**2.2.4 Microdilution method**

Antimicrobial activity was tested by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) by using microdilution plate method with resazurin (Sarker et al., 2007). The 96-well plates were prepared by dispensing 100 μL of nutrient broth into each well. A 100 μL from the stock solution of tested compound (concentration 2000 μg/mL) was added into the first row of the plate. Then, twofold, serial dilutions were performed by using a multichannel pipette. The obtained concentration range was from 1000 μg/mL to 7.81 μg/mL. A 10 μL of diluted bacterial suspension was added to each well to give a final concentration of 5 x 105 CFU/mL. Finally, 10 μL resazurin solution was added to each well inoculated with bacteria. Resazurin is an oxidation– reduction indicator used for the evaluation of microbial growth. It is a blue non-fluorescent dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells (Banfi et al., 2003). The inoculated plates were incubated at 37 °C for 24 h. MIC was defined as the lowest concentration of the tested substance that prevented resazurin color change from blue to pink. Doxycycline was used as a positive control. Solvent control test was performed to study an effect of 10% DMSO on the growth of microorganism. It was observed that 10% DMSO did not inhibit the growth of microorganism. Also, in the experiment, the concentration of DMSO was additionally decreased because of the twofold serial dilution assay (the working concentration was 5% and lower). Each test included growth control and sterility control. All tests were performed in duplicate and MICs were constant. Minimum bactericidal concentration was determined by plating 10 μL of samples from wells, where no indicator color change was recorded, on nutrient agar medium. At the end of the incubation period the lowest concentration with no growth (no colony) was defined as minimum bactericidal concentration.
