2.4. Determination of Minimum Inhibition Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The estimations were made using the MIC and MBC method described previously elsewhere and by Kowalczyk [23,50]. The MIC determines what drug or compound concentration inhibits bacterial growth. The MBC determines what concentration of the drug has bactericidal activity. MICs were estimated using the microtiter plate method using sterile 48-well plates as previously described [21,50]. The material was also taken from oral cavity of 20 volunteers (hygienists) see (point b chapter 2.3). The bacterial inoculum was diluted in in 0.9% saline each, at a final concentration of 10 mM and 50 μL of each compound diluted to 1 mM was placed in the first line of rows of the plate. Next, to the other wells, 25 μL of sterile TSB medium was added and serial dilutions were performer from 10⁻¹ to 10⁻⁷. Then, 200 μL of inoculated TSB medium containing resazurin indicator (0.02 mg mL⁻¹) was added to all wells. TSB medium was inoculated to the concentration in ratio 1:100 (v:v) of the final suspension (~10⁶ cfu mL⁻¹; about 0.5 McFarland) of all analyzed strains and plates were incubated at 37 °C for 24 h. A color change from blue to pink or yellowish and an increase in turbidity was considered positive, and the lowest concentration at which there was no visible colour change was assumed to be the MIC. Experiment was performed in three independent replicates. The MBC was estimated based on the measurement of dehydrogenase activity in cultures after a 24 h incubation. A 4 mL sample of a dense culture (~10⁸ cfu mL⁻¹) that had been incubated for 24 h in TSB medium at 25 °C was added to a test tube, and tested compounds were added until the mixture reached a final concentration of 10−250 mg mL⁻¹. The growth of the individual strains on the Salistat plates was incubated for 1 h at 30 °C. Then ~0.1 g of CaCO3 and 0.1 mL of a 3% TTC solution were added, mixed and the tubes were sealed with parafilm and incubated for 1 h at 30 °C in the dark. The lowest concentration at which there was no visible red color (due to formazan) was taken as the MBC. Then the MIC and MBC values were calculated.