A fastidious organism is one with complete nutritional requirements, needing additional cellular building-block molecules in order to survive (1). Blood Agar Base No. 2 is a highly nutritive medium. Microorganisms producing haemolysin give visible haemolytic zones on this medium. It also serves as a differential medium for Brucella and Campylobacter species by adding different antibiotic supplements for the respective bacteria (2, 3). Brucella cultures are highly infective and must be handled with care. Incubate preferably in 5-10% carbon dioxide atmosphere. Comparative studies of horse, rabbit and sheep blood showed that sheep blood gave the clearest and most reliable colony and haemolysis characteristics at both 24 and 48 hours of incubation (4). It can be used to prepare Chocolate Agar for the isolation of Haemophilus and Neisseria species.
It can also be used for primary isolation of Haemophilus species, where horse blood is used for enrichment. Better results are obtained by spreading half of the horse blood agar plate with 2 drops of 10% saponin (5). Liver extract and yeast extract helps enhance the growth and haemolytic reactions of fastidious organisms like Streptococci and Pneumococci. Proteose peptone serves as the nitrogen source while liver digest and yeast extract provide essential carbon, vitamin, nitrogen and amino acid sources. Sodium chloride maintains the osmotic equilibrium. Supplementation with blood (5-10%) provides additional growth factors and also serves as basis for determining haemolytic reactions. Haemolytic patterns may vary with the source of animal blood or type of base medium used (6).
Directions: Suspend 21.25 grams in 500 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 40 - 50°C and aseptically add 7% v/vsterile defibrinated blood.