Microbiological diffusion assay II: design and applications

Application of new equipment and new techniques was made to antibiotic diffusion assays. Accumulation of data and computation of potencies were made by an on-line computer. The system was tested by assaying cephalexin with the aid of Bacillus subtilis in an FDA single-dose design modified by reducing the number of standards to two. The influence of the thickness of the base layer and the form of the dose-response line were tested. Zone diameter was measured with a resolution of 0.01 mm. The potency of samples was measured with an error usually less than 3%. An error of 0.1 mm in measuring zone size would cause an error of 3% of potency. The usual calibration line was inadequate for extrapolation beyond a twofold range. A dose-response line derived from the Cooper equation was better for standard curves spanning more than a twofold range of concentrations. Precision was better on the plates with the 20-ml base layer. The two-dose method of assaying gave larger errors than the single-dose method. Large variances in measuring zone diameters are a reason for the low precision of diffusion assays and set an inherent limit on precision.