216 DIFCO MANUAL 



BACTO 



NIACIN ASSAY MEDIUM (B322) 



DEHYDRATED 

 Bacto- Vitamin Free Pyridoxine Hydrochloride . . 0.0004 g, 



Riboflavin 0.0004 g 



j&-Aminobenzoic Acid, Difco . 0.0001 g 



Biotin 0.0000008 g 



Dipotassium Phosphate 1 g 



Monopotassium Phosphate 1 g 



Magnesium Sulfate 0.4 g 



Sodium Chloride 0.02 g 



Ferrous Sulfate 0.02 g 



Manganese Sulfate 0.02 g 



Casamino Acids > 12 g 



Bacto-Dextrose 40 g 



Sodium Acetate 20 g, 



/-Cystine, Difco 0.4 g, 



^^/-Tryptophane 0.2 g, 



Adenine Sulfate 0.02 



Guanine Hydrochloride 0.02 g, 



Uracil ^ 0.02 g, 



Thiamine Hydrochloride . . . 0.0002 g, 

 Calcium Pantothenate 0.0002 g, 



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

 of nicotinic acid or nicotinamide (niacin). It is free from nicotinic acid and its 

 analogs, but contains all the other factors necessary for the growth of Lacto- 

 bacillus arabinosus 17-5 ATCC 8014. The addition of nicotinic acid or its analogs 

 in specified increasing concentrations gives a growth response by L. arabinosus 

 17-5 which may be measured acidimetrically or turbidimetrically. Bacto-Niacin 

 Assay Medium is prepared according to the formula described by Snell and 

 Wright^ and modified by Krehl, Strong, and Elvehjem^ and Barton-Wright.^ The 

 formula duplicates that listed in the U. S. Pharmacopeia,* and National Formu- 

 lary^ and the Official Method of Analyses of the Association of Official Agricul- 

 tural Chemists.* 



Best results, using Bacto-Niacin Assay Medium, are obtained through the use 

 of the following procedure: 



Stock cultures of L. arabinosus 17-5 are prepared by stab inoculation of 

 Bacto-Micro Assay Culture Agar. Following incubation at 35-3 7 °G. for 24-48 

 hours, the tubes are kept in the refrigerator. Transplants are made at monthly 

 intervals. Inoculum for assay is prepared by subculturing from a stock culture of 

 L. arabinosus 17-5 into a tube containing 10 ml. of Bacto-Micro Inoculum Broth. 

 After 24 hours incubation at 35-3 7 °C., the cells are centrifuged, under aseptic 

 conditions, and the supernatant liquid decanted. The cells are resuspended in 10 

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

 with sterile isotonic sodium chloride. The suspension should be just faintly cloudy. 

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



It is essential that a standard curve be set up for each separate assay since 

 conditions of autoclaving, temperature of incubation, etc., which influence the 

 standard curve readings, caimot be duplicated exactly from time to time. The 

 standard curve is obtained by using niacin at levels of 0.0, 0.05, 0.1, 0.2, 0.3, 0.4, 

 and 0.5 micrograms niacin per assay tube (10.0 ml.). Bacto-Niacin Assay Me- 

 dium may be used for both turbidimetric and acidimetric analyses. Turbidimetric 

 readings should be made after 16-18 hours incubation at 35-37°C. Acidimetric 

 determinations are best made following 72 hours incubation at 35-37°C. We have 

 found the most effective assay range, using Bacto-Niacin Assay Medium, to be 

 between 0.05 micrograms and 0.3 micrograms niacin. 



The concentrations of niacin required for the preparation of the standard 

 curve may be prepared by dissolving 0.1 gram of niacin in 1000 ml. of distilled 

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

 by adding 1 ml. to 999 ml. distilled water. Use 0.0, 0.5, 1.0, 2, 3, 4 and 5 ml. per 

 tube. The stock solution of niacin used for preparing the standard curve is stable 

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



