B12 ASSAY AGAR (using E.coli mutant Culture)

B12 Assay Agar is dehydrated medium devoid of Vitamin B12 but containing all the nutrients essential for the growth of E. coli mutant 113-3 Davis ATCC-11105. For the preparation of standard, make sterile solutions of Vitamin B12 (Cyanocobalamine Reference Standard). For the determination of Vitamin B12 content of unknown materials the assay sample should be properly diluted and applied similarly as the dilutions of the standards. Inoculum for the assay is prepared by sub-culturing from a stock culture previously made by stab inoculation. Freshly subcultured cells incubated at 35°C for 24 hours, centrifuged, washed and suspended in 10 ml saline are recommended for this assay. The medium contains glucose which acts as a source of energy. Incorporation of Vitamin B12 in specified increasing amounts gives a growth response that can be measured by the diameter of the zone of growth around the disc or cup containing Vitamin B12. Phosphates present provide ions to the medium. And other salts help in maintaining the osmotic balance. B12 Assay Agar acts as a solidifying agent.

for microbiological assay of Vitamin B12 by plate method using E.coli mutant as test organism