Assay of Folic Acid or Vitamin B9 (By Microbiological Method) : Pharmaguideline

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Assay of Folic Acid or Vitamin B9 (By Microbiological Method)

Learn how to determine the assay of Folic Acid or Vitamin B9 with Streptococcus faecalis (ATCC 8043).

Test organism:

Streptococcus faecalis (ATCC 8043).

Stock culture:

The stock culture of the test organism is prepared by stab inoculation in the folic acid culture agar. After 20 to 24 hours incubation at 35-37° C, the tubes are stored in the refrigerator. Transfers are made daily.

Culture medium:

Folic acid inoculum broth is used. The medium is rehydrated using distilled water and distributed in 10 ml quantities in tubes and autoclaved for 15 min. at 121° C.

Inoculum for assay:

This is prepared by sub-culturing from a stock culture to 10 ml of culture medium. Incubate the culture inoculum for 16- 24 hours at 37° C. Under aseptic conditions, transfer the culture to a centrifuge tube, centrifuge and decant the supernatant. Suspend the cells in 5 ml of sterile saline, mix well, centrifuge and decant the supernatant. Repeat the process twice. Finally, suspend the cells in 5 ml of sterile saline and mix well. *From that add 2-3 drops to remaining saline solution. This suspension is used for inoculating the assay tubes. one drop of inoculum being used to inoculate one assay tube. (*Addition from the inoculum suspension will depend upon culture activity that should be validated by the Microbiologist.)

Assay medium:

Folic acid assays medium is used. The medium is rehydrated using distilled water and warmed in a water bath for dissolution.

Standard preparation:

Stock solution:

Weigh accurately a quantity of working standard equivalent to 50 mg. of folic acid activity and transfer to a 500 ml of volumetric flask. To it add 30 ml of 0.01M NaOH. Allow it to dissolve. Make up the volume to 500 ml with distilled water. 1 ml of the stock solution = 100 mcg. of Folic acid.

Working standard solution:

Prepare fresh for each assay. Pipette 5 ml of the stock solution and make up the volume to 500 ml with distilled water. Further dilute 2 ml of this solution to 1000 ml with distilled water. Final concentration: 1 ml of this solution = 2 mg./ml. of Folic acid.

Preparation of sample:

Weigh accurately a tablet powder containing approx. 1 mg. of folic acid activity. Transfer to a 1000 ml of volumetric flask with the aid of 100 ml of distilled water. Add 30 ml of 0.01 M NaOH. Allow it to dissolve for half an hour by keeping in incubator at 35°C-38°C for 15 –20 minutes. [Shake the sample solution intermittently during incubation]. Cool and make up the volume. Allow to settle. Dilute the aliquot of the supernatant with distilled water to get final concentration of folic acid activity as per standard, i.e. 2mg/ml.


Preparation of Folic Acid Tubes - To duplicate 18 x 150 mm test tubes, add 0.0, 0.5, 1.0, 2, 3.0, 4.0 ml respectively of the standard Folic Acid solution (2 ng/ml). To each of these tubes add distilled water to bring the volume to 5 ml. Finally, add 5 ml of assay medium to each tube.

Preparation of Sample Tubes:

To duplicate tubes, add 1.0, 2.0, 3.0 ml of sample solution (diluted to a concentration that approximates that of standard solution). To each of these tubes add distilled water to bring the volume to 5 ml. Finally add 5 ml of assay medium to each tube Plug the tubes with non-absorbent cotton or cover them with metal caps. Autoclave for 10 minutes at 121°C. Cool as rapidly as possible. Take precautions to keep sterilization and cooling conditions uniform throughout the assay. After cooling the tubes to room temperature, aseptically, add 1 drop of inoculum to each tube except blank. Shake well the whole rack. Incubate 18 - 24 hours at 37°C. After the incubation period termination should be done at 121°C and determine the growth response turbid metrically. Measure turbidity of the tubes on UV-Spectrophotometer at 650 nm with uninoculated blank medium. Mix the contents of each tube well prior to reading. From the standard turbidity readings, plot a dose response curve on linear graph paper & estimate the potency of the sample by interpolation.

                                  Avg. Potency x (Dill. Factor) x Std Conc.
  mg of folic acid  =  ---------------------------------------------------
                                                100  x  1000
                                     Avg. Potency x 1000 x 500 x 2
                            =  ------------------------------------------------
                                    100  x Actual wt. taken x 1 x 1000

              mg of folic acid per Tabs. mg of folic acid x Avg. wt of tablet

                           Actual mg of folic Acid per Tablet.
  % of L.A.  =  ---------------------------------------------   X100

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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