VII, ESTIMATION 465 



The standard response curxe starts at 0.0015 7 of A'itamin B12 per milli- 

 liter and goes up to 0.5 7 per milliliter. 



Tlie Englena gracilis method has been studied further by Robbins ei al}^ 

 Casein hydrolyzate or oL-alanine and a supplement of the B vitamins were 

 added to the basal medium of Hutner et al}^ Better growth response at the 

 higher kn'els of \'itamin Bio was apparent when the assays were heated at 

 100° instead of autoclaving. These authors, as well as using the conven- 

 tional tube method, de\'eloped a method utilizing a medium solidified with 

 agar. 



Filter paper disks dipped in vitamin B12 test solutions were placed on the 

 solid medium seeded with Euglena gracilis, and the areas of growth around 

 the disk were measvu'ed. This disk method was rather insensitive, requiring 

 extracts containing at least 2 m7 of vitamin B12 per milliliter. An incubation 

 period of 7 days at 25° to 28° was used by Robbins et al.*^ for both disk 

 and tube methods. 



In a recent paper, Robbins et a/."*" report the application of the Euglena 

 gracilis method to a \'ariety of natural products. Satisfactory assays were 

 obtained on natural materials, in contrast to the experience of Picken and 

 Bauriedel,'''^ who found the E. gracilis tube method applicable only to 

 relatively pure materials. Robbins et al}^ found oysters and clams excellent 

 sources of vitamin B12. Pure cultures of blue-green algae produce vitamin 

 B12 and were suggested to be the primary source of vitamin B12 for aquatic 

 animals. Clams contained vitamin B12 in a bound form from which the 

 vitamin can be released by treatment with 0.1 A' HCl. 



The Euglena gracilis method requires an incubation time of at least 4 

 days,^° and sometimes as long as 7 days."*® This is a major disadvantage 

 when a large number of assays are required. On the other hand, from the 

 report of Hutner and the chromatographic studies of Picken and Bauriedel," 

 it appears that the Euglena gracilis method is the most specific method for 

 vitamin B12. 



e. Escherichia coli 



Davis and ^^lingioli'*^ isolated Ij}' means of the penicillin method'^ several 

 ultraviolet-induced mutants of E. coli which require vitamin B12. One 

 mutant in particular, Xo. 113-3, showed a rapid and reproducible response 

 and was suggested as a possible assay organism. The vitamin Bi2-requiring 

 mutants of E. coli require about 0.5 7 of vitamin B12 per milliliter of medium 

 and hence are slightly less sensitive than L. leichmannii (4797) and other 

 lactobacilli (Table XII). E. coli 113-3 differs from the lactobacilli in tliat it 

 does not respond to thymidine or alkaline hydrolyzates of deso.xynucleic 



" J. C. Picken and W. R. Bauriedel, Pruc. Soc. Exptl. Biol. Med. 76, 511 (1950). 

 " W. J. Robbins, A. Hervey, and M. E. Stebbins, Bull. Torrey Botan. Club 78, 363 

 (1951). 



