Columbia Agar Base is used as the base for media containing blood and for selective media formulations, which incorporates various combinations of antimicrobial agents as additives. Sheep blood allows detection of hemolytic reactions and supplies the X-factor (hemin) necessary for the growth of many bacterial species but lacks V-factor (Nicotinamide Adenine Dinucleotide), since it contains NADase, which destroys the NAD. Therefore, Haemophilus influenzae , which requires both the X and V-factors, will not grow on this medium. Fildes found that Nutrient Agar supplemented with a digest of sheep blood supplied both of these factors and the medium would support the growth of H. influenzae (2, 3). The inclusion of bacitracin makes the enriched Columbia Agar Medium selective for the isolation of Haemophilus species from clinical specimens, especially from upper respiratory tract (4). Columbia Blood Agar Base w/ 1 % Agar is used as a base for preparing media containing blood and for selective media formulations in which different combinations of antimicrobial agents are used as additives.
Columbia Agar Base supplemented with sheep, rabbit or horse blood derives its superior growth-supporting properties from the combination of peptones prepared from pancreatic digest of casein, peptic digest of animal tissue and beef extract. Cornstarch serves as an energy source and also neutralizes toxic metabolites. Sheep blood permits the detection of haemolysis and also provides heme (X-factor), which is required for the growth of many bacteria. As these media have a relatively high carbohydrate content, beta-haemolytic Streptococci may exhibit a greenish haemolytic reaction, which may be mistaken for alpha haemolysis. Confirmatory tests of all the presumptive colonies are needed.
Columbia Agar Base with added sterile serum provides an efficient medium for Corynebacterium diphtheriae virulence test medium. After following the established technique for C. diphtheriae , lines of toxin-antitoxin precipitation are clearly visible in 48 hours. Many pathogens require carbon dioxide; therefore, plates may be incubated in an atmosphere containing approximately 3-10% CO2.
Precaution: Brucella cultures are highly infective and must be handled carefully; incubate in 5-10% CO2. Campylobacter species are best grown at 42°C in a microaerophillic atmosphere. Plates with Gardenerella supplements plates should be incubated at 35°C for 48 hours containing 7% CO2 (5).
Corn starch serves as an energy source and also neutralizes toxic metabolites. Sheep blood permits the detection of haemolysis and also provides heme (X factor) which is required for the growth of many bacteria. However it is devoid of V factor (Nicotinamide adenine dinucleotide) and hence Haemophilus influenzae which needs both, X and V factors will not grow on this medium. Hemin stimulates growth of various fastidious organisms. As this medium has a relatively high carbohydrate content, beta-haemolytic Streptococci may exhibit a greenish haemolytic reaction which may be mistaken for the alpha haemolysis. Carry out confirmatory tests of all the colonies.
Directions: Suspend 44.01 grams in 1000 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 45-50°C before adding heat sensitive compounds.