DEHYDRATED CULTURE MEDIA 227 



Medium the most effective assay range has proved to be between 0.2 and 0.8 

 microgram pyridoxine. 



The concentrations of pyridoxine required for the preparation of the standard 

 curve may be prepared by dissolving 0.1 gram of pyridoxine in 1000 ml. of dis- 

 tilled water, giving a stock solution of 100 micrograms per ml. Dilute the stock 

 solution by adding 1 ml. to 499 ml. distilled water. Use 0.0, 1.0, 2, 3, 4 and 5 ml. 

 per tube. This stock solution of pyridoxine used for preparing the standard curve 

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



To rehydrate the basal medium, dissolve 50 grams of Bacto-Pyridoxine Assay 

 Medium in 1000 ml. of distilled water. Distribute in 5 ml. quantities in 50 ml. 

 Erlenmeyer flasks. Five (5) 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 5 ml. of rehydrated 

 medium, increasing amounts of the standard or the unknown and sufflcient dis- 

 tilled water to give a total volume of 10 ml. per flask. Sterilize in the autoclave 

 for 15 minutes at 15 pounds pressure (121°C.). The flasks are then ready for 

 inoculation. Oversterilization of the medium will give unsatisfactory results. 



One hundred grams of Bacto-Pyridoxine Assay Medium will make 4 liters of 

 final medium. 



ij. Biol. Chem., 150:17:1943. 2 Analyst, 70:283:1945. 



BACTO 



CHOLINE ASSAY MEDIUM (B460) 



DEHYDRATED 



Saccharose, Difco 40 g. 



Ammonium Nitrate 2 g. 



Biotin 0.00001 g. 



Potassium Sodium Tartrate 11.4 g. 



Monopotassium Phosphate 2 g. 



Magnesium Sulfate 1 g. 



Sodium Chloride 0.2 g. 



Calcium Chloride 0.2 g. 



Sodium Borate 0.0007 g. 



Ammonium Molybdate 0.0005 g. 



Ferrous Sulfate 0.0011 g. 



Cuprous Chloride 0.0003 g. 



Manganese Sulfate 0.00011 g. 



Zinc Sulfate 0.0176 g. 



Bacto-Choline Assay Medium is a complete dehydrated medium for the micro- 

 biological assay of choline. It is a slight modification of the medium described by 

 Horowitz and Beadle.^ Bacto-Choline Assay Medium is free from choline but 

 contains all the other factors necessary for the growth of Neurospora crassa 34486 

 ATCG. The addition of choline in specified increasing concentrations gives a 

 growth response by N. crassa 34486 which may be determined gravimetrically. 



The following procedure for the preparation of the inoculum and performance 

 of the test is recommended for the assay of choline using Bacto-Choline Assay 

 Medium: 



Remove one loop of spores from a 48-hour culture of A^. crassa 34486 on Bacto- 

 Neurospora Culture Agar and suspend in 100 ml. sterile saline. Add one drop of 

 this spore suspension to each flask. Incubate at 25 °C. for 3 days. At the end of 

 the incubation period, steam the flasks at 100°C. for 5 minutes. Remove all the 

 mycelium from the flask using a stiff wire needle or glass rod, press dry between 



