232 DIFCO MANUAL 



BACTO 



METHIONINE ASSAY MEDIUM (B423) 



DEHYDRATED 



The composition of Bacto-Methionine Assay Medium is as shown above, but 

 in addition it contains 0.5 g. /-lysine hydrochloride, 0.5 g. isoleucine and 0.5 g. 

 cf/-leucine. Bacto-Methionine Assay Medium, prepared according to the formula 

 given by Steel, Sauberlich, Reynolds and Baumann,^ is a complete dehydrated 

 medium for the assay of c?/-methionine. It is free from methionine but contains 

 all the other growth factors and amino acids necessary for the growth of Leu- 

 conostoc mesenteroides P-60 ATCC 8042. The addition of c?/-methionine in speci- 

 fied increasing concentrations gives a growth response by L. mesenteroides P-60 

 which may be measured acidimetrically or turbidimetrically. 



The following procedure is recommended for the use of Bacto-Methionine 

 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 incubation at 

 35-37°C., the tubes are stored in the refrigerator. 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 35-37°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 c?/-methionine at levels of 0.0, 6.0, 12, 18, 24, 

 30, 36, 48 and 60 micrograms per assay tube ( 10 ml.). 



The concentrations of methionine required for the preparation of the standard 

 curve may be prepared by dissolving 1.2 grams ^/-methionine in 1000 ml. dis- 

 tilled water. This is the stock solution (1200 micrograms 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 is stable for 2 months when 

 stored at 2-6° G. under toluene. 



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

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

 at 35-37°C.; acidimetric determinations are made after 72 hours incubation. The 

 most effective assay range, using Bacto-Methionine Assay Medium, has been 

 found to be between 6 and 48 micrograms c?/-methionine. 



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

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

 slight precipitate 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 

 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 autoclaved for 10 minutes at 15 pounds pressure 

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



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

 of final medium. 



ij. Biol. Chem., ?95:533:i949. 



