Reduced incubation time for inhibition zone formation based on diffusion and growth mechanism elucidation

Abstract

Microbiological agar diffusion methods are widely employed for clinical, pharmacological and industrial purposes. Usually, inhibition zone sizes are defined after a few hours of incubation; however, they can only be visualized after overnight microbial growth. Thus, the aim of this work was to determine the critical parameters and optimize them using the tool Solver (Microsoft Excel®), in order to elucidate the mechanism of inhibition zone formation and develop a microbiological assay for linezolid with a reduced incubation time. A Box–Behnken design was used to determine the critical parameters, such as the critical concentration (C′), critical population (N′), critical time (T′), diffusion coefficient (D), lag phase time (L), and generation time (G). Optimized critical parameters were coherent with the mean values of experimental parameters and were contained within the confidence interval 95%, validating the results obtained with the tool Solver. Furthermore the formation of inhibition zones was elucidated with 97.7% certainty. Increased inoculum sizes reduce critical time, which allow us to use TTC to visualize inhibition zones after 6 hours. The value of the critical concentration obtained with reduced incubation time (8.90 mg L⁻¹) was consistent with the experimental value with standard incubation time (8.62 ± 0.53 mg L⁻¹; CI95% 7.77 to 9.45 mg L⁻¹). Therefore our method was able to reduce incubation from 18 to 24 hours to approximately 6 hours.