Blood Agar Plates (BAP) & Bile Esculin Agar

This is a differential medium. It is a rich, complex medium that contains 5% sheep red blood cells. BAP tests the ability of an organism to produce hemolysins, enzymes that damage/lyse red blood cells (erythrocytes). The degree of hemolysis by these hemolysins is helpful in differentiating members of the genera Staphylococcus, Streptococcus and Enterococcus.

• Beta-hemolysis is complete hemolysis. It is characterized by a clear (transparent) zone surrounding the colonies. Staphylococcus aureus, Streptococcus pyogenes and Streptococcus agalactiae are b-hemolytic (the picture on the left below shows the beta-hemolysis of S. pyogenes).
• Partial hemolysis is termed alpha-hemolysis. Colonies typically are surrounded by a green, opaque zone. Streptococcus pneumoniae and Streptococcus mitis are a-hemolytic (the picture on the right below shows the a-hemolysis of S. mitis).
• If no hemolysis occurs, this is termed gamma-hemolysis. There are no notable zones around the colonies. Staphylococcus epidermidis is gamma-hemolytic.

What type of hemolysis is seen on each one of the following plates?

Bile Esculin Agar

This is a medium that is both selective and differential. It tests the ability of organisms to hydrolyze esculin in the presence of bile. It is commonly used to identify members of the genus Enterococcus (E faecalis and E. faecium).

The first selective ingredient in this agar is bile, which inhibits the growth of Gram-positives other than enterococci and some streptococci species. The second selective ingredient is sodium azide. This chemical inhibits the growth of Gram-negatives.

The differential ingredient is esculin. If an organism can hydrolyze esculin in the presence of bile, the product esculetin is formed. Esculetin reacts with ferric citrate (in the medium), forming a phenolic iron complex which turns the entire slant dark brown to black. The tube on the far right was inoculated with E. faecalis (positive). The tube in the center was inoculated with a bilie esculin negative organism and the tube on the left was uninoculated.