B₁₂ Inoculum Broth

Intended Use

B₁₂ Inoculum Broth is used for preparing the inoculum of L. 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 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

Peptone provides the nitrogen and amino acids in B₁₂ Inoculum Broth. Yeast extract is the vitamin source in the formulas. Tomato juice is added to create the proper acidic environment. Dextrose is the carbon source, and polysorbate 80 acts as an emulsifier. Dipotassium phosphate acts as the buffering agent in B₁₂ Inoculum Broth.

User Quality Control

Identity Specifications
B₁₂ Inoculum Broth
Dehydrated Appearance: Tan, homogeneous, tendency to clump.
Solution:                        3.2% solution, soluble in purified water upon
                                     boiling. Solution is medium to dark amber, opalescent
                                     when hot, clear when cooled to room temperature.
Prepared Appearance:     Medium amber, clear.
Reaction of 3.2%
Solution at 25°C:            pH 6.8 ± 0.1
Cultural Response
B₁₂ Inoculum Broth
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 16-24 hours.

Formula

B₁₂ Inoculum Broth
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
Dipotassium Phosphate................................................. 2.0 g
Polysorbate 80............................................................. 0.1 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:
    Difco™ B12 Inoculum Broth – 32 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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