INFLUENCE OF INOCULUM SIZE ON ANTIBIOTIC ASSAYS BY THE AGAR DIFFUSION TECHNIQUE WITH KLEBSIELLA PNEUMONIAE AND STREPTOMYCIN

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Abstract
The time required for the edge of an inhibition zone to be formed varies with the inoculum size and growth rate of the test organism and is the time required for the inoculum to reach a critical population. Before this population is reached, an inhibitory concentration of streptomycin diffusing through the agar, is able to inhibit all the cells within the diffusion zone. Once the critical population is reached (for K. pneumoniae 158 x 107 viable organisms per ml), the concentration of diffusing streptomycin is lowered to a sub-inhibitory level by absorption onto the cells and growth occurs to form the zone edge. Inhibition zones are formed with somewhat larger inocula due to diffusion of inhibitory concentrations of the antibiotic during the lag phase of growth. With inocula equal to or greater than 6.78 x 107 organisms per ml no inhibition zones are formed. Populations of this size are, by absorption, able to maintain the diffusing streptomycin at sub-inhibitory levels thus allowing growth to occur right up to the source of diffusion, at the end of the lag phase of growth. The bearing of these observations on assay procedure is discussed.