Pantothenate Assay Medium is prepared according to the formulation of the U.S. Pharmacopeia (1) for microbiological assay of pantothenic acid or its salts using Lactobacillus plantarum ATCC 8014 as the test organism.
This medium contains several nutrients and salts which are required for the growth of the test organism. The nutrients include amino acids, carbohydrates, purine, pyrimidine bases, salts and vitamins but not the pantothenic acid. Since the pantothenate is required for the growth of Lactobacillus plantarum assay strain, growth of the organism will occur only if the materials being assayed contain pantothenate. Exact concentration of pantothenate in the test material can be calculated by comparing results with standard curve of pantothenate.
Assay / Procedure: Stock cultures of the test organism Lactobacillus plantarum ATCC 8014, are prepared in triplicate or more by stab inoculation of Pantothenate Culture Agar USP (M135). Following incubation for 16-24 hours at any selected temperature between 30°C and 37°C but held constant to within ± 0.5°C, the tubes are stored at 2-8°C. Prepare a fresh stab of stock culture every week and do not use a culture older than 1 week for transferring to broth for inoculation.
Inoculum for the assay is prepared by subculturing from a suitable stock culture of Lactobacillus plantarum ATCC 8014 on Pantothenate Culture Agar USP (M135) into a tube containing 10 ml of sterile single strength Pantothenate Assay Medium supplemented with pantothenate. The medium is prepared by dissolving 36.5 grams of the dehydrated medium and 20 mcg of pantothenate in 1000 ml of distilled water. Distribute in tubes and sterilize by autoclaving for 15 minutes at 15 lbs pressure (121°C). After 18-24 hours incubation at 30-37°C the cells are centrifuged under aseptic conditions and the supernatant liquid is decanted. The cells are resuspended in 10 ml sterile 0.85% sodium chloride. The cell suspension is then diluted 1:100 with sterile 0.85% sodium chloride. The cell suspension so obtained is the inoculum. Inoculate each tube aseptically with 1 drop of the inoculum.
It is essential that a standard curve be set up for each 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 solution at concentration of 0.0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 and 0.1 mcg pantothenic acid per assay tube (10 ml). Turbidimetric determinations are made after 16-24 hours incubation at any selected temperature between 30°C and 37°C, but held constant to within ± 0.5°C. Acidimetric determinations are made after 72 hours incubation at 30-37°C. A standard curve is then plotted and the unknown is determined by extrapolation.
Storage and Shelf-life:
Store below 8°C in tightly closed container and use freshly prepared medium. Use before expiry date on the label.
Reference:
1. U.S. Pharmacopeia, National Formulary, 2002, 25/NF 20, U.S. Pharmacopoeial , Convention, Rockville, MD.