DEHYDRATED CULTURE MEDIA 233 



BACTO 



LYSINE ASSAY MEDIUM (B422) 



DEHYDRATED 



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

 addition it contains 0.2 g. of cf/-methionine, 0.5 g. c?/-isoleucine and 0.5 g. c^Meu- 

 cine. Bacto-Lysine Assay Medium, prepared according to the formula given by 

 Steel, Sauberlich, Reynolds and Baumann,^ is a complete dehydrated medium 

 for the assay of /-lysine. It is free from lysine but contains all the other growth 

 factors and amino acids necessary for the growth of Leuconostoc mesenteroides 

 P-60 ATCC 8042. The addition of /-lysine in specified increasing concentrations 

 gives a linear growth response by L. mesenteroides P-60, which may be measured 

 acidimetrically or turbidimetrically. 



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

 35-3 7 °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 35-3 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 

 solution. 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 /-lysine at levels of 0.0, 30.0, 60, 90, 120, 150, 

 180, 240 and 300 micrograms per assay tube (10 ml.). 



The concentrations of lysine required for the preparation of the standard curve 

 may be prepared by dissolving 6.0 grams /-lysine in 1000 ml. distilled water. This 

 is the stock solution (6000 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-Lysine Assay Medium may be used for both turbidimetric and acidi- 

 metric 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-Lysine Assay Medium, has been found to 

 be between 30 micrograms and 240 micrograms of /-lysine. 



To rehydrate the medium, suspend 105 grams of Bacto-Lysine 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 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-Lysine Assay Medium will make 1 .9 liters of final 

 medium. 



ij. Biol. Chem., 177:533 = 1 949- 



