Response to and Assay of Vitamin B12 by a Mutant of Escherichia coli.

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Abstract
E. coli (strain 113-3), which responds to vit. B12 or methionine, was used for a turbidimetric assay of B12. Under the conditions employed, half-max. response occurs with 18 2ug. B12 and 2.5 [mu]g. DL methionine per ml. A number of factors influences the B12-induced stimulation of this culture. The amt. of growth varies directly with the degree of aeration. No growth occurs anaerobically unless excess quantities of both B12 and methionine are added to the medium. KCN, Na thioglycolate and ascorbic acid, when autoclaved (with B12) in the basal medium in concns. greater than 1, 10 and 5 ug./ml., respectively, antagonize the growth-promoting action of B12- Aseptically, cyanide and ascorbate are inhibitory at a level of 0.5 and 5 [mu]g./ml., respectively, and thioglycolate is not antagonistic at 500 [mu]g./ml. Autoclaving at 120[degree]C for 5 min. does not inactivate B12, but destroys 30% of the activity of B12a. Cyanide and thioglycolate prevent B12a destruction by heat. Aseptically, B12a is 70% as active as B12. The B12 content of a dried streptomyces fermen-tation concentrate assays the lowest when autoclaved in the unsupplemented basal medium and the highest when tested aseptically or when autoclaved with KCN (1[mu]g./ml.).