230 



DIFCO MANUAL 



at 35-3 7°C. The most effective assay range, using Bacto-CF Assay Medium, has 

 been found to be between 0.6 and 4.8 millimicrograms of citrovorum factor. 



To rehydrate the medium, suspend 110 grams of Bacto-CF Assay Medium in 

 1000 ml. distilled water, and heat to boiling for 2-3 minutes. The slight precipi- 

 tate 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 then autoclaved for 10 minutes at 15 pounds pressure 

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



One hundred grams of Bacto-CF Medium will make 1.8 liters final medium, 



^ J. Biol. Chem., 176:165:1948. 2 Personal Communication, 1951. 



AMINO ACID ASSAY MEDIA 



Media for the microbiological assay of the amino acids, leucine, methionine, 

 lysine and isoleucine have been prepared. A complete description of each medium 

 is given in the individual discussions. The formulation of the media per liter is 

 as follows : 



Bacto-Dextrose 50 g, 



Sodium Acetate 40 g, 



Ammonium Chloride 6 g 



^/-Alanine 0.4 g 



i-Arginine Hydrochloride .... 0.484 g 



Bacto-Asparagine 0.8 



i-Aspartic Acid 0.2 



/-Cystine, Difco 0.1 



/-Glutamic Acid 0.6 



Glycine 0.2 



/-Histidine Hydrochloride ... 0.124 g 



^/-Phenylalanine 0.2 g 



/-Proline 0.2 g 



£?/-Serine 0.1 g 



^/-Threonine 0.4 g 



J/-Tryptophc ne 0.08 g 



/-Tyrosine 0.2 g 



^f/- Valine 0.5 g 



Adenine Sulfate 0.02 g 



Guanine Hydrochloride 0.02 g. 



Uracil 0.02 g. 



Xanthine 0.02 g. 



Thiamine Hydrochloride .... 0.001 g. 



Pyridoxine Hydrochloride . . . 0.002 g. 



Pyridoxamine Hydrochloride 0.0006 g. 



Pyridoxal Hydrochloride . . . 0.0006 g. 



Calcium Pantothenate 0.001 g. 



Riboflavin 0.001 g. 



Nicotinic Acid 0.002 g. 



p-Aminobenzoic Acid, Difco . 0.0002 g. 



Biotin 0.000002 g. 



Folic Acid 0.00002 g. 



Monopotassium Phosphate .... 1.2 g. 



Dipotassium Phosphate 1.2 g. 



Magnesium Sulfate 0.4 g. 



Ferrous Sulfate 0.02 g. 



Manganese Sulfate 0.04 g. 



Sodium Chloride 0.02 g. 



BACTO 



LEUCINE ASSAY MEDIUM 



DEHYDRATED 



(B421) 



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

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

 g. £?/-isoleucine. Bacto-Leucine Assay Medium, prepared according to the formula 

 given by Steel, Sauberlich, Reynolds and Baumann,^ is a complete dehydrated 

 medium for the assay of /-leucine and c?/-leucine. It is free from leucine but con- 

 tains all the other growth factors and amino acids necessary for the growth of 

 Leuconostoc mesenteroides P-60 ATCC 8042. The addition of /-leucine or <i/-leu- 

 cine in specified increasing concentrations gives a growth response by L. meseri' 

 teroides P-60, which may be measured acidimetrically or turbidimetrically. 



