DEHYDRATED CULTURE MEDIA 



229 



BACTO 



GF ASSAY MEDIUM 



DEHYDRATED 



(B456) 



Bacto-Vitamin Free 



Casamino Acids 10 



Bacto-Dextrose 50 



Sodium Acetate 40 



Ammonium Chloride 6 



/-Cystine, Difco 0.2 



Cysteine 0.2 



^//-Tryptophane 0.2 



^//-Alanine 0.2 



Glycine 0.2 



Adenine Sulfate 0.02 



Guanine Hydrochloride 0.02 



Uracil 0.02 



Xanthine 0.02 



Thiamine Hydrochloride .... 0.001 



Pyridoxine Hydrochloride . . 0.002 g 



Pyridoxamine Hydrochloride . 0.006 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. 



Sodium Chloride 0.02 g 



Ferrous Sulfate 0.02 g, 



Manganese Sulfate 0.04 g 



Bacto-CF Assay Medium is a complete dehydrated medium for the assay of 

 citrovorum factor. It is free from citrovorum factor but contains all the other 

 factors necessary for the growth of Leuconostoc citrovorum 8081 ATCC. The 

 addition of citrovorum factor in specified increasing concentrations gives a growth 

 response by L. citrovorum 8081 which may be measured acidimetrically or turbi- 

 dimetrically. Bacto-CF Assay Medium is a modification of the formula given by 

 Sauberlich and Baumann^ and William F. Faloon.- 



The following procedure is recommended for the use of Bacto-CF Assay 

 Medium : 



Stock cultures of the test organism, L. citrovorum 8081, are prepared by stab 

 inoculation of Bacto-Micro Assay Culture Agar. After 24-48 hours incubation at 

 35-3 7 ''C, the tubes are kept in the refrigerator. Transplants are made at monthly 

 intervals, in triplicate. 



Inoculum for assay is prepared by subculturing from a stock culture to 10 ml. 

 of Bacto-Micro Inoculum Broth. After 16-24 hours incubation at 35-37°C., the 

 cells are centrifuged under aseptic conditions, and the supernatant liquid de- 

 canted. Suspend the cells from the culture in 10 ml. of sterile isotonic sodium 

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

 chloride solution. One drop of this latter suspension is used to inoculate the assay 

 tubes (10 ml.). 



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 citrovorum factor at levels of 0.0, 0.6, 1.2, 1.8, 

 2.4, 3.0, 3.6, 4.8 and 6.0 millimicrograms per assay tube (10 ml.). 



The concentration of citrovorum factor required for the construction of the 

 standard cur\'e may be prepared by dissolving the contents of 1 ampul leucovorin 

 (citrovorum factor) in 1000 ml. distilled water. This is the stock solution (3000 

 micrograms per ml.). Dilute the stock solution by adding 4 ml. to 96 ml. distilled 

 water. Dilute this solution further by adding 1 ml. to 99 ml. distilled water. Use 

 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4 and 5 ml. per tube. The stock solution is stable 

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



Bacto-CF Assay Medium may be used for both acidimetric and turbidimetric 

 analysis. Acidimetric determinations are made after 72 hours incubation at 

 35-37°C. Turbidimetric readings should be made after 20-24 hours incubation 



