B12 Assay Agar (Using E.coli Mutant culture) is prepared as per the formula described by Harrison et al (1).Assay Agar is vitamin B-12 free dehydrated medium containing all other nutrients and vitamins essential for the growth and cultivation of E.coli mutant 113-3 Davis ATCC-11105 except vitamin Inclusion of vitamin B12 in specified increasing amount gives a growth response that can be measured by the diameter of the zone of growth around the disc or cup containing vitamin B12. 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.