Influence of inoculum size on antibiotic assays by the agar diffusion technique with Klebsiella pneumoniae and streptomycin.

It is widely known that the inoculum size of the test organism greatly influences the size of inhibition zones in the agar diffusion assay technique, but little quantitative work on this subject has been reported. Mitchison and Spicer (1949), compared different size inocula of the "Mayo" strain of Staphylococcus aureus in the assay of streptomycin, and concluded that greater sensitivity was experienced using inocula as small as were consistent with accurate readings of the zone edge of growth. Cooper and Woodman (1946), and Linton (1949), showed that zones of inhibition were formed at a definite time after commencement of incubation of the seeded agar, thus relating the growth rate of the test organism with the formation of the zone edge. This critical time was shown to be the time required for a constant number of generations of the test organism to develop from a standard inoculum and any factors which influenced the growth rate were shown to correspondingly alter this critical period (Cooper and Linton, 1952; Cooper and Gillespie, 1952; Linton, 1954). Subsequent work has demonstrated that this constant number of generations was the number of cell divisions necessary to reach a critical density of population from a standard inoculum and that the critical time varied in order to allow this density to be reached. When the inoculum size was altered, the number of generations required and the critical time varied accordingly, but the critical population required for positioning the zone edge remained constant (Linton, 1955; Cooper, 1955; Cooper et al., 1958). These studies resulted in a theoretical concept of the formation of the inhibition zones in agar diffusion assays, and this was found to apply over a wide range of temperature and inocula size. Most of this had been worked out with the "Mayo" staphylococcus and streptomycin. Its application to other systems remained to be investigated. Results obtained with Klebsiella pneumoniae and streptomycin have now extended