Blood Agar, Hemolysis, and Hemolytic Reactions
Blood agar, hemolysis, and hemolytic reactions
Blood agar is a solid growth medium that contains red blood cells. The medium is used to detect bacteria that produce enzymes to break apart the blood cells. This process is also termed hemolysis. The degree to which the blood cells are hemolyzed is used to distinguish bacteria from one another.
The blood agar medium is prepared in a two-step process. First, a number of ingredients are added to water, including heart infusion, peptone, and sodium chloride. This solution is sterilized. Following sterilization , a known amount of sterile blood is added. The blood can be from rabbit or sheep. Rabbit blood is preferred if the target bacterium is from the group known as group A Streptococcus. Sheep blood is preferred if the target bacterium is Haemophilus parahaemolyticus.
Blood agar is a rich food source for bacteria. So, it can be used for primary culturing, that is, as a means of obtaining as wide a range of bacterial growth from a sample as possible. It is typically not used for this purpose, however, due to the expense of the medium. Other, less expensive agars will do the same thing. What blood agar is uniquely suited for is the determination of hemolysis.
Hemolysis is the break down of the membrane of red blood cells by a bacterial protein known as hemolysin, which causes the release of hemoglobin from the red blood cell. Many types of bacterial posses hemolytic proteins. These proteins are thought to act by integrating into the membrane of the red blood cell and either punching a hole through the membrane or disrupting the structure of the membrane in some other way. The exact molecular details of hemolysin action is still unresolved.
The blood used in the agar is also treated beforehand to remove a molecule called fibrin, which participates in the clotting of blood. The absence of fibrin ensures that clotting of the blood does not occur in the agar, which could interfere with the visual detection of the hemolytic reactions.
There are three types of hemolysis, designated alpha, beta and gamma. Alpha hemolysis is a greenish discoloration that surrounds a bacterial colony growing on the agar. This type of hemolysis represents a partial decomposition of the hemoglobin of the red blood cells. Alpha hemolysis is characteristic of Streptococcus pneumonia and so can be used as a diagnostic feature in the identification of the bacterial strain.
Beta hemolysis represents a complete breakdown of the hemoglobin of the red blood cells in the vicinity of a bacterial colony. There is a clearing of the agar around a colony. Beta hemolysis is characteristic of Streptococcus pyogenes and some strains ofStaphylococcus aureus.
The third type of hemolysis is actually no hemolysis at all. Gamma hemolysis is a lack of hemolysis in the area around a bacterial colony. A blood agar plate displaying gamma hemolysis actually appears brownish. This is a normal reaction of the blood to the growth conditions used (37° C in the presence of carbon dioxide). Gamma hemolysis is a characteristic of Enterococcus faecalis.
Hemolytic reactions can also display some synergy. That is, the combination of reactions produces a reaction that is stronger than either reaction alone. Certain species of bacteria, such as group B Strep (n example is Streptococcus agalactiae ) are weakly beta-hemolytic. However, if the bacteria are in close proximity with a strain of Staphylococcus the betahemolysins of the two organisms can combine to produce an intense beta hemolytic reaction. This forms the basis of a test called the CAMP test (after the initials of its inventors).
The determination of hemolysis and of the hemolytic reactions is useful in distinguishing different types of bacteria. Subsequent biochemical testing can narrow down the identification even further. For example, a beta hemolytic reaction is indicative of a Streptococcus. Testing of the Streptococcus organisms with bacitracin is often the next step. Bacitracin is an antimicrobial that is produced by the bacterium Bacillus subtilis. Streptococcus pyogenes strains are almost uniformally sensitive to bacitracin. But other antigenic groups of Streptococcus are not bacitracin sensitive.
See also Laboratory techniques in microbiology; Staphylococci and staphylococcal infections; Streptococci and streptococcal infections