456 VITAMIN Bi2 



units of liver extracts used for treatment of pernicious anemia. In 1948^^ 

 Shorb tested crystalline vitamin B12 and found that it was responsible for 

 the LLD activity of liver extracts. At this time the L. lactis Dorner assay 

 for vitamin B12 was subject to a great deal of variation. For example, a 

 60 % increase in the response to vitamin B12 as compared to liver extract 

 was apparent })y simply extending the time of incubation. This organism 

 required the presence of clarified tomato juice (TJ factor)!^"^^ which was 

 later found by Caswell el alr^ to be replaceable by a combination of fumaric 

 acid and sodium ethyl oxalacetate in an amino acid medium containing 

 DL-alanine. Shorb demonstrated (personal communication cited m ref. 21) 

 that D-alanine and tomato juice were interchangeable if oxalacetate was 

 in the medium. The factor as it exists in tomato juice is pyridoxamine 

 phosphate.-^ Assays for vitamin B12 with L. lactis Dorner are further com- 

 plicated by the requirement of the organism for carbon dioxide^^ When 

 carbon dioxide is present the requirement for vitamin B12 is eliminated 

 by anaerobic conditions obtained by the addition of reducing substances 

 to the medium.-^ Shive et al?'^ early reported that ascorbic acid added 

 aseptically may eliminate the requirement of L. lactis Dorner for vitamin 

 B12. Thymidine and other desoxyribosides stimulate growth of L. lactis 

 Dorner and other test organisms of the lactic acid bacteria group under 

 circumstances in which growth is also promoted by vitamin B12, as sho\vn 

 by Shive et al.-^ and Wright et alP Both these groups of investigators 

 report that an emulsifying agent, Tween 80, is also required for satisfactory 

 growth. According to Shorb and Briggs,^^ L. lactis Dorner readily undergoes 

 dissociation on the usual yeast extract-glucose transfer medium, resulting 

 in cultures which do not require vitamin B12 or which respond erratically. 

 However, the vitamin B12 requirement is relatively stable if the transfer 

 medium consists of skim milk, tomato juice, and yeast extract. Because of 

 the many difficulties attending the use of L. lactis, this organism is not 

 generally used in the conventional tube-assay technique. For routine assays 

 L. lactis Dorner is used rather widely in cup-plate procedures'-^ utilizing 

 solid media. 



13 M. S. Shorb, Science 107, 397 (1948). 



20 M. C. Caswell, L. K. Koditschek, and D. Hendlin, /. Biol. Chem. 180, 125 (1949). 

 2> D. Hendlin, M. C. Caswell, V. J. Peters, and T. R. Wood, J. Biol. Chem. 186, 647 

 (1950). 



22 L. K. Koditschek, D. Hendlin, and H. B. Woodruff, /. Biol. Chem. 179, 1093 (1949). 



23 R. D. Greene, A. J. Brook, and R. B. McCormack, J. Biol. Chem. 178, 199 (1949). 

 2^ W. Shive, J. M. Ravel, and R. E. Eakin, J. Am. Chem. Soc. 70, 2614 (1948). 



" L. D. Wright, H. R. Skeggs, and J. W. HufT, J. Biol. Chem. 175, 475 (1948). 



26 M. S. Shorb and G. M. Briggs, J. Biol. Chem. 176, 1463 (1948). 



"^ A. L. Bacharach and W. F. J. Cuthbertson, Analyst 73, 334 (1948). 



