DEHYDRATED CULTURE MEDIA 217 



To rehydrate the basal medium, suspend 75 grams of Bacto-NIacin 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 then autoclaved for 10 minutes at 15 pounds pres- 

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



One hundred grams of Bacto-Niacin Assay Medium will make 2.6 liters of 

 final medium. 



ij. Biol. Chem., 139:675: i94i' Revision: 737: 1950. 



2 Ind. Eng. Chem. Anal. Ed., 15:471:1943. ^ National Formulary, 9th Edition: 746:1950. 



s Biochem. J., 38:314:1944- « A.O.A.G., 7th Edition:782: 1950. 



* Pharmacopeia of the United States, XIV 



BACTO 



THIAMINE ASSAY MEDIUM (B326) 



DEHYDRATED 



Photolyzed Peptone 22 g. Niacin 0.0002 g. 



Bacto-Vitamin Free Pyridoxine Hydrochloride . . 0.0002 g. 



Casamino Acids 5 g. j&-Aminobenzoic Acid, Difco 0.0002 g. 



Bacto-Dextrose 40 g. Folic Acid 0.0000005 g. 



Sodium Acetate 15 g. Biotin . 0.0000008 g. 



/-Cystine, Difco 0.2 g. Dipotassium Phosphate 1 g. 



Adenine Sulfate 0.02 g. Monopotassium Phosphate 1 g. 



Guanine Hydrochloride 0.02 g. Magnesium Sulfate 0.4 g. 



Uracil 0.02 g. Sodium Chloride 0.02 g. 



Riboflavin 0.0002 g. Ferrous Sulfate 0.02 g. 



Calcium Pantothenate 0.0002 g. Manganese Sulfate 0.02 g. 



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

 assay of thiamine. It is free from thiamine but contains all the other factors 

 necessary for the growth of Lactobacillus jermentum 36 ATCC 9833. The addi- 

 tion of thiamine, in certain increasing concentrations, gives a growth response 

 which may be measured turbidimetrically. Bacto-Thiamine Assay Medium is 

 prepared according to the formula given by Sarett and Cheldelin.^ 



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

 Medium: 



Stock cultures of the test organism, L. jermentum 36, are prepared by stab 

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

 at 35-3 7 °G., the tubes are kept in the refrigerator. Transplants are made at 

 monthly intervals, in triplicate. 



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

 of Bacto-Micro Inoculum Broth. After 16-18 hours incubation at 35-3 7 °C., the 

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

 canted. The cells are resuspended in 10 ml. sterile isotonic sodium chloride. One- 

 half (0.5) ml. of this cell suspension are then added to 100 ml. of sterile saline. 

 One drop of this suspension is then used to inoculate the assay tubes. 



The tubes for the standard curve, which should be set up each time an assay 

 is run, contain 0.0, 0.005, 0.01, 0.015, 0.02, 0.03, 0.04 and 0.05 microgram of 

 thiamine hydrochloride per tube (10 ml.). It is essential that a standard curve 

 be run with each assay since conditions of heating, temperature of incubation, 

 etc., which influence the standard curve readings cannot be duplicated exactly 



