2.9. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination of D. angustifolia, Gold Nanoparticles, Honey against clinically isolated MRSA and VRSA isolates

The antimicrobial effectiveness of D. angustifolia extract, honey, and AuNPs against the eight chosen isolates (four isolates MRSA + VRSA and another four isolates MRSA + VSSA) were analyzed through determination of the minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC), and MBC/MIC ratio values through application of the broth micro-dilution method using 96-well micro-titer plates as follows, to each 50 μl of the antibacterial agent dilution vertically, 50 μl of the adjusted bacterial concentration inoculums (5 × 10⁵ CFU/mL) was added horizontally in the 96-well micro-titer plates, and the growth control wells contained MH broth medium with tested bacterial concentrations in order to check the bacterial viability while the sterility control wells contained only a sterile MH broth in order to check the sterility of the medium used. The plates were then covered to ensure that the bacteria were not dehydrated. The plates were then incubated at 37 °C for 18–20 h. The lowest concentration of each antibacterial agent that inhibited the bacterial growth was then considered as the MIC [28]. After the MIC determination, aliquots of 100 μL from each well that does not show any bacterial growth after incubation were streaked onto BHI agar plates followed by incubation at 37 °C for 20 h. The lowest concentration that kills 100% of the initial bacterial population showing no colonies on the BHI agar was recorded as the MBC.