458 VITAMIN Bi2 



absence of reducing agents. Vitamin Bi2b showed about 30 % of the activity 

 of vitamin B]2 in the L. ladis Dorner assay, but the two forms showed 

 equal activity upon the addition of KCN. 



Since the L. lactis Dorner tube method used by Cooperman et al.^° ap- 

 pears to represent a practical and useful method utilizing this organism, 

 it wall be described in some detail. The basal medium used was essentially 

 that of Caswell et al}^ except that several of the pure amino acids were 

 omitted and the amount of acid -hydroly zed casein doubled. Tween 80 and 

 ammonium acetate were also included. 



The detailed composition of the basal medium is given in Table X. 



Culture and Inoculum. The stock culture of L. lactis Dorner (8000) was 

 transferred weekly on the liver-tryptone agar of Nymon and Gortner.*^ 



TABLE X 

 Composition of Double-Strength Basal Medium 



Inoculum was prepared by transferring from agar stab to a broth con- 

 sisting of the Difco riboflavin assay medium supplemented with clarified 

 skim milk (casein removed at pH 4.2). Cell growth obtained from 10 ml. 

 of this broth was washed twice with sterile saline (centrifuge) and resus- 

 pended to give a reading of 70 on the Evelyn colorimeter using the No. 

 660 filter. One drop of this suspension was added per assay tube. 



The assay was carried out using 5 ml. total volume, as this was found 

 by Caswell et alP to eliminate the frequent occurrence of maximum growth 

 in tubes without vitamin B12 when the 10 ml. total volume is used. The 

 assay tubes were autoclaved 8 minutes, incubated 40 hours at 37", and 

 titrated with 0.05 N NaOH. 



The major advantage of this L. lactis Dorner tube method is the elimina- 

 tion of the necessity for preparing clarified tomato juice and enzymatic 



=2 M. C. Nymon and W. A. Gortner, J. Biol. Chem. 163, 277 (1946). 



