464 VITAMIN Bi2 



acid Avas chosen as a reducing agent, since it also gave a growth response 

 similar to non-reducing dicarboxylic acids. By direct comparison it was 

 shown to be superior to thioglycoUic acid for this organism. The method of 

 inoculation finally adopted is worthy of special attention. The organism is 

 carried in stock culture in skim milk supplemented with 1 % Difco tryptose. 

 For assay purposes the organism is transferred daily in this milk medium. 

 The inoculum is prepared by suspending 0.1 ml. of a 24-hour milk culture 

 in 10 ml. of sterile saline from which a second dilution of 1 to 10 is made. 

 One drop of this second dilution is added to each assay tube. This has 

 given good reproduceability wdth consistently less blank trouble than the 

 U.S.P. method,*^ however, it is less sensitive (see Table XII). 



d. Euglena gracilis 



Hutner et al^^ have shown that Euglena gracilis var. hacillaris exhibited 

 a quantitative response to vitamin B12. The unique advantage of the method 



TABLE XIII 



Basal Medium for Euglena gracilis 



Per 1 1. final medium 

 (pH 6.5) 



NH4H2PO4 



Potassium citrate monohydrate 



MgS04-7H20 



Sodium butyrate 



Monosodium glutamate 



CaClz 



FeS04-7H20 



MnS04-H20 



CoS04-7H20 



ZnCh 



Na2Mo04-2H20 



CuS04-5H20 



Thiamine chloride 



is that, for E. gracilis, thymidine up to 10 7 per milliliter does not support 

 growth. This is in contrast to the lactobacilli which grow in the absence of 

 vitamin B12 on levels of desoxyribosides as low as 0.2 7 per milliliter of 

 medium. The Euglena gracilis method utilizes a relatively simple medium, 

 as shown in Table XIII. The organism is grown in small 100 X 13-mm. 

 test tubes which must be exposed continually to light. To provide the 

 required illumination the racks of tubes were placed on glass plates over 

 large fluorescent lights. The temperature ranged from 25° to 31°. Tempera- 

 tures of 32° or over caused inhibitory effects. 



