Blood agar is an enriched medium used for isolation and cultivation of fastidious bacteria. Fastidious organisms, such as streptococci, do not grow well on ordinary growth media. Blood agar is also used to differentiate bacteria on the basis of their hemolytic properties, especially within the genera Streptococcus, Enterococcus, and Aerococcus.

Principle Several species of Gram-positive cocci produce exotoxins called hemolysins that radially diffuses outwards from the colony causing complete or partial lysis of the red cells (RBC) in the medium and complete lysis of hemoglobin within the cells to colorless products. Blood agar, which is a mixture of tryptic soy agar and sheep blood, allows differentiation of bacteria based on their ability to hemolyze RBCs.

Four types of hemolysis are produced in Sheep blood agar by Streptococci namely;

1. α-hemolysis,
2. β-hemolysis
3. γ-hemolysis.
4. alpha prime or wide zone alpha hemolysis.

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

1. Alpha hemolysis: Partial destruction of the RBC resulting in a greenish-gray or brownish discoloration of agar medium around the colonies. In α hemolysis RBC hemoglobin is reduced 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 diseases.

2. Beta Hemolysis: Complete lysis of Red Blood Cells and hemoglobin, and results in clearance 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 with 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 Hemolysis: Clostridium perfringens is readily identified in the laboratory by its characteristic “double zone” hemolysis also known has target hemolysis.

Hemolysins produced by streptococci are called streptolysins. Streptolysins come in two forms-type 0 and type S. Streptolysin 0 is oxygen labile and expresses maximal activity under anaerobic conditions. Streptolysin S is oxygen stable, but it can expresses optimally under anaerobic conditions as well. The easiest way to provide an environment favorable for streptolysins on blood agar is to perform what is called the streak-stab technique. In this procedure, the blood agar plate is streaked for isolation and then stabbed with a loop. The stabs encourage streptolysin activity due to the reduced oxygen concentration of the subsurface environment

Composition of Blood Agar:
• 0.5% Peptone
• 0.3% beef extract/yeast extract
• 1.5% agar
• 0.5% NaCl
• Distilled water
• 5% Sheep Blood
• pH should be from 7.2 to 7.6 (7.4)

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 the blood agar 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. The blood should be warm to room temperature at the time of dispensing to molten agar base.
Dispense 15 ml amounts to sterile Petri plates aseptically
4. Label the medium with the date of preparation and give it a batch number and Store the plates at 2-8°C.

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

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.