## 3 Results
### CLSM Imaging of the Biofilms and Quantitative Analysis  
In this study, the biofilms were stained with fluorescent dyes and viewed with a CLSM, and the CLSM images of the 120 h biofilms are shown in Figure 7.  
Figure 5: Data fitting of the 120 h biomass as a function of exposure time.  
Figure 6: Data fitting of the coefficients \(a\) and \(b\) as functions of the temperature rise.  
As can be seen in Figure 7, for the biofilms in the control group, the majority of the cells were alive (green fluorescence), regardless of the exposure time, suggesting that immersing with normal temperature water has no bactericidal effect.  
For the biofilms treated with 50 \({}^{\circ}\)C water, when the exposure time was short (less than 10 min), only a few bacteria in the outer layer were inactivated (red fluorescence). As the exposure time increased, more cells were killed. When the exposure time was long (more than 30 min), the majority of the cells were dead and only few interior cells, which were protected by the biofilm layer, were alive.  
A similar trend, that more cells were killed as the exposure time increased, was shown in the CLSM pictures of biofilms treated at 60, 70, and 80 \({}^{\circ}\)C. However, as the temperature increased, the exposure time required to inactivate the majority of the cells decreased. Specifically, for the biofilms thermal shocked at 80 \({}^{\circ}\)C, even for a very short exposure time (1 and 2 min), the majority of the cells were inactivated and only a few bacteria in the bottom layers remained alive. When the exposure time increased, nearly all of the cells were killed, suggesting a significant bactericidal effect of the thermal shock at such a temperature.  
Additionally, when comparing biofilms thermally-shocked for the same exposure time, but at different temperatures, it can be seen that as the temperature increased, more cells were killed, suggesting an enhanced bactericidal effect as the temperature increased.  
Regarding the structure of the biofilms, matured, porous, and dense structures of the biofilms were observed in the control group, suggesting that immersing at a normal temperature has no inhibiting effect on the biofilm formation. As the temperature and the exposure time increased, it could be seen that the biofilms became thinner and more black area could be observed, suggesting that the thermal shock decreased the volume and surface coverage rate of the biofilms.  
Figure 7: CLSM images of the 120 h biofilms thermally-shocked with different temperatures and exposure times.  
Furthermore, Imaris was used to calculate the biovolume of the live cells and dead cells according to the amount of green and red fluorescence, and the ratio of the live cell biovolume to the dead cell biovolume at 120 h is shown in Figure 8.  
As can be seen in Figure 8, in the control group, the ratios were all close to 100 regardless of the exposure time, suggesting immersing with normal temperature water barely had any bactericidal effects. In the thermal shock groups, the ratio decreased significantly as both the temperature and exposure time increased, suggesting an enhanced bactericidal effect as the temperature and exposure time increased. The minimum ratio was close to 0.01, suggesting a significant bactericidal effect of the thermal shock.  
In summary, it could be concluded from the CLSM figures and the quantitative analysis that as the temperature and exposure time increased, more cells were killed and the amount of the biofilms decreased, leading to an enhanced biofilm inhibition effect.