A Microbiological Assay for Vitamin B12 Using Lactobacillus leichmannii.

Abstract

A microbiological assay using L. leichmannii which is highly sensitive to vit. B12 and which yields consistent results was developed. The assay time is 16 hrs. Crystalline amino acids are used as the N source and the unknown factors found to be required by this organism for rapid and maximum growth are supplied by a norit-ad-sorbed tomato juice filtrate which is free of vit. B12 and thymi-dine. Instead of adjusting the medium to the usual pH 6.6-6.8, it has been found that taking it to pH 5.5 establishes more optimum conditions for L. leichmannii. More growth is obtained with vit. B12 when the medium is in a reduced state. This growth increase becomes marked if the vit. B12 is supplied in the form of a natural supplement. A level of 800 mg. of FeSO4.7H2O and 2 g. of cysteine HC1 has been found to keep a liter of double-strength basal medium in the necessary reduced condition. Thymidine will promote growth on this medium. However, 24,000 times more of this substance than vit. B12 is required to produce the same growth response. Due to this wide ratio it is highly improbable that interference from thymidine will be encountered in the assay of most materials for vit. B12 activity. The ability of the method to produce repeatable results was determined by running several assays on successive days on the same sample of unknown against crystalline vit. B12. Excellent replicability was found and values obtained from a crude liver fraction were found to be just as repeatable as those from a vit. B12 concentrate. Digestion of animal products with pancreatin at pH 6.8 for 20 hrs. at 37[degree]C was found most suitable for the release of vit. Bl2 activity. The best values were obtained from vegetable products by incubating them with a mixture of takadiastase and papain at pH 4.5 for 40 hrs. at 37[degree]C.