Combinations of three antimicrobial agents. (i). Experimental three-dimensional checkerboard dilution method for quantitative analysis of the in vitro combined effect of three antimicrobial agents

We have established an experimental three-dimensional checkerboard dilution method for quantitative analysis of the in vitro combined effect of three antimicrobial agents. We designed this method through a modification of the checkerboard dilution method used for two antimicrobial agents. In this experiment, we used a micro-broth dilution method using the Dynatech MIC-2000 system. A two-fold serial dilution of a third agent was added to the checkerboard containing the first and second agents. Then, 0.0015 ml of a bacterial suspension was inoculated and incubated for 24 hours at 37°C. The results obtained were plotted on a three-dimensional cubic graph so as to describe a spherical surface, and the volume under the surface (VUS) was calculated. By comparing the VUS, with the relative proportion of the three agents fixed at a certain ratio, it was possible to compare the efficacy of triple antimicrobial combinations. For example, by application of the three-dimensional checkerboard method, we could evaluate the effect of the combination of a combined agent with a single agent, or the combination of equal amounts of three agents.