Gradient Plate Bioassay for Tyrothricin and its Application to Dentifrices

Abstract

A bioassay for tyrothricin, based on a procedure used by Szybalski in bacterial resistance studies, was developed. Replacement of the tube dilution assay with this procedure represented economy in time and equipment, with a resultant increase in productivity. The procedure involved preparation of special agar plates which provided graded concentrations of tyrothricin along a horizontal axis. A culture dish was inclined, and a base layer of agar, without antibiotic, was poured to cover the bottom of the dish and allowed to harden. A second layer of agar, containing 1 ppm of tyrothricin, was poured and allowed to harden with the dish in a level position. Diffusion of the antibiotic from the top layer into the bottom layer yielded a concentration gradient. A third thin layer of agar seeded with Streptococcus faecalis was poured on the surface. After incubation, a bacterial growth front, representing the minimal effective concentration (MEC), developed. The MEC is expressed as distance (in millimeters) from the edge of the plate. Unknowns were directly related to a standard preparation for calculation of tyrothricin concentration.