Product Code TM 2086
Folic Acid Assay Medium is prepared according to the formula described by Capps et al and is recommended for the determination of folic acid content of the pharmaceutical products. Folic acid is required for the growth of Enterococcus hirae. Hence growth of this organism will occur only if the sample being assayed contains folic acid. The exact folic acid concentration in the test sample can be determined by comparing the growth obtained to that of known standard concentrations of folic acid (standard curve).
Stock cultures of Enterococcus hirae ATCC 8043 are prepared by stab inoculation of Micro Vitamin Test Culture Agar. Following incubation at 35-37?C for 24 hours, the tubes are stored in the refrigerator. Transplants are made at monthly intervals. Inoculum for assay is prepared by sub-culturing from a stock culture of Enterococcus hirae ATCC 8043 into a tube containing 10 ml of Micro Vitamin Test Inoculum Broth. After 24 hours incubation at 35-37?C, the cells are centrifuged under aseptic conditions, and the supernatant liquid is decanted. The cells are resuspended in 10 ml of sterile 0.85%NaCl. The cell suspension is then diluted 1:100 with sterile 0.85% NaCl. One drop of this later suspension is 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 folic acid at levels of 0, 2, 4, 6, 8 and 10 ng per assay tube (10 ml). Tubes are refrigerated for 15-30 minutes to stop growth before reading. Turbidimetric readings should be read after 16-18 hours incubation at 35-37?C and acidimetric after 72 hours at 35-37?C. To prepare stock solution of folic acid, 20 mg folic acid is used.
Preparation of Folic Acid Concentrations:
Dissolve 20 mg dried folic acid in 100 ml distilled water containing 20 ml ethanol. Adjust the pH of the solution to 10.0 with 0.1 N NaOH to dissolve the acid and then adjust pH to 7.0 with 0.05 N HCl. This solution contains 200 mcg folic acid per ml. Dilute 1 ml of this solution with 999 ml of distilled water to get 200 ng per ml and finally, dilute 1 ml of this solution with 999 ml of Folic Acid Buffer A to get a standard solution containing 0.2 ng folic acid per ml. use 0.0, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 ml per assay tube. Extreme care should be taken to avoid contamination of media or glassware used for the assay. Detergent free clean glassware should be used. Even small amount of contamination by foreign material can be lead to erroneous results.
for microbiological assay of Folic Acid using Enterococcus hirae ATCC 8043 as the test organism
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