2.7.1. Minimal Inhibitory Concentration (MIC) of Silver Nanoparticles

In order to determine the MICs of the obtained AgNPs against tested bacterial strains, we used the microdilution method, as previously described [20]. To obtain binary dilutions from the TS, 200 μL TS was added in the first column of the microplate. One-hundred microliters of sterile water were added in all the other wells of the microplate. One-hundred microliters from the first column were transferred with a multichannel pipette into the second column of the microplate. These steps were repeated until the last column, from which, the last 100 μL was discarded. Ten microliters of 0.5 McFarland bacterial suspension were mixed with 9990 μL of Mueller-Hinton broth medium 2X and 100 μL from this bacterial inoculum were dispensed in each well of the microplate. Additionally, wells with culture medium only, culture medium with TS (negative controls), and culture medium with bacterial inoculum (growth control) were prepared. The microplates were incubated at 37 ◦C for 24 h. MIC was considered in the last well in which no bacterial growth was noted. For TS with a high degree of turbidity, resazurin was used as an indicator of bacterial growth [23]. After the plates were incubated for 24 h at 37 ◦C, 3 μL 0.015% resazurin was added to each well, and the plates were further incubated for 2–4 h. A color change of resazurin, from purple to pink, indicated a bacterial growth. The last well in which the resazurin color did not change was considered MIC.
