A Microbiological Assay of Penicillin in Animal Feeds

R. C. Kersey, F. V. Leghorn
1953 Applied microbiology  
The universal acceptance of the animal growth-promoting properties of some antibiotics and their wide usage in formulated commercial feeds requires that simple, rapid and reliable analytical methods be available for control purposes. Of the several antibiotics used in poultry rations, penicillin exerts its growth-promoting properties at levels considerably lower than other antibiotics (2 grams penicillin per ton), and thus, the analytical problems are accentuated. For penicillincontaining
more » ... lincontaining feeds, two problems are presented (a) a method of preparing stable extract from feed samples and (b) a method of measuring quantitatively the small amount of penicillin in the extract. From the voluminous information available on the properties of penicillin, it is well known that the antibiotic is incompatible with many solvents. In this report, only two salts of penicillin are considered, procaine and dibenzyl ethylene diamine (DBED), since these are the only forms currently employed in commercial feeds. EXPERIMENTAL Assay. Micrococcus pyogenes var. aureus ATCC 9144 was employed as the test organism in a disc-plate assay and maintained on nutrient agar described in table 1. Daily inoculum was prepared by transferring the organism to broth of the same composition and incubating at 37 C. for 18 hours. To increase the sensitivity of the test organism to as little as 0.05 units of penicillin per ml, a single layer of assay medium was used in the plate. Fifty ml of assay agar, described in table 1 was melted and cooled to 45 to 48 C. and seeded with 0.5 ml of the above described inoculum. It is extremely important that the plate be prepared on a level surface as variations in the thickness of the agar affect the sensitivity of the test and thus reproducibility of the zone sizes. An agar depth of 1.3 mm has been found most satisfactory. Flat pyrex glass plates, 12" x 62a" with 34" pyrex strips cemented to sides and ends of plate were used in place of the conventional Petri dishes. Filter paper discs 2" diameter (No. 740E, Schleicher and Schuell, Keene, N. H.) served as reservoirs for the antibiotic. Zones of inhibition were measured by vernier calipers, millimeter rule or by projection of the zones against a calibrated screen. Extraction. Twenty-gram samples of feed containing penicillin were mixed with 150 ml of the extracting agent and allowed to stand one hour with frequent agitation. The mixture was diluted with 1 per cent phosphate buffer, pH 6.6, to contain approximately 0.075 units/ml. For feeds containing 2 grams penicillin/ ton, it was convenient to conduct the extraction and subsequent dilutions in liter volumetric flasks. A 1 unit/ml stock solution of penicillin G sodium was prepared in phosphate buffer, pH 6.6. The standard curve was prepared by further diluting this standard so that solutions containing 0.05, 0.06, 0.07, 0.09, 0.10 and 0.12 units/ml were obtained. Filter paper discs were saturated with the standard solution series, the excess solution was removed from the disc by bringing it in contact withthe lip of the test tube containing the diluted standards. One disc of each dilution of standard was placed on the surface of the agar plate. Six replicate discs were saturated with the formamide-buffer feed extract and carefully placed on either side of the standard discs. Two samples were assayed per plate. A flat pyrex glass cover was placed on the plate and it was then incubated for 18 to 24 hours at 37 C. A typical assay plate is pictured in figure 1 . The inhibition zones of the standard discs were measured and a dose response curve plotted on two cycle semi-log paper with the dose as the ordinate, while the zone diameter in millimeters was plotted as the abscissa. The six readings obtained from the sample discs were averaged and the potency calculated from the standard curve. This value times the dilution, divided by the weight of the sample gave the potency of the sample on a weight basis. RESULTS Various solvents were compared for extraction efficiency, compatibility with penicillin, and their ability to reject non-specific substances influencing the growth of the test organism. Water, phosphate buffer pH 6.6, methanol, dimethyl formamide and formamide (99 per cent purity) were investigated as extracting agents for procaine and DBED from feeds. Incomplete extractions or other interferences were encountered with all solvent systems except formamide. Extractions with formamide were complete. The use of methanol was suggested in a preliminary report of Esposito and Williams (1952a) for extracting penicillin from feeds. However, in view of several reports of the instability of penicillin in the presence of primary alcohols (Chain, 1949; Clark, et al, 1949) some of these experiments were repeated. Table 2 150 on March 22, 2020 by guest http://aem.asm.org/ Downloaded from
doi:10.1128/aem.1.3.150-152.1953 fatcat:jkkk367dbrffveskzkqvo4grta