Highly Sensitive Paper-Disc Assays for Detecting Penicillin in Milk

A modified Bacillus subtilis disc-plate assay could detect 0.015 I.U. of penicillin G per ml of milk, whereas the lower detection limit of the standard assay was 0.050 I.U. per ml. Likewise, a modified Bacillus stearothermophilus disc-plate assay could detect 0.003 I.U. of penicillin G per ml of milk, whereas the lower detection limit of the standard assay was 0.005 I.U. per ml. Increased sensitivities were accomplished by preloading assay discs with "critical" concentrations so that minute quantities of antibiotic above the "critical" concentrations would produce zones of inhibition. Only a few alterations in routine laboratory procedure were required to perform the assays. Use of these assays should assure a milk supply that would not cause allergic reactions in humans or significantly inhibit dairy starter cultures. The general principal of "critical" concentration might have applications other than those that we have described, to increase the sensitivity of radial-diffusion analyses for biologically active compounds.