*3.2. Core + S. Mutans Biofilm Showed a Net-Like Structure and Increased the Depth of the Biofilms*

To have an integrated understanding of the biofilm, the SEM and CLSM were enrolled for observation of the biofilm formation, EPS synthesis, and biofilm structure in situ. The 72-h-old biofilms exhibited different morphologies under the SEM; the Core + *S. mutans* group biofilms with the existence of both EPS and bacillus represented a thick and netlike structural; the *S. mutans* group biofilms with rich EPS looked like honeycombs; the Core groups, however, without EPS, could only be detected with loose and thin biofilms (Figure 2a). Furthermore, consistent with the observation from SEM, the Core + *S. mutans* group biofilm images taken by CLSM showed a net-like structure that might improve the stability of the biofilm in complex environments (Figure 2b). The depth of the biofilms was significantly increased in the Core + *S. mutans* biofilm (*p* < 0.05) and EPS was not produced or rarely produced in the core biofilm (Figure 2c–g). The depth of the biofilm and EPS showed a significant increase in the Core + *S. mutans* group.

**Figure 2.** Biofilm structure by SEM and EPS distribution by CLSM. (**a**) Structure of the 72 h Core + *S. mutans* biofilm, *S. mutans* biofilm, and the core biofilm (n = 3). (**b**) Double-labeling of 72 h biofilms. Green, bacteria (SYTO 9); red, EPS (Alexa Fluor 647) (n = 3). (**c**) The distributions of EPS and bacteria at different heights of the Core + *S. mutans* biofilm. (**d**) The distributions of EPS and bacteria at different heights of the *S. mutans* biofilm. (**e**) The distributions of EPS and bacteria at different heights of the core biofilm. (**f**) The depth of the biofilm. (**g**) Quantification of EPS /bacteria biomass of the 72 h biofilms. \*\* *p* < 0.01.
