§ 436.512 - Procaine penicillin G-novobiocin-neomycin-dihydrostreptomycin in oil.

(a) Potency—(1) Penicillin G content. Proceed as directed in § 440.180d (b)(1)(i)(a) of this chapter, using the novobiocin-resistant strain of Staphylococcus aureus (ATCC 12692),1 except prepare the sample as follows: Place the equivalent of one dose of sample in a blending jar, add 1.0 milliliter of polysorbate 80 and a quantity of 1 percent potassium phosphate buffer, pH 6.0, sufficient to make a total of 500 milliliters. Blend for 5 minutes with a high‐speed blender and make appropriate dilutions, using 1 percent potassium phosphate buffer, pH 6.0. Its content of penicillin G is satisfactory if it contains not less than 85 percent of the number of units that it is represented to contain.

(2) Novobiocin content. Proceed as directed in § 440.180d(b)(3)(i), with the following exceptions:

(i) Prepare the sample as follows: Place the equivalent of one dose of sample in a blending jar, add 1.0 milliliter of polysorbate 80 and a quantity of 0.1M potassium phosphate buffer, pH 8.0, sufficient to make a total of 500 milliliters. Blend for 5 minutes with a high-speed blender. To an aliquot, add sufficient penicillinase to inactivate the penicillin, further dilute with 10 percent potassium phosphate buffer, pH 6.0 (solution 6) to give a final concentration of 0.5 microgram novobiocin per milliliter (estimated), and allow to stand for 1/2-hour at 37° C. before filling the plates.

(ii) Aseptically add to the seed agar used for this assay, at the time the bacterial suspension is added, a slurry of Dowex 50 WX-4, Na+ type 200-400 mesh, sufficient to make a total concentration of 2 percent. Prepare the slurry by adding 50 grams of the resin to 30 milliliters of distilled water and sterilize for 15 minutes at 15 pounds pressure. Mix the slurry thoroughly before adding. Its content of novobiocin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(3) Neomycin content. Proceed as directed in § 436.517(b)(1) of this chapter, using the Staphylococcus epidermidis (ATCC 12228) 1 procedure, except:

(i) Prepare the sample as follows: Place the equivalent of one dose of sample in a blending jar, add 1.0 milliliter of polysorbate 80 and a quantity of 0.1M potassium phosphate buffer, pH 8.0, sufficient to make a total of 500 milliliters. Blend for 5 minutes with a high-speed blender. To an aliquot, add sufficient penicillinase to inactivate the penicillin, further dilute with 0.1M potassium phosphate buffer, pH 8.0, to give a final concentration of 1.0 microgram neomycin per milliliter (estimated), and allow to stand for 1/2-hour at 37° C. before filling the plates.

(ii) Aseptically add to the seed agar used for this assay, at the time the bacterial suspension is added, a slurry of Dowex 1-X8, Cl type 200-400 mesh, to make a total concentration of 1 percent. Prepare the slurry by adding 50 grams of the resin to 30 milliliters of distilled water and sterilize for 15 minutes at 15 pounds pressure. Mix the slurry thoroughly before adding. Its content of neomycin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(4) Dihydrostreptomycin content. Proceed as directed in § 436.105 except prepare the sample by placing the equivalent of one dose in a blender, add 1.0 milliliter of polysorbate 80 and a quantity of 0.1M potassium phosphate buffer, pH 8.0, sufficient to make a total of 500 milliliters. Blend for 5 minutes with a high-speed blender. To an aliquot, add sufficient penicillinase to inactivate the penicillin, further dilute with 0.1M potassium phosphate buffer, pH 8.0, to give a final concentration of 1.0 microgram dihydrostreptomycin per milliliter (estimated), and allow to stand for 1/2-hour at 37° C. before filling the plates. Its content of dihydrostreptomycin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(b) Moisture. Proceed as directed in § 436.500(c).