Professional Documents
Culture Documents
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.
NaCl
Agar
Distilled water
Combine the ingredients and adjust the pH to 7.3. Boil to dissolve the agar, and sterilize by
autoclaving.
1. Prepare the blood agar base as instructed by the manufacturer. Sterilize by autoclaving at
121°C for 15 minutes.
3. When the agar base is cooled to 50°C, add sterile blood agar aseptically and mix well
gently. Avoid formation of air bubbles. You must have warmed the blood to room
temperature at the time of dispensing to molten agar base.
(Note: If you are planning to prepare a batch of blood agar plates, prepare few blood
agar plates first to ensure that blood is sterile).
5. Label the medium with the date of preparation and give it a batch number (if necessary).
6. Store the plates at 2-8°C, preferably in sealed plastic bags to prevent loss of moisture.
The shelf life of thus prepared blood agar is up to four weeks.
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).
1. S. pyogenes: Beta-hemolysis
2. S. pneumoniae: Alpha-hemolysis
3. Satellitism of H. influenzae
Optochin and Bacitracin Sensitivity of
the isolates in Blood Agar
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.
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.
Double Zone hemolysis produced by
Clostridium perfringens