DEHYDRATED CULTURE MEDIA 225 



BACTO 



FOLIC ACID ASSAY MEDIUM (B318) 



DEHYDRATED 



Bacto- Vitamin Free Riboflavin 0.002 g 



Casamino Acids 12 g 



Bacto-Dextrose 40 g 



Sodium Citrate 20 g 



/-Cystine, Difco 0.2 g 



^/-Tryptophane 0.2 g 



Adenine Sulfate 0.02 g 



Guanine Hydrochloride 0.02 g 



Uracil . . 0.02 g 



Thiamine Hydrochloride .... 0.002 g. 

 Pyridoxine Hydrochloride . . . 0.004 g. 



Niacin 0.002 g 



/>-Aminobenzoic Acid, Difco 0.0002 g 



Biotin 0.0000008 g 



Calcium Pantothenate 0.0004 g 



Dipotassium Phosphate 1 g 



Monopotassium Phosphate 1 g 



Magnesium Sulfate 0.4 g 



Sodium Chloride 0.02 g 



Ferrous Sulfate 0.02 g 



Manganese Sulfate 0.02 g 



Bacto-Folic Acid Assay Medium is a complete dehydrated medium for the 

 assay of folic acid. It is free from folic acid but contains all the other factors 

 necessary for the growth of Streptococcus lactis R. 8043 ATCG [Streptococcus 

 jecalis R.) . The addition of folic acid in specified increasing concentrations gives 

 a growth response by S. lactis R. 8043 after 18 hours incubation at 35-37°C. 

 which may be measured turbidimetrically. Bacto-Folic Acid Assay Medium is 

 prepared according to the formula described by Capps, Hobbs and Fox,^ modi- 

 fied by the use of sodium citrate instead of sodium acetate. 



The following procedure for the preparation of the inoculum and performance 

 of the test is recommended for the assay of folic acid using Bacto-Folic Acid 

 Medium : 



Stock cultures of S. lactis R. 8043 are prepared by stab inoculation of the 

 Bacto-Micro Assay Culture Agar. Following incubation at 35-37°C. for 24-48 

 hours, the tubes are stored in the refrigerator. Transplants are made at monthly 

 intervals. Inoculum for assay is prepared by subculturing from a stock culture of 

 S. lactis R. 8043 into a tube containing 10 ml. of the Bacto-Micro Inoculum 

 Broth. After 24 hours incubation at 35-3 7 °C., the cells are centrifuged, under 

 aseptic conditions, and the supernatant liquid decanted. The cells are resuspended 

 in 10 ml. of sterile isotonic sodium chloride. The cell suspension is then diluted 

 1-100 with sterile isotonic sodium chloride. The suspension should be just faintly 

 cloudy. One drop of this latter suspension is then used to inoculate each of the 

 assay tubes. 



It is essential that a standard curve be constructed each time an assay is run, 

 since conditions of autoclaving, temperature of incubation, etc., which influence 

 the standard curve readings, cannot be duplicated exactly from time to time. A 

 standard curve is obtained by using the folic acid at levels of 0.0, 0.001, 0.002, 

 0.004, 0.006, 0.008, 0.009 and 0.01 microgram per assay tube (10 ml.). 



The concentrations of folic acid required for the construction of the standard 

 curve may be prepared by dissolving 0.1 gram folic acid in 1000 ml. of distilled 

 water (100 micrograms per ml.). Add 0.05N sodium hydroxide drop by drop to 

 effect complete solution of the folic acid, being careful not to get more alkaline 

 than pH 7.0. Dilute the stock solution by adding 1 ml. to 999 ml. distilled water. 

 This solution is diluted further by adding 4 ml. to 996 ml. distilled water. Use 

 0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.25 and 2.5 ml. of this final solution per tube. The 

 stock solution of folic acid used for preparing the standard curve is stable for 2 

 months when stored at 2-6° C. under toluene. 



Following inoculation, tubes are incubated at 35-37°C. for 18-24 hours and 

 placed in the refrigerator for 15-30 minutes in order to stop growth. The growth 

 can then be measured by any suitable nephelometric method, and the curve con- 



