Plot No. C40, Road No. 21Y, MIDC, Wagle Industrial Estate,
Thane(W)-400604, MH, India.
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Intended Use:
Recommended for microbiological assay of Monensin using Bacillus subtilis as test organism.
Composition**
| Ingredients | Gms / Litre |
|---|---|
| Dipotassium hydrogen phosphate | 0.690 |
| Potassium dihydrogen phosphate | 0.450 |
| Yeast extract | 2.500 |
| Dextrose (Glucose) | 10.000 |
| Agar | 15.000 |
| Final pH (at 25°C) | 6.0±0.2 |
**Formula adjusted, standardized to suit performance parameters
Directions
Suspend 28.64 grams in 1000 ml purified / distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Mix well and pour into sterile Petri plates.
Principle And Interpretation
Antibiotic Assay Medium L is formulated in accordance with AOAC (3) for the microbiological assay of Monensin in feeds, using Bacillus subtilis (ATCC 6633) as the test organism.
Use single inoculated agar layer. Optimum concentration of suspension of Bacillus subtilis, is determined before assay by preparing trial plates. Usually 0.5 ml suspension is used per 100 ml of seed agar, to obtain appropriate inhibition zones (17.5± 2.5 mm with 0.5µg/ml). For actual assay add appropriate amount of suspension to sterile, molten medium, mix and pour 6 ml into sterile Petri plate. Cover and refrigerate for about 1 hour before use.
For the standard graph or response lines prepare dilution using 50% methanol to obtain 0.25, 0.5, 1.0 and 2.0 µg monensin/ ml. Reference concentration is 0.5 µg/ml. To obtain standard curve 10 seeded agar plates are used placed with cylinders. Different standard concentrations are filled in it. Incubate at 16-18 hours at 35-37°C and measure diameters of zones of inhibition. Weigh 20gram finished feed and 5 gram premix and add it to chromatographic column. Elute with 9:1 methanol water. 200 ml elute is again diluted with 50% methanol to 0.5µg monensin /ml. This is called assay solution. Use 5 plates for each assay solution. Fill the alternate cylinders with reference concentration and assay solution after incubation at 35-37°C for 16-18 hours, measure diameters of zones of inhibition to nearest 0.1 mm. Average 10 reading of reference concentration and 10 reading of reference concentration and 10 readings of assay solution.
If assay solution gives larger average than reference concentration add difference between them to reference point on standard curve. If assay solution gives smaller value than reference concentration, substract difference between them from reference point. Using corrected value of assay solution determine amount of antibiotic.
Type of specimen
Pharmaceutical sample
Specimen Collection and Handling
For pharmaceutical sample samples follow appropriate techniques for handling specimens as per established guidelines (3). After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations :
- Freshly prepared medium plates must be used or it may result in erroneous results.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.
Quality Control
Appearance
Cream to yellow homogeneous free flowing powder
Gelling
Firm, comparable with 1.5% Agar gel
Colour and Clarity of prepared medium
Yellow coloured clear to slightly opalescent gel forms in Petri plates.
Reaction
Reaction of 2.86% w/v aqueous solution at 25°C. pH: 6.0±0.2
pH
5.80-6.20
Cultural Response
Cultural characteristics observed after an incubation at 35 - 37°C for 16 - 18 hours.
| Organism | Inoculum (CFU) | Growth | Recovery | Anibiotic assayed |
|---|---|---|---|---|
| Bacillus subtilis subsp. spizizenii ATCC 6633 (00003*) | 50-100 | luxuriant | >=70% | Inhibition zones with Monensin |
Key: (*) Corresponding WDCM numbers.
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and use freshly prepared medium. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques (1,2).
Reference
- Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
- Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual Clinical Microbiology, 11th Edition. Vol. 1.
- Williams. (Ed.), 2005, Official Methods of Analysis of AOAC International, 19th ed., AOAC, International, Washington D. C.
| Product Name | Antibiotic Assay Medium L- AOAC |
|---|---|
| SKU | M991 |
| Product Type | Regular |
| Physical Form | Powder |
| Origin | Animal Free (Microbial) |
| Packaging type | HDPE |
| References | 1. Williams. (Ed.), 2005, Official Methods of Analysis of AOAC International, 19th ed., AOAC, International, WashingtonD. C. |
| Customized Product Available | No |
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