Background/Objectives: The increasing prevalence of antimicrobial-resistant bacteria highlights the need for improved methodologies to evaluate antimicrobial activity beyond conventional minimum inhibitory concentration testing. While resazurin-based assays are widely used for minimum inhibitory concentration determination due to their simplicity and sensitivity, minimum bactericidal concentration assessment still relies on labor-intensive colony-forming unit counting. The objective of this study was to develop and validate a resazurin-based microwell assay capable of determining both the minimum inhibitory concentration and the minimum bactericidal concentration without routine plate counting, thereby simplifying bactericidal evaluation. Methods: A two-step resazurin-based fluorescence assay was designed and performed in microplates. After determining the minimum inhibitory concentration using resazurin as a metabolic indicator, well-showing inhibited bacterial growths were subjected to a regrowth phase by transferring aliquots into fresh antimicrobial-free medium containing resazurin. This additional step allowed discrimination between reversible metabolic inhibition and irreversible bacterial death. The method was evaluated using ciprofloxacin and chloramphenicol against four bacterial species: Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Minimum bactericidal concentration values obtained using this assay were compared with those obtained through conventional colony counting on agar plates. Results: Minimum bactericidal concentration values obtained using the two-step fluorescence assay were fully concordant with the conventional colony-forming unit counting method for all tested antibiotics and bacterial species. Conclusions: The proposed two-step resazurin-based microwell assay represents a rapid, reliable, and less labor-intensive alternative for the determination of both the minimum inhibitory concentration and the minimum bactericidal concentration, with potential applications in clinical and industrial microbiology laboratories.
Calvo et al. (2026) studied this question.