THE VIRTUAL EDGE: Lab 16 Enzyme-Mediated Biochemical Activities of Bacteria: Physiological Testing II

Staphylococcus and Streptococcus species:

In the previous lab, BAP and MSA plates were inoculated with Staphylococcus and Streptococcus spp..

Blood agar is a rich, non-selective medium that supports the growth of most bacteria. However, it is differential based on the ability of the organism to produce enzymes called hemolysins, which lyse red blood cells (RBC). Three patterns of hemolysis can be observed on a blood agar plate.

1. Alpha ( a ) -hemolysis: incomplete lysis of RBC - Greenish, cloudy zone around the colony.

2. Beta ( b)-hemolysis: complete lysis of RBC - A clear zone with a clean edge around the colony.

3. Gamma ( g)-hemolysis: no lysis of RBC - No change in the blood agar around the colony.

MSA contains a high concentration of salt (NaCl), and therefore, selects for the growth of microbes that can tolerate high salt concentrations. Staphylococcus species are halotolerant, whereas Streptococcus species and many other organisms are inhibited by high concentrations of NaCl. MSA also differentiates on the basis of mannitol fermentation. Microorganisms that can ferment mannitol create acidic byproducts, which decrease the pH of the medium and cause the pH indicator, phenol red, to turn yellow.

Expected results:

Staphylococcus aureus

  • Growth on MSA, ferments mannitol causing a color change to yellow
  • b-Hemolysis on BAP

Staphylococcus epidermidis

  • Growth on MSA, does not ferment mannitol (medium remains red)
  • g-Hemolysis on BAP

Streptococcus pneumoniae

  • Limited or no growth on MSA
  • a-Hemolysis on BAP

Streptococcus pyogenes

  • Limited or no growth on MSA
  • b-Hemolysis on BAP

 

Gram-positive cocci are often isolated from clinical samples. The catalase test is a particularly important test used to determine whether the Gram + cocci is a staphylococci or a streptococci. Catalase is an enzyme that converts hydrogen peroxide to water and oxygen gas. The test is easy to perform; bacteria are simply mixed with H 2O 2. If bubbles appear (due to the production of oxygen gas) the bacteria are catalase positive. If no bubbles appear, the bacteria are catalase negative. Staphylococcus and Micrococcus spp. are catalase positive, whereas Streptococcus and Enterococcus spp. are catalase negative. If a Gram-positive cocci is catalase positive and presumed to be a staphylococci, the coagulase test is often performed. This test assays for the presence of coagulase, an enzyme that coagulates blood plasma, and can differentiate between Staphylococcus aureus (coagulase positive) and Staphylococcus epidermidis (coagulase negative). If, in contrast, a Gram-positive cocci is catalase negative and thought to be a streptococci, antibiotic susceptibility tests can differentiate between pathogenic streptococci (Streptococcus pneumoniae and Streptococcus pyogenes), and the normal floral streptococci (e.g. Streptococcus mitis).


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Rachel Watson, M.S.
AG 5010
766-3524
Cell: 307-760-2942
rwatson@uwyo.edu

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