Determination of MIC and MBC of AI-AgNPs

Minimum inhibitory concentration (MIC) was measured as the lowest concentration of AI-AgNPs sufficient to inhibit the growth of bacteria and was tested by broth micro dilution method as recommended by the guidelines of CLSI (2012). The MIC test was performed in a 96 well microtiter plate as described by Klancnik et al. (2010). Bacterial suspensions grown overnight in MHB were adjusted to a concentration of 1 × 10⁶ CFU/mL. From a stock solution of AI-AgNPs (50 mg/mL) serial two-fold dilutions were prepared in MHB. This diluted series were inoculated with bacterial suspensions along with untreated control samples. The final volume in each well was 100 μL. The plates were sealed to prevent evaporation and incubated for 24 h at 37°C. Bacterial growth was measured at 600 nm in a microplate reader (Tecan-Spark). Minimum bactericidal concentration (MBC) endpoint is defined as the lowest concentration of antibacterial agent that completely kills the bacterial population. For determination of MBC, aliquots from each well of overnight microtiter plate were seeded on MHA plates without AI-AgNPs and incubated for 24 h at 37°C.