For selective and nutritive for Clostridium species. COLUMBIA AGAR is a highly nutritive and selective medium for the cultivation of fastidious organisms, especially when used as a base for blood chocolate agar. It can also be used as a selectiveisolation medium by adding antimicrobial agents. It is a nutritiously rich general purpose medium developed by Ellner et al5 from Columbia University. Columbia Agar conforms to harmonized USP/EU/JP requirements.1,2,3,4 Nitrogen, vitamins, and amino acids are provided by a combination of Pancreatic digest of Casein, Peptic digest of Meat and Pancreatic digest of Heart. Maize starch is included to supply a carbon source. Yeast extract provides B-complex vitamins. Sodium chloride maintains the osmotic balance of the medium. Agar acts as a solidifying agent. Supplementation with blood (5 - 10%) provides additional growth factors for fastidious microorganisms, and aids in determining hemolytic reactions. Hemolytic patterns may vary with the source of animal blood and the type of basal medium used.6 In general, blood agar bases are relatively free of reducing sugars, which have been reported to adversely influence the hemolytic reactions of hemolytic streptococci. Inoculate Columbia Agar (TMH 116) medium with a small number (not more than 100 cfu) of the appropriate microorganism and incubate under anaerobic conditions at 30 - 35°C for 18 - 48 hours. For isolation purpose, streak withinoculating loop on Columbia Agar (TMH 116) and incubate under anaerobic conditions at 30-35ºC for 48 hours. For Columbia Agar, supplemented with blood, examine the hemolytic reactions after 18 - 24 and 48 hoursincubation. There are four types of hemolysis on blood agar media described as7: 1. Alpha hemolysis (α) is the reduction of hemoglobin to methemoglobin in the medium surrounding the colony. This produces a green discoloration of the medium. 2. Beta hemolysis (β) is the lysis of red blood cells, producing a clear zone surrounding the colony. 3. Gamma hemolysis (γ) indicates no hemolysis. No destruction of red blood cells occurs and there is no change in the medium. 4. Alpha-prime-hemolysis (α’) is a small zone of complete hemolysis that is surrounded by an area of partial lysis.