*2.6. CLSM*

Confocal laser scanning microscopy (CLSM) (LSM780, Carl Zeiss, Jena, Germany) was used in this study to detect the live and dead cells in the biofilms [46–49]. The 304SS coupons were immersed in a culture medium without inoculation, or with *D. desulfuricans* inoculum in the absence or presence of 25 ppm BDMDAC at 37 ◦C at different time intervals up to 28 days. After cleaning the surface once with phosphate-buffered saline, the biofilms were stained with LIVE/DEADTM *Bac*LightTM bacterial viability kit (L7012, Thermo Fisher Scientific, Waltham, MA, USA). After staining, the coupons were observed under a fluorescence microscope. Live cells and dead cells appeared green and red, respectively, in the biofilm. The three-dimensional (3D) scanning images obtained by CLSM were used to measure biofilm thicknesses [46,47].

#### **3. Results and Discussion**

#### *3.1. Bactericidal Assay of BDMDAC*

To test the antimicrobial activity of BDMDAC, the growth of *D. desulfuricans* was monitored in different concentrations of BDMDAC. The cell growth in 10 ppm BDMDAC did not differ from control (0 ppm) (Figure 1). When the BDMDAC concentration was elevated to 15 and 20 ppm, cell growth was slowed, with doubling time increasing from 12 to 18 h. Furthermore, when 25 and 50 ppm BDMDAC was added to the SRB solution, the cells did not grow. Therefore, the minimum inhibition concentration of BDMDAC for *D. desulfuricans* is 25 ppm.

To validate the long-term antimicrobial effect, *D. desulfuricans* was inoculated in a medium containing 25 ppm BDMDAC, which was then incubated for 4 weeks at 37 ◦C. As shown in Figure 2, no growth was observed during this period. Thus, BDMDAC has a favorable inhibitory effect on the growth of *D. desulfuricans.*

**Figure 1.** Effects of BDMDAC concentration on the growth curve of *D. desulfuricans* as a function of time in hours.

**Figure 2.** Visual inspection of the *D. desulfuricans*-inoculated medium with and without BDMDAC after (**a**) 7 days and (**b**) 28 days of incubation.
