466 VITAMIN Bi2 



acid.^^ Methionine, however, does interfere in the assay, 20 7 of methionine 

 per miUihter of medium giving full growth. Burkholder'^^ has tested the 

 E. coli mutant 113-3 for vitamin B12 assay, using a medium containing, in 

 addition to the salts and carbohydrate recommended by Davis and Mingi- 

 oli,^' five amino acids and thioglycollate. He recommends that the assay 

 tubes be shaken during the incubation. The organism responded "in a 

 similar fashion" to vitamins B12 and Bi2a. Assays of blood serum proteins, 

 animal tissue extracts, and various preparations of bacteria and algae 

 yielded satisfactory results in comparison to duplicated assays by Euglena 

 gracilis. 



Shive has referred to a sulfanilamide inhibition assay for vitamin B12, 

 using wild type E. coli}^ Vitamin B12 and methionine are interchangeable 

 in reversing the inhibition of sulfanilamide. The details of this method 

 have not been published, although the method has been used extensively 

 by the Texas workers. 



2. Cup-Plate Methods for Assay of Vitamin B12 



Cup-plate methods in general have certain advantages over tube 

 methods. Perhaps the chief advantage is the adaptability of the cup-plate 

 technique to the routine assay of large numbers of samples. In the assay 

 of vitamin B12 by tube methods using lactobacilli, the degree of aeration, 

 the accumulation of carbon dioxide and peroxides, and the oxidation- 

 reduction potential have given assay troubles. In the cup-plate procedure 

 or any procedure using solid media these factors are readily controllable. 

 The first complete cup-plate method for vitamin B12 was described by 

 Foster ei al}^ These workers used a stabilized culture of L. lactis Dorner, 

 A.T.C.C. No. 10,697, and a chemically defined medium using crystalline 

 amino acids. The inclusion of 2 % sodium chloride in the growth medium 

 was found to eliminate the response of the organism to thymidine and 

 desoxynucleic acid. One-half per cent ascorbic acid also failed to cause 

 interference in the test. The authors state that impure materials containing 

 various LLD-active substances do not necessarily give equal values by the 

 cup-plate and tube methods, using the same organism. 



Cuthbertson and Lloyd^^ have studied a variety of factors influencing 

 the response of L. lactis to vitamin B12 in the cup-plate procedure. The 

 thickness of the solid medium, i.e., the oxygen concentration, the concen- 

 tration of interfering desoxyribosides which must be less than 5 7 per milli- 



^9 B. D. Davis, Proc. Natl. Acad. Sci. U. S. 35, 1 (1949). 



^^ W. Shive, Presented at N. Y. Academy of Sciences, Symposium on Antimetab- 

 olites, Feb. 11, 1949. 

 " J. C. Foster, J. A. Lally, and H. B. Woodruff, Science 110, 507 (1949). 

 62 W. F. J. Cuthbertson and J. T. Lloyd, /. Gen. Microbiol. 5, 416 (1951). 



