In-vitro Methods for Determining Minimal Lethal Concentrations of Antimicrobial Agents

To determine the minimal lethal concentration of an antimicrobial agent, broth dilution tests may be performed and then a sample from each tube showing no visible growth may be subcultured onto a drug-free agar medium. By counting the number of viable cells recovered from each tube, the minimal concentration of drug required to kill 99.9% of the cells in the initial inoculum can be determined. Studies were undertaken to determine the circumstances under which false-negative tests could occur as a result of continued inhibition of growth by the antimicrobic carried over in the sample. Drug carryover did significantly reduce the number of viable cells recovered from broth containing relatively high concentrations of antimicrobic: the larger the sample, the greater the effect of drug carryover. The effect was minimal with samples of 10 microliter or less. Furthermore, the effect of drug carryover was reduced by spreading the sample over the surface of an agar medium. The relative precisions of four methods for performing subcultures with such small-volume samples were determined; coefficients of variation were 16-25%. Sampling errors inherent in the procedure for subculturing broth tubes should be taken into consideration when interpreting results of individual tests, and replicate subcultures will improve the reliability of the colony counts.