## 4 Discussion  
This longitudinal study evaluated changes in the antibacterial efficacy of CAP and CHX over a period of 6 days using QLF-D-based red fluorescence intensity (\(\mathrm{Ratio_{R/G}}\)) analysis. The porphyrins and extracellular and intracellular polysaccharides produced by bacterial metabolism in response to the blue light (405 nm) emitted from the QLF-D camera \begin{table}
\begin{tabular}{l l l l} \hline \hline
**Treatments** & **N** & **Total Bacteria** & **Aciduric Bacteria** \\ \hline Distilled water & 19 & 7.19 (0.52) \({}^{\mathrm{a}}\) & 5.83 (0.48) \({}^{\mathrm{a}}\) \\ Cold atmospheric plasma & 19 & 5.96 (0.66) \({}^{\mathrm{b}}\) & 4.89 (0.60) \({}^{\mathrm{b}}\) \\ Chlorhexidine gluconate & 19 & 6.44 (0.80) \({}^{\mathrm{b}}\) & 4.61 (0.68) \({}^{\mathrm{b}}\) \\ \multicolumn{3}{c}{\(p\)-values \({}^{\mathrm{t}}\)} & \textless{}0.001 & \textless{}0.001 \\ \hline \hline \end{tabular} All data are displayed as the mean (standard deviation). \({}^{\mathrm{t}}\)\(p\)-value derived using one-way ANOVA. \({}^{\mathrm{a,b}}\) Different letters in a single column represent significant differences between groups, as determined by [PERSON]’s post hoc analysis (\(\alpha\) = 0.05).  
\end{table} 
Table 1: Total bacterial and aciduric bacterial counts after antibacterial treatment (unit, \(\mathrm{log}\)\(\mathrm{CFUs/mL}\)). Figure 7: Representative confocal laser scanning micrographs (magnification 100\(\times\)) displaying live (green) and dead (red) bacterial staining (CAP, cold atmospheric plasma; CHX, chlorhexidine gluconate; DW, distilled water). manifests as red fluorescence [29; 30]. Thus, stronger red fluorescence indicates a higher concentration of these substances within the biofilm, signifying increased bacterial activity and enhanced biofilm pathogenicity [23; 31]. This is a non-invasive method that enables the monitoring of biofilm changes over time [32]. The most significant finding of this study was the reduction in biofilm pathogenicity observed in the CAP group. The Ratio\({}_{\text{R/G}}\) in the CAP group exhibited the greatest decrease from the baseline (\(p<0.001\); Figure 6) and was consistently lower than that in the CHX group at each treatment interval (adjusted \(p<0.001\)). Thus, CAP may be more effective compared with CHX in controlling biofilm growth. The efficacy of CAP translates to a substantial reduction in bacterial activity within the biofilm, signifying a decline in the pathogenic potential of biofilms [26]. Consistent with these findings, another study previously reported that argon-based CAP application for 2 min led to a notable decrease in the number of cells in a 2-day salivary biofilm compared with 0.12% CHX [15]. However, they only completed a single CAP treatment session, and biofilm alterations were not monitored continuously. Oral biofilms become structurally complex over time, and their resistance to antimicrobials intensifies with genetic transfer and adaptation among bacteria [33]. In the present study, the difference in antibacterial efficacy between CHX and CAP progressively increased as the oral microcosm biofilms underwent repeated CAP exposure over a period of 6 days (Figure 6). This may be attributed to the inability of CHX to penetrate the deeper layers of mature biofilms, unlike CAP [34]. The proportion of EPSs increases as the age of the oral biofilm increases, resulting in a more intricate internal structure [35], which can act as a physical barrier that limits chemical transport within and outside the biofilm, thereby decreasing bacterial susceptibility to antimicrobial treatments [34; 36]. Moreover, EPSs contain negatively charged phosphates that can strongly adsorb cationic compounds, such as CHX, thereby impeding CHX penetration into the biofilm [37]. Although biofilm age has been reported to be inversely related to the penetration rate of antimicrobial substances [38], the findings of the present study suggest that the efficacy of CAP in inhibiting oral biofilm growth is superior to that of CHX.