B12 Culture Agar (L. leichmannii Maintenance Medium), 500 g

B12 Culture Agar recommended by USP for cultivation and maintenance of Lactobacillus leichmannii ATCC 7830 ( Lactobacillus delbrueckii subsp.lactis ATCC 7830) which is used as a test bacterium during the microbiological estimationof vitamin B12 (1). Lactobacillus species have very exacting nutritional requirements for amino acids and vitamins. This restricts them to nutritionally compete in the environment. Lactobacillus species grow poorly on non-selective media. Kulp (2) found that the growth of Lactobacillus acidophilus was enhanced with tomato juice, while investigating the use of tomato juice on bacterial development, which was reported earlier by Mickle and Breed (3) for the microbiological assay of vitamins. Peptic digest of animal tissue serves as a source of nitrogen and amino acids. Yeast extract is the vitamin source. Tomato juice is added to create the proper acidic environment. Dextrose is the carbon source and Polysorbate 80 acts as an emulsifier. Monopotassium phosphate provides buffering capacity. Stock cultures of Lactobacillus leichmannii ATCC 7830 are prepared by stab inoculation of 3 or more tubes. These stab cultures are made at least 3 times in a week. Do not use the culture for preparing assay inoculum if it is over 4 days old. Before using a fresh culture for assay, make at least 10 successive transfers of the culture in 15 days period. Incubate the culture for 16-24 hours at 35°C but hold constant within 0.5°C. After incubation, store at 2-8°C. Storage and Shelf-life: Store below 30°C in tightly closed container and the prepared medium at 2 - 8°C. Use before expiry date on the label. References: 1. The United States Pharmacopoeia, 2006, USP 29/ NF 24, The United States Pharmacopoeial Covention, Rockville, MD. 2. Kulp and White, 1932, Science 76:17. 3. Mickle and Breed, 1925, Technical Bulletin 110, NY State Agriculture Ex. station, Geneva, N.Y.