Difco™ Antibiotic Medium 19
Approximate Formula* Per Liter
Beef Extract : 2.4 g
Yeast Extract : 4.7 g
Peptone : 9.4 g
Dextrose : 10.0 g
Sodium Chloride : 10.0 g
Agar : 23.5 g
*Adjusted and/or supplemented as required to meet performance criteria.
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Selection of Media "Difco™ Antibiotic Medium 19" for the Microbiological Assay of Antibiotics:
ANTIBIOTIC / ASSAY METHOD / ORGANISM / ATCC
Amphotericin B / Cylinder Plate / Saccharomyces cerevisiae / 9763
Candicidin / Turbidimetric / Saccharomyces cerevisiae / 9763
Nystatin / Cylinder Plate / Saccharomyces cerevisiae / 2601
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Directions for Preparation from Dehydrated Product
1. Suspend the powder in 1 L of purified water: Difco™ Antibiotic Medium 19 – 60 g.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. To raise the pH of Antibiotic Medium 11 to 8.3 ± 0.1, cool the base to 45-50°C and add NaOH.
5. Test samples of the finished product for performance using stable, typical control cultures.
Cylinder Plate Assay
Use 20 × 100 mm glass or plastic Petri dishes with sufficient depth so that cylinders used in the assay will not be pushed into the medium by the cover.
Use stainless steel or porcelain assay cylinders having the following dimensions (± 0.1 mm): 8 mm outside diameter, 6 mm inside diameter and 10 mm long. Carefully clean the cylinders to remove all residues, using an occasional acid bath (i.e., with approximately 2N nitric acid or with chromic acid). Four or six cylinders are generally used per plate, evenly spaced on a 2.8 cm radius.
To assure accurate assays, work on a level surface to obtain uniformly thick base and seed layers in the Petri dish. Allow the base layer to solidify and then overlay the seed layer containing a proper concentration of the test organism. The amount of medium in the layers varies for different antibiotics, with most assays specifying a 21 mL base layer and a 4 mL seed
layer. In any case, dishes with flat bottoms are required to assure complete coverage of the bottom of the dish when small amounts of base medium are used. Tilt the plate to obtain even coverage of the base layer by the seed layer and allow it to solidify in a level position. Plates should be used the same day as prepared.
Turbidimetric Assay
Use glass or plastic test tubes (i.e., 16 × 125 mm or 18 × 150 mm) that are relatively uniform in length, diameter and thickness and substantially free from surface blemishes. Tubes that will be placed in the spectrophotometer should be matched and free of scratches or blemishes.2 Clean the tubes thoroughly
to remove all antibiotic residues and traces of cleaning solution and, prior to subsequent use, sterilize tubes that have been previously used. Prepare working dilutions of the antibiotic reference standards in specific concentrations. To a 1 mL quantity of each solution in a suitable tube, add 9 mL of inoculated broth, as required. Prepare similar solutions of the assay materials containing approximately the same amounts of antibiotic activity and place in tubes. Incubate the tubes for 3-4 hours at the required temperature, generally in a water bath. At the end of the incubation period, stop growth by adding 0.5 mL of 1:3 formalin. Determine the amount of growth by measuring light transmittance with a suitable spectrophotometer. Determine the concentration of the antibiotic by comparing the growth btained with that given by reference standard solutions.
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