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Blood Agar
Blood Agar
Blood contains inhibitors for certain bacteria such as Neisseria and Haemophilus genera and the
blood agar must be heated to inactivate these inhibitors and to release essential growth factors
(e.g., V factor) . Heating of blood agar converts it into chocolate agar (heated blood turns a
chocolate color) and supports the growth of these bacteria.
Blood agar consists of a base containing a protein source (e.g. Tryptones), soybean protein
digest, sodium chloride (Nacl), agar and 5% sheep blood.
Combine the ingredients and adjust the pH to 7.3. Boil to dissolve the agar, and sterilize by
autoclaving.
1. The pH of the blood agar range from 7.2 to 7.6 at room temperature.
2. Inoculate the plates with 5 hour broth cultures of Streptococcus pyogenes and S.
pneumoniae. Inoculate also a plate with H. influenzae and streak with S. aureus
(i.e. Satellitism Test).
3. Incubate the plates in a carbon dioxide enriched atmosphere at 35-37°C overnight.
4. Check for the growth characteristics of each species
1. S. pyogenes: Beta-hemolysis
2. S. pneumoniae: Alpha-hemolysis
3. Satellitism of H. influenzae
1. Isolation, identification (with the use of either Optochin disc or Bacitracin disc and
testing the sensitivity of the isolate) and antimicrobial susceptibility of Streptococci.
2. Determine the type of hemolysis, if any.
Certain bacterial species produce extracellular enzymes that lyse red blood cells in the blood agar
(hemolysis). These hemolysin (extotoxin) radially diffuses outwards from the colony (or
colonies) causing complete or partial destruction of the red cells (RBC) in the medium and
complete denaturation of hemoglobin within the cells to colorless products.
Four types of hemolysis are produced in Sheep blood agar by Streptococci namely; Alpha
hemolysis, Beta hemolysis, gamma hemolysis and alpha prime or wide zone alpha hemolysis.
How does one know if the colonies they are observing on a plate have caused alpha hemolysis
or beta hemolysis?
Note: To know the type of blood agar hemolysis, the blood agar plate must be held up to a light
source and observed with the light coming from behind (transmitted light).
If either type of hemolysis is present, then one will observe a zone of hemolysis surrounding a
growing colony.
2. Beta Hemolysis: Complete lysis of Red Blood Cells, causing a clearing of blood from
the medium under and surrounding the colonies e.g. Group A beta hemolytic
streptococci-Streptococcus pyogenes and Group B, beta hemolytic streptococci-
Streptococcus agalactiace. For group A streptococci maximal activity of both the
hemolysins; Oxygen labile SLO and oxygen stable SLS hemolysins is observed only in
anaerobic conditions.
3. Gamma or non hemolysis: No hemolysis of RBC. No change of the medium under and
surrounding the colonies.
4. Alpha prime or wide zone alpha hemolysis: A small zone of intact erythrocytes
immediately adjacent to bacterial colony, with a zone of complete red-cell hemolysis
surrounding the zone of intact erythrocytes. This type of hemolysis may be confused with
Beta hemolysis.