4.4. Agar Dilution Susceptibility Testing

Minimum inhibitory concentrations (MICs) were determined by the agar dilution method using chocolate II agar according to CLSI guidelines [33]. Briefly, F. tularensis LVS bacteria were cultivated overnight in TSBc, diluted to an OD600 of 0.3 (approximately 5 × 10⁸ CFU/mL), and then diluted to 1 × 10⁶ CFU/mL. Ten microliters of this suspension (1 × 10⁴ CFU) were plated onto chocolate II agar containing a series of two-fold dilutions of resazurin. Following incubation (37 °C with 5% CO₂ for 48–72 h), the MIC reported for each F. tularensis strain was the lowest concentration of resazurin that completely inhibited the growth of bacteria on the agar plate. No strain differed by more than one dilution in 3–5 tests.