Biology & Biotechnology Department Blood Agar Biology & Biotechnology Department

PURPOSE : Blood agar is used for : isolation and cultivation of many types of fastidious bacteria. It is also used to differentiate bacteria based on their hemolytic characteristics, especially within the genera Streptococcus, Enterococcus, and Aerococcus.

PRINCIPLE : Several species of Gram-positive cocci produce exotoxins called hemolysins able to destroy red blood cells (RBCs) and hemoglobin. Blood agar, which is a mixture of nutrient agar and sheep blood, allows differentiation of bacteria based on their ability to hemolyze RBCs. The three major types of hemolysis are ß hemolysis, a hemolysis, and 'Y hemolysis.

ß hemolysis is the complete destruction of RBCs and hemoglobin, and results in a clearing of the medium around the colonies a hemolysis is the partial destruction of RBCs and produces a greenish discoloration of the agar around the colonies . 'Y hemolysis is actually non-hemolysis and appears as simple growth with no change to the medium.

Hemolysis : is the breakdown of red blood cells. The ability of bacterial colonies to induce hemolysis when grown on blood agar Is used to classify certain microorganisms. This is particularly useful in classifying streptococcal species. A substance that causes hemolysis is a hemolysin

NOTE: Preparation of Blood Agar: Nutrient agar which has been melted and cooled to( 40 to 45°C) add 5% (vol/vol) sterile defibrinated blood that has been warmed to room temperature. Swirl the flask to mix thoroughly, avoiding the formation of bubbles, and dispense into sterile plates, continuing to avoid bubbles and froth on the surface. NOTE: Cooling the agar and warming the blood are essential steps in this procedure. Hot agar can damage red blood cells, and cold blood can cause the agar to gel before pouring.

Bacteria can produce different types of hemolysins :   Beta-hemolysin breaks down the red blood cells and hemoglobin completely. This leaves a clear zone around the bacterial growth. Such results are referred to as β-hemolysis (beta hemolysis). Alpha-hemolysin partially breaks down the red blood cells and leaves a greenish color behind.  This is referred to as α-hemolysis (alpha hemolysis).  The greenish color is caused by the presence of biliverdin, which is a by-product of the breakdown of hemoglobin. 

If the organism does not produce hemolysins and does not break down the blood cells, no clearing will occur.  This is called γ-hemolysis (gamma hemolysis).

Hemolyses of Streptococcus spp. (left) α-hemolysis (S. mitis); (middle) β-hemolysis (S. pyogenes); (right) γ-hemolysis (non-hemolytic, S. salivarius)