B12 ASSAY MEDIUM (using L.leichmannii)

Lactobacillus species grow poorly on non-selective culture media and require special nutrients for their growth. Vitamin assay media are prepared for use in the microbiological assay of vitamins. Three types of media used for the microbiological assay of vitamins are the maintenance media used for carrying the stock culture, the inoculum media for preparation of the inoculum and the assay media for quantitation of the vitamin under test. It was first described by Capp et al and is recommended by USP and AOAC, using Lactobacillus leichmannii ATCC 7830 as the test organism.
Standard curve is constructed with known dilutions of vitamin B12 standards. Inoculum for the assay is prepared by sub-culturing from a stock culture previously made by stab inoculation. Freshly subcultured organisms incubated at 37?C for 24 hours, centrifuged, washed and suspended in 10 ml saline are recommended for the assay. The growth response obtained is turbidometrically or acidimetrically measured. A standard curve is plotted with absorbance as a function of the vitamin B12 concentration. The concentration of vitamin B12 in the test sample is calculated based on the interpretation of the standard curve. 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 may lead to erroneous results. The test organism used for inoculating must be cultured and maintained on media recommended for this purpose.

for microbiological assay of Vitamin B12 by using Lactobacillus leichmannii