DEHYDRATED CULTURE MEDIA 231^ 



The following procedure is recommended for the use of Bacto-Leucine Assay 

 Medium : 



Stock cultures of the test organism, L. mesenteroides P-60, are prepared by 

 stab inoculation of Bacto-Micro Assay Culture Agar. After 24—48 hours incuba- 

 tion at 35-3 7 °C., the tubes are stored in the refrigerator. The transplants are 

 made at monthly intervals, in triplicate. 



Inoculum for assay is prepared by subculturing from a stock culture to 10 ml. 

 of Bacto-Micro Inoculum Broth. After 16-24 hours incubation 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 solu- 

 tion. The cell suspension is diluted 5-100 with sterile isotonic sodium chloride. 

 One drop of this latter suspension is used to inoculate each of the assay tubes 

 (10 ml.). 



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 /-leucine at levels of 0.0, 10.0, 20, 30, 40, 50, 

 60, 80 and 100 micrograms per assay tube (10 ml.). (<i/-leucine may be used at 

 levels of 0.0, 20.0, 40, 60, 80, 100, 120, 160 and 200 micrograms per assay tube.) 



Bacto-Leucine Assay Medium may be used for both turbidimetric and acidi- 

 metric analyses. Turbidimetric readings should be made after 16-20 hours at 

 35-3 7 °C. Acidimetric determinations are made after 72 hours incubation at 

 35-3 7 °C. The most effective assay range, using Bacto-Leucine Assay Medium, 

 has been found to be between 20 micrograms and 80 micrograms /-leucine. 

 (20-160 micrograms of fi?/-leucine). 



The concentrations of leucine required for the construction of the standard 

 curve may be prepared by dissolving 2.0 grams of /-leucine (4.0 g. ^//-leucine) in 

 1000 ml. distilled water giving a stock solution of 2000 micrograms of /-leucine 

 per ml. Dilute the stock solution by adding 1 ml. to 99 ml. distilled water. Use 

 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4 and 5 ml. per tube. The stock solution of leucine 

 used for preparing the standard curve is stable for 2 months when stored at 

 2-6°G. under toluene. 



To rehydrate the medium, suspend 105 grams of Bacto-Leucine Assay Medium 

 in 1000 ml. distilled water, and heat to boiling for 2-3 minutes. The slight pre- 

 cipitate which forms should be evenly distributed by shaking. Five (5) ml. of 

 the medium are added to each tube in the preparation of the tubes for the stand- 

 ard 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 stand- 

 ard or the unknown, and sufficient distilled water to give a total volume of 10 ml. 

 per tube. The tubes are autoclaved for 10 minutes at 15 pounds pressure 

 (121°G.). Oversterilization of the medium will give unsatisfactory results. 



One hundred grams of Bacto-Leucine Assay Medium will make 1.9 liters of 

 final medium. 

 *J. Biol, Chem., 177:533:1949. 



