## 4 Discussion  
This study aims to elucidate the intrinsic factors influencing the antibacterial activity of zinc oxide (ZnO) nanoparticles synthesized via microwave-assisted hydrothermal methods, specifically focusing on the impact of particle size, precursor concentration, and reaction time. The design of the experiments was based on the hypothesis that the smaller the size of ZnO particles, the larger the specific surface area, the higher the number of surface atoms, the higher the absorption rate, and the stronger the adsorption capacity. Therefore, it is easier to reach the surface of the bacteria, causing damage to the cell membrane and large molecules in the cell. Moreover, small-sized ZnO nanoparticles produce more reactive oxygen species, increasing the endochemic effect of bacterial cells and causing their deterioration [6; 32]. Studies with similar aims are found in the literature, most of them recently published. However, they are following different synthesis routes or chemical precursors, especially the sol-gel route, with fewer synthesis parameters to control. [PERSON] et al. reported the sol-gel synthesis of spherical, flower, and sheet morphology ZnO, starting from zinc  
Figure 11: Sensitivity of Gram-positive, Gram-negative, and fungal strains in the presence of ZnO samples. Results are represented as mean \(\pm\) SD; different letters indicate significant differences between each sample (\(p<0.05\)). Figure 12: Cell viability results, assessed by MTT assay and represented as mean \(\pm\) SD; different letters indicate significant differences between each sample (\(p<0.05\)). nitrate and zinc acetate, and concluded that specific surface area and particle core defects are crucial parameters that significantly influence the antimicrobial effectiveness of ZnO nanoparticles [33]. [PERSON] et al. focused on the synthesis of ZnO NPs smaller than 10 nm and concluded that the smaller nanoparticles obtained had bactericidal properties against _S. aureus_ and _E. coli_[32]. [PERSON] et al. tackled the synthesis of ZnO starting from zinc chloride (0.5 mol/L) and using the same method as proposed in our study (microwave-assisted hydrothermal synthesis, with 5 min reaction time) [34]. Their results were promising, obtaining nano-scale pure ZnO particles, but a variation in precursor concentration and reaction time was not performed. [PERSON] et al. extended the study by including zinc nitrate and zinc acetate along with zinc chloride as starting salts at an even larger precursor concentration (1.6 mol/L) and varied the reaction time (5, 10, and 20 min) [35]. Their recommendation was to use Zn(NO\({}_{3}\))\({}_{2}\) as the precursor and concluded that increasing the microwave irradiation time results in more well-defined structures and a system with more uniform particle size and morphology distribution due to the extended time available for growth along the c-axis. However, a direct connection between these synthesis parameters and the antibacterial activity of the obtained ZnO was not made. Starting from the limitations of the previous literature studies, our work investigated the direct role of two synthesis parameters (Zn\({}^{2+}\) precursor concentration and microwave-assisted hydrothermal reaction time) in controlling the particle size and morphology of ZnO and enhancing their antibacterial properties. It was important for our findings to include longer reaction times than presented before (e.g., 30 min, 60 min) and smaller zinc chloride concentrations (starting from 0.3488 mol/L and followed by a serial dilution down to 0.0218 mol/L). Based on the presented results, all syntheses were successful in obtaining ZnO (zincite) as the sole mineral phase, with increased crystallinity when high precursor concentrations and long reaction times were applied. There is also a change in morphology and particle size due to the synthesis parameters' variance.