Practical Three-Hour and Two-Hour Cylinder-Plate Assays for Penicillin

Abstract

IN the course of experiments designed to study cytochemical changes which occur in susceptible cells under the influence of penicillin, we applied various stains and reagents to agar plates prepared as for the standard F D A cylinder-plate assay, which is essentially similar to that recommended by Schmidt and Mover¹. The technical procedures employed and the cytochemical observations are being reported in detail elsewhere, and are interpreted in terms of an equilibrium between sulphhydryl and disulphide groups². It will suffice here to state that thresholds for a number of chemical groupings were shown to exist at the boundaries of zones of inhibition on plates seeded with Staph. aureus NRRL. strain No. 313, or with the rough form of B. subtilis NRRL. strain No. B-558, and afterwards exposed to penicillin. It is well known that these test organisms are most sensitive to penicillin when they are in the logarithmic phase of growth. Therefore, we found it advantageous to pre-incubate the seeded plates for three hours before adding the solutions to be tested³. When this is done, a secondary incubation period of three hours for Staph. aureus or of two hours for B. subtilis is sufficient for the minute colonies developed during primary incubation to react to the diffusion of penicillin so that the thresholds can be located clearly by suitable techniques.