Blood Agar: Composition, Preparation, Uses and Types of Hemolysis

Blood agar is an enriched, bacterial growth medium. Fastidious organisms, such as streptococci, do not grow well on ordinary growth media. Blood agar is a type of growth medium (trypticase soya agar enriched with 5% sheep blood)  that encourages the growth of bacteria, such as streptococci, that otherwise wouldn’t grow.

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.

Beta hemolysis in sheep blood agar.

Blood agar consists of a base containing a protein source (e.g. Tryptones), soybean protein digest, sodium chloride (Nacl), agar and 5% sheep blood. 

Composition of Blood Agar:

Combine the ingredients and adjust the pH to 7.3. Boil to dissolve the agar, and sterilize by autoclaving.

Procedure for the preparation of Blood Agar

  1. Prepare the blood agar base as instructed by the manufacturer. Sterilize by autoclaving at 121°C for 15 minutes.
  2. Transfer thus prepared blood agar base to a 50°C water bath.
  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).
  4. Dispense 15 ml amounts to sterile petri plates aseptically
  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.

Quality control of Blood Agar

  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

      Optochin and Bacitracin Sensitivity of the isolates in Blood Agar

Uses of Blood Agar:

Blood agar has two major uses:

  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. 

Blood Agar and Hemolysis

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.

Hemolysis is best observed by examining colonies grown under anaerobic conditions or inspecting sub-surface colonies.

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.

  1. Alpha hemolysis: Partial lysis of the RBC to produce a greenish-gray or brownish discoloration around the colony. α hemolysis is due to the reduction of RBC hemoglobin to methemoglobin  in the medium surrounding the colony. Many of the alpha hemolytic streptococci are part of the normal body flora. But Streptococcus pneumoniae which is also alpha hemolytic causes serious pneumonia and other deadly infectious disease.

    Various types of Hemolysis

  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

  5. Target HemolysisClostridium perfringens is readily identified in the laboratory by its characteristic “double zone” hemolysis also known has target hemolysis.