B₁₂ Assay Medium

Intended Use

B₁₂ Assay Medium is used for determining vitamin B₁₂ concentration by the microbiological assay technique. Meets United States Pharmacopeia (USP) performance specifications.

Summary and Explanation

Vitamin assay media are used in the microbiological assay of vitamins. Three types of media are used for this purpose:
1. Maintenance Media: For carrying the stock culture to preserve the viability and sensitivity of the test organism for its intended purpose;
2. Inoculum Media: To condition the test culture for immediate use;
3. Assay Media: To permit quantitation of the vitamin under test. They contain all the factors necessary for optimal growth of the test organism except the single essential vitamin to be determined.
B₁₂ Assay Medium is used in the microbiological assay of vitamin B₁₂ according to the procedures of the Vitamin B₁₂ Activity Assay in the USP1 and the Cobalamin (Vitamin B₁₂ Activity) Assay in the Official Methods of Analysis of AOAC International (AOAC).₂ Lactobacillus delbrueckii subsp. lactis ATCC™ 7830 (Lactobacillus leichmannii) is the test organism used in this procedure..

Principles of the Procedure

B₁₂ Assay Medium is a vitamin B₁₂-free dehydrated medium containing all other nutrients and vitamins essential for the cultivation of L. delbrueckii subsp. lactis ATCC 7830. To obtain a standard curve, USP Cyanocobalamin Reference is added in specified increasing concentrations giving a growth response that can be measured titrimetrically or turbidimetrically.

User Quality Control

Identity Specifications
B₁₂ Assay Medium
Dehydrated Appearance: Very light to light beige, homogeneous, with
                                    a tendency to clump.
Solution:                       4.25% (single strength) solution, soluble in
                                   purified water upon boiling for 2-3 minutes.
                                   Solution is light amber, clear, may have a slight
                                   precipitate.
Prepared Appearance:    Very light to light amber, clear, may have a
                                   slight precipitate.
Reaction of 4.25%
Solution at 25°C:           pH 6.0 ± 0.1
Cultural Response
B₁₂ Assay Medium
Prepare the medium per label directions. The medium supports the growth of Lactobacillus delbrueckii subsp. lactis ATCC™ 7830 when prepared in single strength and supplemented with cyanocobalamin (vitamin B12). The medium should produce a standard curve when tested with a cyanocabalamin reference standard at 0.0 to 0.25 ng per 10 mL. Incubate tubes with caps loosened at 35-37°C for 16-24 hours. Read the percent transmittance using a spectrophotometer at 530 nm.

Formula

B₁₂ Assay Medium
Approximate Formula* Per Liter
Vitamin Assay Casamino Acids................................... 15.0 g
Dextrose................................................................. 40.0 g
Asparagine................................................................ 0.2 g
Sodium Acetate........................................................ 20.0 g
Ascorbic Acid.............................................................. 4.0 g
L-Cystine................................................................... 0.4 g
DL-Tryptophan............................................................ 0.4 g
Adenine Sulfate...................................................... 20.0 mg
Guanine Hydrochloride............................................. 20.0 mg
Uracil..................................................................... 20.0 mg
Xanthine................................................................ 20.0 mg
Riboflavin................................................................ 1.0 mg
Thiamine Hydrochloride............................................. 1.0 mg
Biotin ..................................................................... 10.0 μg
Niacin....................................................................... 2.0 mg
p-Aminobenzoic Acid.................................................. 2.0 mg
Calcium Pantothenate................................................ 1.0 mg
Pyridoxine Hydrochloride............................................ 4.0 mg
Pyridoxal Hydrochloride.............................................. 4.0 mg
Pyridoxamine Hydrochloride..................................... 800.0 μg
Folic Acid................................................................ 200.0 μg
Monopotassium Phosphate........................................... 1.0 g
Dipotassium Phosphate................................................ 1.0 g
Magnesium Sulfate...................................................... 0.4 g
Sodium Chloride....................................................... 20.0 mg
Ferrous Sulfate......................................................... 20.0 mg
Manganese Sulfate................................................... 20.0 mg
Polysorbate 80............................................................. 2.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Precautions

Great care must be taken to avoid contamination of media or glassware in microbiological assay procedures. Extremely small amounts of foreign material may be sufficient to give erroneous results. Scrupulously clean glassware free from detergents and other chemicals must be used. Glassware must be heated to 250°C for at least 1 hour to burn off any organic residues that might be present. Take precautions to keep sterilization and cooling conditions uniform throughout the assay.

Directions for Preparation from Dehydrated Product

1. Suspend 8.5 g of the powder in 100 mL of purified water.
2. Heat with frequent agitation and boil for 2-3 minutes to completely dissolve the powder.
3. Dispense in 5 mL amounts into tubes, evenly dispersing the precipitate.
4. Add standard or test samples.
5. Adjust the tube volume to 10 mL with purified water. 6. Autoclave at 121˚C for 5 minutes.

Procedure

Follow assay procedures as outlined in USP¹ or AOAC.² Use levels of B₁₂ in the preparation of the standard curve according to these references. It is essential that a standard curve be constructed each time an assay is run. Autoclave and incubation conditions can influence the standard curve reading and cannot always be duplicated. Generally satisfactory results are obtained with B₁₂ at the following levels: 0.0, 0.025, 0.05, 0.075, 0.1, 0.125, 0.15, 0.2 and 0.25 ng per assay tube (10 mL).
Stock cultures of L. delbrueckii subsp. lactis ATCC 7830 are prepared by stab inoculation into 10 mL of B₁₂ Culture Agar or Lactobacilli Agar AOAC. After 16-24 hours incubation at 35-37°C, the cultures are kept refrigerated. The inoculum for assay is prepared by subculturing a stock culture of L. delbrueckii subsp. lactis into 10 mL of B₁₂ Inoculum Broth. For a complete discussion on B₁₂ Culture Agar and B₁₂ Inoculum Broth, refer to USP.¹

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 and 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. For successful results to these procedures, all conditions of the assay must be followed precisely.
3. Aseptic technique should be used throughout the assay procedure.
4. The use of altered or deficient media may cause mutants having different nutritional requirements and will not give a satisfactory response.

*Store at 2-8° C.

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