Standardization of a fluorescence-based microplate assay for rapid antibacterial screening of Escherichia coli and...

The sustained increase in bacterial resistance has driven the development of rapid and objective methodologies for the evaluation of new antimicrobial agents. In this context, two fluorometric microplate methods were standardized based on the microdilution assay and the reduction of the resazurin redox indicator, using the reference strains E. coli ATCC 25922 and S. aureus ATCC 25923. Critical assay parameters were optimized in 96-well plates, including inoculum concentration, incubation time, final volume, and the resazurin reading window, to ensure linearity and reproducibility. Validation was performed by calculating the minimum inhibitory concentration (MIC) using the antibiotics meropenem, amikacin, and rifampicin. In S. aureus, the MIC values obtained using the conventional and fluorometric methods were consistent for the three antibiotics evaluated. In E. coli, agreement was observed only for amikacin, whereas for meropenem and rifampicin, the fluorometric method demonstrated the limitations of the visual method when the survival rate was <30%. Overall, the results indicate that the standardized fluorometric methodology constitutes a quantitative, objective, and reproducible alternative to the conventional method, representing a useful tool for the preclinical evaluation of new compounds.