Folic Acid Casei Medium

Intended Use

Recommended for the microbiological assay of folic acid in blood serum using Lactobacillus casei ATCC 7469 as the test organism.

Composition

Final pH (at 25°C): 6.7±0.1

**Formula adjusted, standardized to suit performance parameters
# Equivalent to Vitamin free casein acid hydrolysate

Directions

Suspend 9.4 grams in 100 ml purified/distilled water containing 50 mg ascorbic acid. Heat to boiling to dissolve the medium completely. For assay dispense in 5 ml amount of medium per assay tube. After adding standard and test solution adjust volume in test tube to 10 ml with distilled water. Sterilize by autoclaving at 15 lbs pressure (121°C) for 5 minutes. Standard curve is obtained using Folic acid at levels of 0,0.1,0.2,0.4,0.6,0.8 and 1 ng per assay tube (10 ml).

Principle And Interpretation

Normal persons have a normal serum folic acid level of 9.9mg/ml. This level is greatly altered in case of abnormalities and also when the body is under a diseased state. Folic Acid Casei Medium is used for the microbiological assay of folic acid in blood serum using Lactobacillus rhamnosus ATCC 7469 as the test organism. This medium is based on the formulation of Flynn et al (1), modified by Baker et al (2) and Waters and Mollin (3). The test organism used in vitamin assay generally requires three media, i.e. a culture maintenance medium, a inoculation medium and a test medium. The latter is usually a chemically defined medium that contains all the ingredients and nutrients essential for growth of the test organisms except the material under study. Similarly Folic Acid Casei Medium contains all the essential nutrients for the growth of L.rhamnosus casei except folic acid. Therefore additions of folic acid in specified increasing concentrations gives a similar increase in the growth response of L.rhamnosus.

Technique

Stock cultures of Lactobacillus rhamnosus ATCC 7469 are prepared by stab inoculation of Lactobacilli Agar AOAC (M366). Following incubation at 35-37°C for 18-24 hours the tubes are stored in a refrigerator. Transfers are made at monthly intervals. Inoculum for assay is prepared by subculturing from stock culture of Lactobacillus rhamnosus ATCC 7469 into a tube containing 10 ml of Micro Vitamin Test Inoculum Broth (M133) or Lactobacilli Broth (M367). After 24 hours incubation at 35-37°C, the cells are centrifuged, under aseptic conditions, and the supernatant liquid is decanted. The cells are then resuspended in 10 ml of sterile single strength Folic Acid Casei Medium, resedimented as before and washed one more time. Finally, the washed cells are resuspended in 10 ml of sterile single strength Folic Acid Casei Medium (M543) and diluted 1:100 with the same medium. One drop of this suspension is used to inoculate each of the assay tubes. 0.85% NaCl can be used instead of the single strength basal medium to wash and dilute the inoculum. A standard curve for each assay should be prepared, since the conditions of sterilization, 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 0.0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 ng per assay tube (10 ml).

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 0.1N NaOH to dissolve the acid and then adjust pH to 7.0 with 0.05N HCl. This solution contains 200 mcg folic acid per ml. Dilute 1 ml of this solution with 999 ml of distilled water to get 200ng per ml and finally, dilute 1 ml of this solution with 999 ml of Folic Acid Buffer A (M544) 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 ml per assay tube.

Preservation of Serum Specimen

Allow the blood specimen to clot so as to separate the serum. Separate the serum into a clean dry tube and centrifuge to remove any blood cells present. Take care to avoid haemolysis of erythrocytes. Dispense 5 ml of serum sample into clean dry test tubes and add 25 mg ascorbic acid to each tube. Keep the tubes frozen below -20°C till assay.

Preparation of Serum Specimens

Thaw the serum containing ascorbic acid. Add 5 ml of this sample to 45 ml of rehydrated Folic Acid Buffer A (M544). Incubate this serum - buffer solution at 37°C for 90 minutes and then autoclave at 15 lbs pressure (121°C) for 2.5 minutes. Remove the coagulated protein by centrifuging and transfer the supernatant to a clean, dry tube. This clear solution obtained is used as a sample in the folic acid assay.

Procedure for determination of Total Folic Acid concentration in specimens

Use 0.5, 1.0, 1.5 ml or other volumes of the prepared serum extracts as described earlier. Fill each tube with 5 ml Folic Acid Casei Medium and sufficient distilled water to give total volume of 10 ml per tube. Sterilize tubes at 15 lbs pressure (121°C) for 5 minutes. Cool the tubes and add one drop of inoculum to each assay tube. Turbidimetric reading should be made after 18-24 hours incubation at 35-37°C. Tubes are refrigerated for 15-30 minutes to stop growth before reading. The turbidometric readings are recorded at 620 nm. The amount of folic acid in the test samples can be determined by interpreting the results with the values obtained on the standard curve taking into consideration the dilution of sample. 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 erroroneus results.

Type of specimen

Clinical samples - Serum

Specimen Collection and Handling

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (4,5). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic Use only. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. Further biochemical tests should be carried out for confirmation.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance: Off-white to yellow homogeneous free flowing powder

Colour and Clarity of prepared medium: Light amber coloured, clear solution which may be a slight precipitate.

Reaction: Reaction of 9.4% w/v aqueous solution at 25°C. pH: 6.7±0.1

pH: 6.60-6.80

Cultural Response: Microbiological assay of folic acid is carried out using Lactobacillus rhamnosus ATCC 7469. After an incubation at 35-37°C for 16-18 hours.

Growth: Good growth is obtained. Gradual increase in growth with increasing concentration of standard folic acid 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1ng per assay tube was recorded as equivalent increase in absorbance at 620 nm.

Storage and Shelf Life

Store between 2-8°C in a tightly closed container. Use freshly prepared medium. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (4,5).

Reference

1. Flynn, Williams, ODell and Hogan, 1951, Anal. Chem., 23, 180.
2. Baker, Herbert, Frank, Pasher, Hunter, Wasserman and Sobotka, 1959, Clin. Chem., 5, 275.
3. Waters and Mollin, 1961, J. Clin. Path., 14, 335.
4. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
5. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.