Pantothenate Assay Medium (Powder)

Pantothenate Assay Medium is used for determining the concentration of pantothenic acid and its salts by the microbiological assay technique. It is a modification of the formula described in The United States Pharmacopeia1 for the microbiological assay of calcium pantothenate using Lactobacillus plantarum ATCC 8014 as the test organism. Appearance: White to light beige, homogenous, free flowing powder, Solubility (4.8%): Light tan, clear, may have a slight precipitate, pH (4.8%): As Reported, Principles of the Procedure: Pantothenate Assay Medium is a dehydrated medium free from pantothenic acid or pantothenate but containing all other nutrients and vitamins essential for the cultivation of L. plantarum ATCC 8014. The addition of calcium pantothenate in specified increasing concentrations gives a growth response that can be measured turbidimetrically or titrimetrically. Procedure: Prepare stock cultures of L. plantarum ATCC 8014 in triplicate by stab inoculation of Lactobacilli Agar AOAC. Incubate cultures for 18-24 hours at 35-37°C. Store the tubes at 2-8°C. Prepare a fresh stock culture every week. Do not use a culture older than 1 week for this assay. Inoculum: Subculture from a stock culture of Lactobacillus plantarum ATCC 8014 to 10 ml of sterile single-strength Pantothenate Assay Medium supplemented with 0.02ug pantothenate. Incubate for 18-24 hours at 35-37°C. Centrifuge the cells under aseptic conditions and decant the supernatant. Wash the cells three times with 10ml sterile 0.85% saline. After the third wash, resuspend the cells with sterile 0.85% saline and adjust to a turbidity of 40-45% transmittance when read on a spectrophotometer at 660 nm. Aseptically inoculate each assay tube with one drop of the cell suspension. Standard Curve: It is essential that a standard curve be constructed each time an assay is run. Autoclave and incubation conditions can influence the standard curve readings and cannot always be duplicated. The standard curve is obtained by using calcium pantothenate solution at levels of 0.0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08 and 0.1ug per assay tube (10 ml). Turbidimetric determinations are made after 18-24 hours incubation at 35-37°C. Construct a standard curve and determine the concentration of the unknown by interpolation from the standard curve. , The concentration of pantothenic acid required for the preparation of the standard curve may be prepared by dissolving 50 mg dried calcium pantothenate in a solution containing approximately 500ml purified water, 10ml 0.2N acetic acid and 100ml 0.2N sodium acetate. Dilute to 1,150ml with additional water to make the calcium pantothenate concentration 43.47ug per ml, one ml equals 40ug pantothenic acid. This solution is diluted by adding 25 ml to a solution containing 500ml purified water, 10ml 0.2N acetic acid and 100ml 0.2N sodium acetate. Dilute to 1L with purified water to make a solution solution containing 1.0ug panthothenic acid per ml. The standard solution is made by diluting 2ml of the stock solution to 100ml with purified water. This solution contains 0.02ug pantothenic acid per ml. Use 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 and 5.0 ml per assay tube. Prepare the stock solution fresh daily. Expected Results: 1. Prepare a standard concentration response curve by plotting the response readings against the amount of standard in each tube, disk or cup., 2. Determine the amount of vitamin at each level of assay solution by interpolation from the standard curve., 3. Calculate the concentration of vitamin in the sample from the average of these values. Use only those values that do not vary more than ±10% from the average. Use the results only if two-thirds of the values do not vary more than ±10%. Limitations of the Procedure: 1. The test organism used for inoculating an assay medium must be cultured and maintained on media recommended for this purpose., 2. Aseptic technique should be used throughout the assay procedure., 3. The use of altered or deficient media may cause mutants having different nutritional requirements that will not give a satisfactory response., 4. For successful results to these procedures, all conditions of the assay must be followed precisely., , Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.