234 DIFCO MANUAL 



BACTO 



ISOLEUCINE ASSAY MEDIUM (B437) 



DEHYDRATED 



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

 addition, it contains 0.2 g. of c?/-methionine, 0.5 g. of c?/-leucine and 0.5 g. /-lysine. 

 Bacto-Isoleucine Assay Medium, prepared according to the formula by Steel, 

 Sauberlich, Reynolds and Baumann,^ is a complete dehydrated medium for the 

 assay of cf/-isoleucine. It is free from isoleucine but contains all the other growth 

 factors and amino acids necessary for the growth of Leuconostoc mesenteroides 

 P-60 ATCC 8042. The addition of df/-isoleucine in specified increasing concentra- 

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

 acidimetrically. 



The following procedure is recommended for the use of Bacto-Isoleucine 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. 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 decanted. 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 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 ^/-isoleucine at levels of 0.0, 12.0, 24, 36, 48, 

 60, 72, 96 and 120 micrograms per assay tube (10 ml.). The concentrations of 

 isoleucine required for the construction of the standard curve may be prepared 

 by dissolving 2.4 grams of ^/-isoleucine in 1000 ml. distilled water. This is the 

 stock solution (2400 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. 



Tubes are incubated at 35-3 7 °G. for 72 hours following inoculation. The 

 growth response is then measured by acidimetric determinations. The most effec- 

 tive assay range, using Bacto-Isoleucine Assay Medium, has been found to be 

 between 12 and 96 micrograms cf/-isoleucine. 



To rehydrate the medium, suspend 105 grams of Bacto-Isoleucine 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°G.). Oversterilization of the medium will give unsatis- 

 factory results. 



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

 final medium. 



1 J. Biol. Chem., i77:333:»949« 



