224 DIFCO MANUAL 



ml. distilled water. This is the stock solution A containing 1.6 micrograms per 

 ml. To obtain solution B containing 0.8 micrograms dilute 10 ml. of A with 10 

 ml. of distilled water. To obtain solution C containing 0.4 micrograms dilute 

 10 ml. of B with 10 ml. distilled water. To obtain solution D containing 0.2 

 micrograms dilute 10 ml. of C with 10 ml. distilled ^vater. To obtain solution E 

 containing 0.1 micrograms dilute 10 ml. of D with 10 ml. distilled water. To 

 obtain solution F containing 0.05 micrograms dilute 10 ml. of E with 10 ml. 

 distilled water. These solutions are stable for 2 months when stored at 2-6° C. 

 under toluene. 



In determining the Vitamin B;l2 content of unknown materials, the assay 

 samples are appropriately diluted and similarly applied. In the assay of some 

 preparations, better defined, clearer cut zones may be obtained by the addition of 

 0.5 to 1.0 per cent of sodium chloride to Bacto-C. S. Vitamin B^o Agar, since 

 added sodium chloride, with some unknown samples, makes the assay more 

 specific for vitamin B^o- Sodium chloride, under these conditions, must be added 

 to the medium in preparing the standard curve. Incubate inoculated plates at 

 35-3 7 °C. for 24 hours and measure size of zone of growth produced. The diame- 

 ter of the growth zone is indicative of the amount of Vitamin B^o present. 



To rehydrate the medium, suspend 90 grams Bacto-C. S. Vitamin B^g Agar in 

 1000 ml. of cold distilled water. Heat to boiling to dissolve the medium com- 

 pletely. Distribute in flasks and sterilize in the autoclave for 15 minutes at 15 

 pounds pressure (121°C,). 



One hundred grams of Bacto-C. S. Vitamin B12 Agar are sufficient for 1.1 

 liters of medium. 



1 Paper read at American Chemical Society, 

 Philadelphia, 1950. 



