VII. ESTIMATION 459 



digests of casein. The composition of these preparations may vary from 

 l)at('h to hatch. This method in its present form must he apphed to a variety 

 of natural materials ];efore it can he completely evaluated. 



b. Lactobacillus leichmamiii 313 (7830) 



Development. The most thoroughly tested method for vitamin B12 is the 

 method adopted by the Pharmacopeia of the United States,^^ utilizing the 

 organLsm L. Icichmannii 313 (7830). This organism was observed to respond 

 to thjonidine*^ • ^* whereas thymine was inactive. The details of this assay 

 are given in the section entitled Standardization of Activity (p. 472). 



HoffmaiHi et al.^^ demonstrated that for L. leichmannii 313 thymidine 

 and crystalline vitamin B12 were interchangeable and that this organism 

 may be useful as a sensitive test organism for vitamin B12. Thymidine and 

 \'itamin B12 were also shown to be interchangeable for the growth of L. 

 lactis Dorner (8000). 2^- ^^ A report by Kitay et al.^^ indicated that L. leich- 

 mannii 313 responded to the desoxyribosides of adenine, hypoxanthine, 

 and cytosine as well as to intact desoxy ribonucleic acid and thymidine. 



Hofi'mann et al}" first published a detailed method usmg L. leichmannii 

 313 (7830) for the assay of vitamin B12. The medium used, in addition to 

 the usual \'itamins, minerals, carbohydrate, and buffers present in media 

 for lactic acid bacteria, contained thioglycollic acid, Tween 80, and en- 

 zymatic digest of casein. The addition of thioglycollic acid to the medium 

 promoted much hea\'ier growth at submaximal levels of liver extract or 

 vitamin B12. Cysteine and ascorbic acid had similar effects but were some- 

 what inferior to thioglycollic acid. Thus these three reducing agents caused 

 a several-fold increase in growth response to both vitamin B12 and liver 

 extract. This was interpreted as indicating that reducing agents protected 

 vitamin B12, pure or contained in liver extract, from destruction during 

 autocla\-ing. Newer information published by Broquist et al?^ suggests that 

 the protective effect may be of importance only for vitamin Bi2b and that 

 the effect on vitamm B12 is rather an activation or change of vitamin B12 

 to a fonn more potent for the organism. The effect of reducing agents on 

 microbial growth promotion of vitamin B12 and vitamin Bi2b will be dis- 

 cussed in Section X. 



" E. E. Snell, E. Kitay, and W. S. McNutt, J. Biol. Chetn. 175, 473 (1948). 

 3< L. D. Wright, H. R. Skeggs, and J. W. Huff, J. Biol. Chem. 175, 475 (1948). 

 " C. E. Hoffmann, E. L. R. Stokstad, A. L. Franklin, and T. H. Jukes, J. Biol. 



Chem. 176, 1465 (1948). 

 3« E. Kitay, W. S. McNutt, and E. E. Snell, /. Biol. Chem. 177, 993 (1949). 

 " C. E. Hoffmann, E. L. R. Stokstad, B. L. Hutchings, A. C. Dornbush, and T. H. 



Jukes, J. Biol. Chem. 181, 635 (1951). 

 38 H. P. Broquist, E. L. R. Stokstad, and T. H. Jukes, Proc. Soc. Exptl. Biol. Med. 



76, 806 (1951). 



