462 VITAMIN Bi2 



a test organism for vitamin B12. The medium was not completely chemically 

 defined, as it contained an enzymatic digest as well as an acid hydrolyzate 

 of casein. Growth was measured turbidimetrically after 24 hours. Reducing 

 agents were not added to the medium. Ascorbic acid and air permitted 

 growth of the organism in the absence of vitamin B12, but the effects could 

 be minimized by using an autoclaving period of 15 minutes in the assay 

 procedure. Thymidine was interchangeable with vitamin B12. Further in- 

 vestigations on the use of L. leichmannii (4797) for vitamin B12 assay were 

 reported by Peeler et al}^ These workers substituted crystalline amino 



TABLE XI 

 Composition of Double-Strength Medium 



The preparation of the acid hydrolyzed casein requires special care, since repeated carbon treatment of 

 the hydrolyzates results in inadequate growth of the organism. One hundred grams of Labco \ntamin-free 

 casein are refiuxed for 8 to 10 hours with 1000 ml. of 6 A'^ HCl. The HCl is removed under reduced pressure, 

 the volume is restored with water, and the evaporation under vacuum is repeated. The hydrolyzate is diluted 

 to 800 ml., adjusted to pH 3.0, and treated after filtration with 10 g. of Darco G60 for 30 minutes. 



acids for the casein hydrolyzates used by Skeggs et alP On the amino acid 

 basal medium the maximum growth obtainable with excess vitamin B12 

 corresponded to a galvanometer reading of about 50% light transmission. 

 After the addition of charcoal-treated tomato juice to furnish unidentified 

 factors, a growth corresponding to 13 % light transmission was obtained 

 with adequate vitamin B12. Thioglycollic and ascorbic acids did not duplicate 

 the effect of the charcoal-treated tomato juice. Considerably more growth 

 was obtained at all levels of vitamin B12 by increasing the amount of 

 FeS04 -71120 to 0.4 mg. per milliliter and the cysteine to 1 mg. per milliliter, 

 amounts quite in excess of the usual. The best initial pH of the basal me- 

 dium for best response to vitamin B12 was reported to be pH 5.5. Although 



^0 H. T. Peeler, H. Yacowitz, and L. C. Norris, Proc. Soc. Exptl. Biol. Med. 72, 515 

 (1949). 



