DEHYDRATED CULTURE MEDIA 215 



Stock cultures of L. casei e 7469 are prepared by stab inoculation into 10 ml. 

 of Bacto-Micro Assay Culture Agar. After 24-48 hours incubation at 35-37°C., 

 the stock cultures are kept in the refrigerator. Transplants are made at monthly 

 intervals, in triplicate. Inoculum for assay is prepared by subculturing from a 

 stock culture of L. casei e 7469 into 10 ml. of Bacto-Micro Inoculum Broth. 

 Following incubation for 24 hours at 35-37° C, the culture is centrifuged, under 

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

 in 10 ml. sterile isotonic sodium chloride. The cell suspension is then diluted 1-20 

 with sterile isotonic sodium chloride. One drop of this latter suspension is then 

 used to inoculate each of the assay tubes. 



Bacto-Riboflavin Assay Medium may be used for both turbidimetric and 

 acidimetric determinations. Turbidimetric readings should be made after 18-24 

 hours incubation at 35-3 7 °C., whereas acidimetric determinations are best made 

 after 72 hours incubation at 35-3 7 °C. We have found the most effective assay 

 range, using Bacto-Riboflavin Assay Medium, to be between 0.025 microgram 

 and 0.15 microgram riboflavin. 



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. 

 The standard curve is obtained by using riboflavin at levels of 0.0, 0.025, 0.05, 

 0.075, 0.1, 0.15, 0.2 and 0.3 microgram riboflavin per assay tube (10 ml.). 



The concentrations of riboflavin required for the preparation of the standard 

 curve may be prepared by dissolving 0.1 gram of riboflavin in 1000 ml. of 

 distilled water by heating, giving a stock solution of 100 micrograms per ml. 

 Dilute the stock solution by adding 1 ml. to 999 ml. distilled water. Use 0.0, 0.25, 

 0.5, 0.75, 1.0, 1.5, 2.0 and 3 ml. of the diluted stock solution per tube. The stock 

 solution of riboflavin used for preparing the standard curve is stable for 2 months 

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



To rehydrate the basal medium, suspend 48 grams of Bacto-Riboflavin Assay 

 Medium in 1000 ml. of distilled water and heat to boiling for 2-3 minutes. The 

 flask should be well shaken to distribute evenly the slight precipitate which forms. 

 Five (5) ml. of the medium are added to each tube in the preparation of the 

 tubes for the standard curve and to each tube containing material under assay. 

 For the assay, each tube must contain 5 ml. of rehydrated medium, increasing 

 amounts of the standard or the unknown and sufficient distilled water to give a 

 total volume of 10 ml. per tube. The tubes are then autoclaved for 10 minutes 

 at 15 pounds pressure (121°C.). Oversterilization of the medium will give un- 

 satisfactory results. 



One hundred grams of Bacto-Riboflavin Assay Medium will make 4 liters of 

 final medium. 



ilnd. Eng. Chem. Anal. Ed., 11:346:1939. s National Formulary, 9th Edition: 763: 1950. 



8 Pharmacopeia of the United States Revision * A.O.A.G., 7th Edition: 779:1950. 

 XIV: 753: 1 950. 



