Plot No. C40, Road No. 21Y, MIDC, Wagle Industrial Estate, Thane(W)-400604, MH, India
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B12 Assay Medium
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$122.10
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SKU:
MM036
Recommended for microbiological assay of vitamin B12 using Lactobacillus leichmannii ATCC 7830 as the test organism in accordance with Indian Pharmacopoeia 2018.
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Intended Use
B12 Assay Medium is recommended for microbiological assay of Vitamin B12 by using Lactobacillus leichmannii ATCC 7830 as the test organism in accordance with Indian pharmacopoeia.
Composition
| Ingredients | Gms / Litre |
|---|---|
| Acicase, vitamin free | 10.000 |
| Dextrose | 40.000 |
| Asparagine | 0.200 |
| Sodium acetate | 20.000 |
| Ascorbic acid | 4.000 |
| L-Cystine | 0.400 |
| L-Tryptophan | 0.200 |
| Adenine sulphate | 0.020 |
| Uracil | 0.020 |
| Xanthine | 0.020 |
| Riboflavin (Vitamin B2) | 0.001 |
| Thiamine hydrochloride | 0.001 |
| Biotin | 0.00001 |
| Niacin | 0.002 |
| p-Amino benzoic acid (PABA) | 0.002 |
| Calcium pantothenate | 0.001 |
| Pyridoxine hydrochloride | 0.004 |
| Pyridoxal hydrochloride | 0.004 |
| Pyridoxamine dihydrochloride | 0.0008 |
| Folic acid | 0.0002 |
| Potassium dihyrogen phosphate | 1.000 |
| Dipotassium hydrogen phosphate | 1.000 |
| Magnesium sulphate | 0.400 |
| Sodium chloride | 0.020 |
| Ferrous sulphate | 0.020 |
| Manganese sulphate | 0.020 |
| Polysorbate 80 | 2.000 |
| Guanine hydrochloride | 0.020 |
| Final pH (at 25°C) | 5.50-6.00 |
Formula adjusted, standardized to suit performance parameters
Directions
Suspend 7.94 grams in 100 ml distilled water. Heat if necessary to dissolve the medium completely. Mix well to distribute the slight precipitate evenly. For the assay, dispense 5 ml medium to each assay tube (containing increasing amounts of standard or the unknown). Total volume of 10 ml per tube is adjusted by addition of distilled water. Sterilize by autoclaving at 15 lbs pressure (121°C) for 5 minutes. Cool the medium immediately. Generally satisfactory results are obtained with Vitamin B12 (Cyanocobalamin) at levels 0, 0.025, 0.05, 0.075, 0.1, 0.125, 0.15, 0.2 ng per assay tube (10 ml).
Principle And Interpretation
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.
Vitamin B12 Assay Medium is a Vitamin B12 free medium containing all other vitamins and nutrients essential for the growth of Lactobacillus leichmannii ATCC 7830. It was first described by Capp et al (1) and is recommended by USP (2), AOAC (3) and IP (4) using Lactobacillus leichmannii ATCC 7830 as the test organism.
Standard curve is constructed with known dilutions of vitamin B12 standards (2, 3,4).
Inoculum for the assay is prepared by subculturing 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
Quality Control
Appearance: Cream to yellow homogeneous having a tendency to form soft lumps which can be easily broken down to powder form.
Colour and Clarity of prepared medium: Light amber coloured clear solution that may contain a slight precipitate.
Reaction: Reaction of 7.9% w/v aqueous solution at 25°C. pH : 5.50-6.00
pH: 5.50 -6.00
Growth: Gradual increase in growth with increasing Cyanocobalamin reference standard levels of 0.0, 0.025, 0.050, 0.075, 0.1, 0.125, 0.150 and 0.2 ng per assay tube is recorded as equivalent increase in absorbance at 620nm.
Cultural Response: MM036: Microbiological assay of Vitamin B12 is carried out using Lactobacillus leichmannii ATCC 7830 after an incubation at 35-37°C for 18-24 hours.
Storage and Shelf Life
Store below 8°C in tightly closed container, preferably in dessicators and use freshly prepared medium. Use before expiry date on the label.
| Product Name | B12 Assay Medium |
|---|---|
| SKU | MM036 |
| Product Type | Regular |
| Physical Form | Powder |
| Origin | Animal |
| Packaging type | Glass Bottle |
| References | 1. Capps B. E., Hobbs M. H. H. and Fox S. H., 1949, J. Biol. Chem., 178:517. 2.The United States Pharmacopoeia, 2006, USP29/NF24, The United States Pharmacopeial Convention, Rockville, MD. 3.H. Williams, (Ed.), 2005, Official Methods of Analysis of the Association of Official Analytical Chemists, 19th Ed., AOAC,Washington, D.C 4.Indian Pharmacopoeia, 2018, Ministry of Health and Family Welfare, Govt. of India |
| Customized Product Available | No |
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