Figure 3.

Changes in the distribution of live and dead bacteria with changes in the VCM concentration administered at various time points of biofilm formation on fluorescence staining using LIVE/DEAD BacLight. The cultures were observed under a fluorescence microscope (8x magnification). Numbers in the figures represent the VCM concentrations (μg/mL). In the immediate group, most bacteria were alive, stained green, when the VCM concentration was 2 μg/mL or lower, similarly to those in the control group. Dead bacteria, stained red, surrounded by live bacteria appeared as spots at 4 μg/mL, and the rate of live bacteria decreased and the rate of dead bacteria increased at 8 μg/mL. Most bacteria were dead at 16 μg/mL or higher (a). In the 2 hr group, most bacteria were alive when the VCM concentration was 4 μg/mL or lower. Dead bacteria surrounded by live bacteria appeared as spots at 8 μg/mL, and the rate of live bacteria decreased and the rate of dead bacteria increased at 16 μg/mL. Most bacteria were dead at 32 μg/mL or higher (b). In the 4 hr group, most bacteria were alive when the VCM concentration was 4 μg/mL or lower. Dead bacteria surrounded by live bacteria appeared as spots at 8 μg/mL, these spots of dead bacteria expanded at 16 μg/mL, and the rate of live bacteria decreased at 32 μg/mL. Most bacteria were dead at 62 μg/mL or higher (c). In the 8 hr group, most bacteria were alive when the VCM concentration was 16 μg/mL or lower. The rate of live bacteria decreased and the rate of dead bacteria increased at 32 μg/mL, and most bacteria were dead at 64 μg/mL or higher (d).