DEHYDRATED CULTURE MEDIA 223 



BACTO 



G. S. VITAMIN Bio AGAR (B399) 



DEHYDRATED 



Bacto- Vitamin Free Calcium Pantothenate 0.002 g 



Casamino Acids 10.0 g. Niacin 0.002 g 



Bacto-Soytone, Vitamin Free . . 5.0 g. Pyridoxine 0.004 g 



Bacto-Dextrose 20.0 g. Pyridoxal 0.004 g 



Sodium Acetate 12.0 g. Biotin 0.00000001 g 



Ribose Nucleic Acid 1.0 g. <i/-Tryptophane 0.2 g, 



Sodium Thioglycollate, Difco . . 1.7 g. Potassium Sulfate 20.0 g 



/-Cystine, Difco 0.2 g. Monopotassium Phosphate .... 1.0 g, 



Adenine Sulfate 0.0176 g. Dipotassium Phosphate 1-0 g 



Guanine Hydrochloride .... 0.0124 g. Magnesium Sulfate 0.4 g, 



Uracil 0.01 g. Sodium Chloride 0.02 g 



Xanthine 0.01 g. Ferrous Sulfate 0.02 g 



Folic Acid 0.001 g. Manganese Sulfate 0.02 g 



Riboflavin 0.002 g. Sorbitan Monooleate Complex . 1.0 g 



Thiamine Hydrochloride .... 0.002 g. Bacto- Agar 15.0 g 



Bacto-C. S. Vitamin B22 Agar is prepared according to the formula given by 

 Cohen and Bennett.^ This medium is suggested for the assay of Vitamin Bjg with 

 Lactobacillus leichmannii ^1^1 ATCG as the test organism. The addition of a 

 solution of crystalline Vitamin 6^2 in specified increasing amounts to cylinders 

 or disks placed on this medium produces proportionately larger zones of growth 

 by L, leichmannii ^191. 



The following procedure is recommended for the use of Bacto-G. S. Vitamin 

 BiaAgar: 



Stock cultures of the test organism, L. leichmannii 4797, are prepared by stab 

 inoculation of Bacto-Micro Assay Culture Agar. Following incubation at 

 35-3 7 °C. for 24-48 hours, the tubes are kept in the refrigerator. Transplants are 

 made at two-week intervals. Inoculum for assay is prepared by subculturing from 

 a stock culture of L. leichmannii 4797 into a tube containing 10 ml. of Bacto- 

 Micro Inoculum Broth. Following incubation for 16-24 hours at 35-3 7 °C., the 

 cells are centrifuged, under aseptic conditions, and the supernatant liquid de- 

 canted. The cells are resuspended in 10 ml. sterile isotonic sodium chloride, 

 centrifuged, the supernatant liquid discarded and made up to 10 ml. volume 

 with sterile isotonic sodium chloride. Under aseptic conditions 10 ml. of washed 

 cells are added per 1000 ml. of sterile melted Bacto-C. S. Vitamin B12 Agar at 

 45-50 °C. Into each of 4 sterile flat bottom petri dishes (95 mm.) are poured 

 25 ml. of the inoculated medium and allowed to solidify. When the surface is 

 dry 6 sterile penicillin assay cups or 13 mm. filter paper disks are distributed 

 evenly on the surface of the medium. 



For the preparation of the standard, prepare sterile solutions of Vitamin B-^g 

 containing 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 micrograms per ml. Use 0.2-0.3 ml. of 

 the standard B12 solutions per cup. When paper disks are used saturate the pads 

 with 0.1 ml. of the above solutions under aseptic conditions. The use of a syringe 

 and needle or a capillary pipette is suggested as a convenience in restricting the 

 solutions to the pad. One or more duplicate plates per test may be employed 

 using 4 to 6 disks per plate and modifying the concentration of Vitamin B12 

 as desired. Using Bacto-C. S. Vitamin B12 Assay Medium experience has shown 

 that the most effective assay range is between 0.1 microgram and 0.8 microgram. 



The concentrations of Vitamin Bj^2 required for the preparation of the standard 

 curve may be prepared by dissolving 1.6 g. of a 0,1 per cent trituration of crystal- 

 line Vitamin B12 with mannitol (1 gram = 1 mg. Vitamin 'Q-^^) ^^ 1000 



