236 DIFCO MANUAL 



vals. Inoculum for assay is prepared by subculturing from a stock culture of 

 L. arabinosus 17-5 into a tube containing 10 ml. of Bacto-Micro Inoculum Broth. 

 After 24 hours incubation at 35-3 7 °C., the cells are centrifuged under aseptic 

 conditions, and the supernatant liquid decanted. The cells are resuspended in 10 

 ml. of sterile isotonic sodium chloride. The cell suspension is then diluted 1-100 

 with sterile isotonic sodium chloride. The suspension should be just faintly 

 cloudy. One drop of this latter suspension is then used to inoculate each of the 

 assay tubes. 



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 /-tryptophane at levels of 0.0, 2.0, 4, 6, 8, 10 

 and 12 micrograms per assay tube (10 ml.). ( cf/-tryptophane may be used at 

 levels of 0.0, 4.0, 8, 12, 16, 20 and 24 micrograms). 



The concentrations of tryptophane required for the preparation of the standard 

 curve may be prepared by dissolving 0.1 gram of /-tryptophane (0.2 g. t/Z-trypto- 

 phane) in 1000 ml. of distilled water giving a stock solution of 100 micrograms of 

 /-tryptophane per ml. Dilute this stock solution by adding 4 ml. to 96 ml. dis- 

 tilled water. Use 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 5 ml. per tube. This stock 

 solution of tryptophane used for preparing the standard curve is stable for 2 

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



Following inoculation, tubes are incubated at 35-3 7 °C. for 72 hours and are 

 assayed by acidimetric methods. Using Bacto-Tryptophane Assay Medium, we 

 have found the most effective assay range to be between 2 and 10 micrograms of 

 /-tryptophane. (4 and 20 micrograms c?/-tryptophane). 



To rehydrate the basal medium, suspend 75 grams of Bacto-Tryptophane 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 sterilized by autoclaving for 10 minutes at 15 pounds pressure 

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



One hundred grams of Bacto-Tryptophane Assay Medium will make 2.6 liters 

 of final medium. 



1 J. Biol. Chem., 155:1:1944' 



