Figure 2.

Morphogenesis, 3D architecture, and quantitative measurement of microcolonies in 72-h multispecies biofilms (1% sucrose condition). The 72-h biofilms of the control group (C. albicans and S. mutans) and experimental groups (with L. plantarum 14917) in 1% sucrose condition were visualized using a two-photon laser confocal microscope. The three-dimensional structure of the biofilms was rendered using Amira software. The green color indicates bacteria and the red color indicates the exopolysaccharides (EPS). L. plantarum 14917 dramatically reduced biofilm formation, compared to the control group (A, B). Biofilm dry weight was significantly reduced with added L. plantarum 14917 (C). *p < 0.05. The biomass of the two biofilm components, bacteria and exopolysaccharides (EPS), was calculated using image-processing software COMSTAT (Heydorn et al., 2000). Both L. plantarum 14917 significantly reduced the biomass of bacteria and EPS (D). The confocal images indicate the cross-sectional and sagittal views of microcolonies formed in the control group (S. mutans and C. albicans duo-species) and with added L. plantarum 14917. Well-formed mushroom-shaped microcolonies were seen in the control group, and the largest size microcolonies were seen in the S. mutans and C. albicans duo-species biofilm. Microcolonies formed with added L. plantarum 14917 were much less structured. Bacterial components were less encapsulated with EPS. The amount of co-localization between bacteria and EPS was calculated using DUOSTAT (E), which was consistent with the findings revealed in the images (*p < 0.05). The surface-attached and free-floating microcolonies were evaluated using COMSTAT and DUOSTAT software. Panel (F) illustrates that biofilms treated by L. plantarum 14917 had significantly reduced microcolony size (p > 0.05; ANOVA, comparison for all pairs using Tukey–Kramer HSD).