474 VITAMIN Bi2 



The solution is adjusted to pH 6.8. The soUd stab medium for stock cultures 

 is obtained by simply adding 1.5 % agar to the culture medium. 



From the fresh culture of L. leichmannii 313 (7830) obtained, for example, 

 from the American Type Culture Collection, to inoculum for the assay 

 tubes, the following procedure is observed: 



1 . At least ten successive transfers over a 2-week period are made in the 

 stab medium incubating 16 to 24 hours at a constant temperature between 

 30° and 37°. 



2. During use of the organism for routine assays it is transferred at least 



TABLE XVI 

 Composition of the Basal Medium for the U.S. P. Vitamin B12 Method 



° Tomato juice preparation — 3 1. of canned tomato juice is centrifuged and then filtered by suspending 10 

 to 20 g. of filter-aid in the solution and also using a filter-aid layerona Biichner funnel. A clear straw-colored 

 solution must be obtained. The solution is treated at pH 3.5 with 50 g. of activated charcoal and filtered, using 

 filter-aid as previously described. 



three times a week, at no time using a stab culture which is more than 4 

 days old. 



3. Transfer from the stab culture 10 ml. of the described liquid culture 

 medium and incubate 16 to 24 hours at a constant temperature between 

 30° and 37°. 



Daily transfer of the stab culture has been found to be advisable. To 

 prepare inoculum the cells are centrifuged from 10 ml. of culture medium 

 and are washed twice with 10-ml. portions of previously sterilized single- 

 strength basal medium, finally suspending in 10 ml. of the basal medium. 

 The published procedure- fails to mention that the suspension should be 

 diluted 1 to 10 in the basal medium at this point. Dilution of 1 to 20 fre- 

 quently has been advisable. One drop of this suspension is added per assay 

 tube. 



The double-strength basal medium contains the ingredients shown in 



