VIII. STANDARDIZATION OF ACTIVITY 475 



Table XVI. This medium is added in 5-ml. portions to 20 X ir)0-mm. 

 Pyrex test tubes and made to 10-ml. volume with test sokitions and water. 

 The assay tubes are autocla\'ed for 5 minutes at 121° to 123°. The first 

 tube in the standard curve contains 0.01 m7 of vitamin B12, the successive 

 tubes containing increasing amounts of vitamin B]2 by increments of 0.01 

 m7 and the last tuiie containing 0.1 my. 



The organism requires 0.01 my of vitamin B12 per milliliter for maximum 

 growth, whereas Euglena gracilis reaches approximately half-maximum 

 growth at this concentration.^ In experiments reported by Robbins ei al.'*- ^ 

 Euglena gracilis required O.l my per milliliter for maximum growth. Thus 

 this present method using L. leichmannii 313 is at least as sensitive as the 

 Euglena gracilis method. It is about ten times as sensitive as methods 

 utilizing other lactobacilli (Table XII). 



The sensitivity of the L. leichmannii 313 assay is a disadvantage under 

 some conditions. In our laboratories, growth in tubes to which no vitamin 

 B12 has been added is a frequent occurrence. The same blank troubles have 

 lieen frequently encountered in other laboratories according to verbal re- 

 ports. It is possi}:)le that reduction in the volume of medium used in the 

 assay tube would help eliminate some of this erratic growth. Caswell et al.^ 

 found that, by reducing the volume of medium from 10 ml. to 5 ml., maxi- 

 mal growth in tubes containing no added vitamin B12 was completely 

 eliminated and greater reproducibility of assay results with L. lactis Dor- 

 ner (8000) was obtained. The U.S. P. method^ recommends titrating the 

 acid produced at 72 hours as a means of measuring growth. Undoubtedly 

 turbidity measurements would prove satisfactory in a considerably shorter 

 period of incubation. The American Association of Official Agricultural 

 Chemists^ has conducted a study of the U.S. P. method^ using L. leichmannii 

 313 except that turbidity measurements of growth are used. Means should 

 be investigated for the elimination of the clarified tomato juice, since the 

 composition of this preparation has been found to vary with different 

 batches. Tomato juice was eliminated from L. lactis Dorner (8000) media 

 by a combination of fumaric acid, sodium ethyl oxalacetate, and DL-ala- 

 nine.^ With the improvements of a shorter assay period by using turbidi- 

 metric measurement of growth and elimination of the tomato juice prepa- 



3 S. H. Hutner, W. Provasoli, C. E. Hoffmann, M. Belt, A. L. Franklin, and T. H. 



Jukes, Proc. Soc. Expil. Biol. Med. 70, 118 (1949). 

 * W. J. Robbins, A. Hervey, and M. E. Stebbins, Bnll. Torrey Botnn. Club 77, 423 



(1950). 

 ' W. J. Robbins, A. Hervej-, and M. E. Stebbins, Bull. Torrri/ Botan. Club 78, 363 



(1951). 

 8 M. C. Caswell, L. K. Koditscliek, and D. Hendlin, J. Biol. Chem. 180, 125 (1949). 

 ^ C. H. Krieger, Personal communication. 



