226 DIFCO MANUAL 



structed from the values obtained. Acidimetric determinations of growth have 

 not been found satisfactory. Using Bacto-Folic Acid Assay Medium, we have 

 found the most effective assay range to be between the levels of 0.002 and 0.01 

 microgram folic acid per tube (10 ml.). 



To rehydrate the basal medium, suspend 75 grams of Bacto-Folic Acid 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°C). Do not sterilize longer than specified above. One hundred grams of 

 Bacto-Folic Acid Assay Medium will make 2.6 liters of final medium. 

 ij. Bact., 55:869:1948. 



BACTO 



PYRIDOXINE ASSAY MEDIUM (B324) 



DEHYDRATED 



Saccharose, Difco 30 g 



Biotin 0.000008 g 



Ammonium Tartrate 10 g 



Sodium Dihydrogen Citrate 4 g 



Monopotassium Phosphate 5 g 



Magnesium Sulfate 1 g 



Sodium Chloride 0.2 g 



Calcium Chloride 0.2 g 



Ferric Chloride 0.01 g 



Zinc Sulfate 0.004 g 



Bacto-Pyridoxine Assay Medium is a complete dehydrated medium for the 

 microbiological assay of pyridoxine. It is patterned after the medium described 

 by Stokes, Larsen, Woodward and Foster^ and modified by Barton-Wright^. 

 Bacto-Pyridoxine Assay Medium is free from pyridoxine but contains all other 

 factors necessary for growth of Neurospora sitophila 299 ATCG 9276. The addi- 

 tion of pyridoxine, in specified concentrations, gives increasing growth response 

 by N. sitophila 299 which may be determined gravimetrically. 



The test is run according to the method described by Stokes, Larsen, Wood- 

 ward and Foster^ and modified by Barton- Wright. ^ Remove one loop of spores 

 from a 48-hour culture of A^. sitophila 299 on Bacto-Neurospora Culture Agar 

 and suspend in 100 ml. sterile saline. Add one drop of this spore suspension to 

 each flask. Incubate at 30° C. for 5 days. At the end of the incubation period 

 steam the flasks at 100°G. for 5 minutes. Remove all the mycelium from the flask 

 using a stiff wire needle or glass rod, press dry between paper towels, and roll into 

 a small pellet. Dry the pellets at 100°C. for 2 hours in a vacuum. A glazed porce- 

 lain spot plate is convenient for handling the mycelium during drying and weigh- 

 ing. A standard curve is then constructed from the weights obtained and the 

 unknown determined by interpolation. 



It is essential that a standard curve be constructed each time as 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 pyridoxine at levels of 0.0, 0.2, 0.4, 0.6, 0.8 

 and 1.0 microgram per assay flask (10 ml.). Using Bacto-Pyridoxine Assay 



