Blood Agar is a general purpose enriched medium used to grow fastidious organisms and to differentiate bacteria based on their hemolytic properties. It is a highly nutritious medium generally used as a basal medium for preparing blood agar by supplementation with blood. It is typically Tryptic Soy Agar or Columbia Agar base with 5% Sheep blood.
The composition of Blood Agar
Composition per liter:
Agar = 15.0 gm
Beef extract = 10.0 gm
Peptone = 10.0 gm
Sodium Chloride (NaCl) = 5.0 gm
Sheep blood, defibrinated = 50.0 mL
pH 7.3 ± 0.2 at 25°C
The principle of Blood Agar
Beef extract and Peptone provides carbon, nitrogen, amino acids and vitamins. Sodium chloridehelps in maintaining the osmotic equilibrium of the medium. Agar is a solidifying agent. Addition of blood makes the medium more nutritious by providing additional growth factors required by fastidious organisms. It also helps in visualizing the haemolytic reactions. However, haemolytic reactions depend on the animal blood used.
Sheep blood gives best results for Group A Streptococci. But sheep blood fails to support growth of Haemophilus haemolyticus since sheep blood is deficient in pyridine nucleotides. However when horse blood is used H. haemolyticus colonies produce haemolysis and mimic Streptococcus pyogenes.
Preparation of Blood Agar
- Add above components (40 gm), except sheep blood, to distilled/deionized water and bring volume to 950.0 mL.
- Mix thoroughly.
- Heat with frequent agitation and boil for 1 min to completely dissolve.
- Autoclave for 15 min at 15 psi pressure at 121°C.
- Cool to 45°- 50°C.
- Aseptically add 50.0 mL of sterile, defibrinated sheep blood.
- Mix thoroughly and pour into sterile Petri dishes.
Uses of Blood Agar
- It is used for the isolation and cultivation of Streptococci, Neisseria and other fastidious microorganisms.
- It is used to differentiate bacteria on the basis of their hemolytic properties (β-hemolysis, α-hemolysis and γ-hemolysis (or non-hemolytic)).
- To determine salinity range of marine Flavobacteria.
- It can also be used for the preparation of Salmonella Typhi antigens.
- Blood Agar Base is recommended by APHA and Standard Methods for testing of food samples.