.

2
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
• Catalase test
• Coagulase test
• Growth and fermentation on Mannitol Salt Agar
• Susceptibility to the antibiotic Novobiocin

3
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 end-products
  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.

4
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/Resistance
• 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.

5
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.

6
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.

7
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.