228 DIFGO MANUAL 



paper towels, and roll into a small pellet. Dry the pellets at 100°C. in a vacuum 

 oven for 2 hours. A glazed porcelain spot plate is convenient for handling the 

 mycelium during drying and weighing. Weigh to the nearest 0.5 mg. A standard 

 curve is then constructed from the weights obtained and the unknown determined 

 by interpolation. In the assay for choline, 125 ml, Erlenmeyer flasks containing 

 a total volume of 20 ml. medium each are used. 



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. The 

 standard curve is obtained by using choline at levels of 0.0, 2.5, 5.0, 10, 20, 30, 

 40 and 50 micrograms per assay flask (20 ml.). Using Choline Assay Medium the 

 most effective assay range has proved to be between 2.5 and 30 micrograms 

 choline. 



The concentration of choline required for the preparation of the standard curve 

 may be prepared by dissolving 1.0 gram choline in 1000 ml. distilled water. This 

 is the stock solution (1000 micrograms per ml.). Dilute the stock solution by 

 adding 1 ml. to 99 ml. distilled water. Use 0.0, 0.5, 1.0, 2, 3, 4, and 5 ml. per 

 tube. The stock solution is stable for 2 months when stored at 2-6° C. under 

 toluene. 



To rehydrate the basal medium, dissolve 57 grams of Bacto-Choline Assay 

 Medium in 1000 ml. of distilled water. Ten (10) ml. of the medium are added 

 to each flask in the preparation of the flasks for the standard curve and to each 

 flask containing material under assay. For the assay, each flask must contain 10 

 ml. of rehydrated medium, increasing amounts of the standard or the unknown 

 and sufficient distilled water to give a total volume of 20 ml. per flask. Sterilize 

 in the autoclave for 10 minutes at 15 pounds pressure (121°C.). The flasks are 

 then ready for inoculation. Oversterilization of the medium will give unsatisfac- 

 tory results. 



One hundred grams of Bacto-Choline Assay Medium will make 3.5 liters of 

 final medium. 

 1 J. Biol. Chem., 130:325:1943. 



