A Differential Microbiological Assay

Although vitamin B12 can be assayed biologically with mice, chicks or rats, the microbiological method of assay is preferred since it is more rapid and economical. One serious shortcoming of many microbiological B12 assay procedures is the lack of specificity when crude materials are assayed. Crude materials can contain inhibitory or stimulatory factors which cause serious errors in assay results. One of the interfering substances in the microbiological assay of vitamin B12 with Lactobacillus leichmannii is pseudovitamin B12. Pfiffner et al. (1951) isolated an organism from bovine rumen contents which produced a new form of vitamin B12 which was completely inactive for chicks, rats, and humans, but was active for L. teichmannii and Lactobacillus lactis var. Dorner. These workers crystallized two major red pigments from an anaerobic fermentation broth containing this rumen organism and named the pigments pseudovitamin B12 and pseudovitamin B12b. The chemical differences between pseudovitamin B12 and vitamin B12 have been described (Dion et al., 1952; Pfiffner et al., 1952). Lewis et al. (1952) reported that a vitamin B12 analog which they designated vitamin B12f appeared to be identical with pseudovitamin B12. Using paper chromatography in conjunction with a bioautographic technique, these workers found that vitamin B12f was present in the fecal matter of the rat, cow, sheep, pig, horse, chicken, guinea pig, and man. Sheep rumen contents were also a source of vitamin B12f. No vitamin B12f was detected in rat or beef liver or in a sample of injectable liver extract. The experimental work described in this paper was conducted in an attempt to develop a simple microbiological procedure for detecting the presence of pseudovitamin B12 in crude samples. In addition, the application of the method in the differential quantitative assay of vitamin B12 and pseudovitamin B12 was also investigated. The observation by 0. D. Bird (personal communica-