BLOOD AGAR Biology Biotechnology Department PURPOSE Blood agar

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

§ 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)