B₁₂ Culture Agar

Intended Use

B₁₂ Culture Agar is used for cultivating Lactobacillus delbrueckii subsp. lactis ATCC™ 7830 used in the Vitamin B₁₂ Activity Assay.
These media meet United States Pharmacopeia (USP) performance specifications.

Summary and Explanation

Vitamin assay media are prepared for use 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.
Lactobacillus species grow poorly on nonselective culture media and require special nutrients. Mickle and Breed² reported the use of tomato juice in culture media for lactobacilli. Kulp,³ while investigating the use of tomato juice on bacterial development, found that growth of Lactobacillus acidophilus was enhanced.
B₁₂ Culture Agar is recommended for maintaining stock cultures of L. delbrueckii subsp. lactis ATCC 7830 (Lactobacillus leichmannii) for use in the Vitamin B₁₂ Activity Assay according to the USP.¹
B₁₂ Inoculum Broth is used for preparing the inoculum of L. delbrueckii subsp. lactis ATCC 7830 in the microbiological assay of vitamin B12 according to the USP.¹

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 Culture Agar
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution:                       4.7% solution, soluble in purified water upon
                                    boiling. Solution is light to medium amber,
                                    opalescent when hot, slightly opalescent with
                                    flocculent precipitate when cooled.
Prepared Appearance:     Light to medium amber, slightly opalescent, may
                                    have a slight flocculent precipitate.
Reaction of 4.7%
Solution at 25°C:            pH 6.8 ± 0.1
Cultural Response
B₁₂ Culture Agar 
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 16-24 hours.

Formula

B₁₂ Culture Agar
Approximate Formula* Per Liter
Tomato Juice (from 100 mL)......................................... 5.0 g
Proteose Peptone No. 3................................................ 7.5 g
Yeast Extract................................................................ 7.5 g
Dextrose.................................................................... 10.0 g
Monopotassium Phosphate.......................................... 2.0 g
Polysorbate 80............................................................. 1.0 g
Agar.......................................................................... 14.0 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.

Directions for Preparation from Dehydrated Product

1. Suspend the powder in 1 L of purified water: B₁₂ Culture Agar – 47 g; Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Dispense 10 mL amounts into tubes.
4. Autoclave at 121°C for 15 minutes.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

For a complete discussion of vitamin assay methodology, refer to appropriate procedures outlined in the USP.¹

Expected Results

For test results of vitamin assay procedures refer to the USP.¹

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 of 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 that will not give a satisfactory response.

*Store at 2-8° C. 

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