Blood Agar: Hemolytic Reactions
When streaked on Blood Agar, many species of bacteria cause hemolysis – i.e., destruction of the erythrocytes (and hemoglobin) in the medium. Hemolytic reactions are generally classified as alpha, beta or gamma according to the appearance of zones around isolated colonies growing on or in the medium:
Beta hemolysis: The colony is surrounded by a white or clear zone in which few or no intact erythrocytes are found. This reaction is best seen when the organism is growing under anaerobic conditions. Beta hemolysis is caused by one or more erythrocyte-lysing enzymes called hemolysins. In the top photo at right which shows Enterococcus durans growing on Blood Agar, the light to clear zones can be seen around the colonies. Where growth occurs in the presence of less oxygen (in stabs into the medium made with the inoculating loop as seen here on a plate of Streptococcus pyogenes), the reaction is more pronounced.
Alpha hemolysis: The colony is surrounded by a zone of intact but discolored erythrocytes that have a greenish color. This appearance is generally due to the action of peroxide produced by the bacteria. The bottom photo shows Streptococcus mitis growing on Blood Agar; some greenish zones of alpha hemolysis are visible. The absence of growth around an optochin disc, as seen here, helps to distinguish S. pneumoniae from other alpha-hemolytic streptococci.
Gamma hemolysis is simply a synonym for negative hemolysis in which there is no change in the medium surrounding the colony as seen here.
Blood Agar: The CAMP Test
Streptococcus agalactiae, a member of the Lancefield Group B streptococci, is one of the causative agents of mastitis in cows. Identifying this organism can be difficult, and the CAMP Test was designed to aid in the identification of this organism. This test relies on the fact that most S. agalactiae strains produce a diffusible, extracellular compound that will, in conjunction with a specific beta-hemolysin of Staphylococcus aureus, cause complete lysis of sheep red blood cells in an agar medium. This test was named after the authors of the original paper.
Remember not to confuse the terms hemolysin (that which causes hemolysis) and hemolysis (also known as hemolytic reaction – i.e., the reaction seen in the blood). Different Greek-letter systems apply to various hemolysins produced by S. aureus and hemolytic reactions.
In this photo, a Blood Agar plate is shown after 24 hours of incubation at 37°C. The vertical streak is a beta-hemolysin-producing strain of Staphylococcus aureus, and at right angles to it are streaks of (1)Enterococcus faecalis, (2)Streptococcus salivarius, (3)S. agalactiae, and (4)E. durans. Note the large area of complete lysis where the extracellular compound of S. agalactiae encounters the beta-lysin of S. aureus.
Reference: Christie, R., N. E. Atkins, and E. Munch-Peterson. 1944. A note on a lytic phenomenon shown by group B streptococci. Aust. J. Exp. Biol. Med. Sci. 22:197-200.
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Department of Bacteriology, U.W.-Madison
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