Development of a Novel Direct Bioautography–Thin-Layer Chromatography Test: Optimization of Growth Conditions for Gram-Negative Bacteria, Escherichia coli

Abstract

Abstract With the aim of developing a TLC-direct bioautography assay using Escherichia coli as test bacteria, various parameters influencing the viability of microorganisms on TLC plates were examined and checked for flumequine standards. The optimal times for preincubation and incubation of bacterial broth were 20 h at 37°C and 2 h at 37°C, respectively. The optimal viscosity of the broth was obtained for 0.05% agarose solution in Mueller-Hinton broth. Various incubation times of the seeded TLC plates were also tested (5 h proved to be optimal). After incubation, the plates were sprayed with 0.2% aqueous [3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyltetrazolium bromide] (MTT) solution and incubated for 0.5 h at 37°C. The precision of the method was evaluated by the repeatability (intraday assay) and intermediate precision (interday assay). The regression coefficients were 0.9977 and 0.9968, respectively, for intraday and interday curves. The calibration curves show good linearity in the range of 0.005–0.50 μg (0.5–50.0 μg/mL). The established LOD of flumequine equaled 0.5 μg/mL, i.e., 5 ng flumequine in the spot. The developed direct bioautography test significantly enhances the sensitivity of the TLC method.