DEHYDRATED CULTURE MEDIA 219 



BACTO 



PANTOTHENATE ASSAY MEDIUM (B323) 



DEHYDRATED 

 Bacto- Vitamin Free Niacin 0.002 g 



Casamino Acids 12 g, 



Bacto-Dextrose 40 g 



Sodium Acetate 20 g 



/-Cystine, Difco 0.2 g 



^/-Tryptophane 0.2 g 



Adenine Sulfate 0.02 g 



Guanine Hydrochloride 0.02 g 



Uracil 0.02 g 



Thiamine Hydrochloride .... 0.002 g 

 Riboflavin 0.002 g 



Pyridoxine Hydrochloride . . . 0.004 g 

 j&-Aminobenzoic Acid, Difco 0.0002 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 



Bacto-Pantothenate Assay Medium is a complete dehydrated medium for 

 the assay of pantothenic acid. It is free from pantothenic acid or pantothenate 

 but contains all the other factors necessary for the growth of Lactobacillus 

 arabinosus 17-5 ATCG 8014. The addition of pantothenate, in specified increas- 

 ing concentration, gives a linear growth response by L. arabinosus 17-5 which 

 may be measured acidimetrically or turbidimetrically. Bacto-Pantothenate Assay 

 Medium is a slight modification of the medium described by Skeggs and Wright.^ 



The following procedure is recommended for the use of Bacto-Pantothenate 

 Assay Medium : 



Stock cultures of the test organism, L. arabinosus 17-5, are prepared by stab 

 inoculation of Bacto-Micro Assay Culture Agar. Following incubation at 35- 

 37°C. 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. Following incubation for 24 hours 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 suspen- 

 sion 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, cannot be duplicated exactly from time to time. The 

 standard curve is obtained by using calcium pantothenate at levels of 0.0, 0.02, 

 0.04, 0.06, 0.08, 0.1, 0.12 and 0.2 microgram per assay tube (10 ml.). Bacto- 

 Pantothenate Assay Medium may be used for both turbidimetric and acidimetric 

 analysis. Turbidimetric readings should be made after 18-24 hours at 35-3 7 °C. 

 Acidimetric determinations are made after 72 hours incubation at 35-37°C. We 

 have found the most effective assay range, using Bacto-Pantothenate Assay 

 Medium, to be between 0.02 microgram and 0.12 microgram calcium pan- 

 tothenate. 



The concentrations of calcium pantothenate required for the preparation of 

 the standard curve may be prepared by dissolving 0.1 gram of calcium pan- 

 tothenate in 1000 ml. of distilled water, giving a stock solution of 100 micrograms 

 per ml. Dilute the stock solution by adding 1 ml. to 99 ml. of distilled water. 

 This solution is further diluted by adding 4 ml. to 96 ml. distilled water to give 

 the final solution. Use 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 5 ml. of this final solu- 

 tion per tube. The stock solution of calcium pantothenate used for preparing the 

 Standard curve is stable for 2 months when stored at 2-6° G. under toluene. 



