*2.3. Biofilm Formation Assay*

Overnight specimens of 0.1 mL of *E. coli* (ATCC# 8739), *S. aureus* (ATCC# 25923) and *S. mutans* (ATCC# 25175) subject isolates were inoculated into 100 mL of sterile Tryptic Soy Broth (TSB-Oxoid, Basingstoke, Hampshire, UK). The group contained a mixture of all three isolates. The sterile acrylic specimens were inoculated into each flask aseptically and incubated at 37 ◦C for 24 h, 36 h, 48 h and 96 h. After incubation, the acrylic specimens were collected and washed with distilled water to remove debris and loosely attached cells (Figure 2). Specimens were placed in phosphate buffered saline (PBS) (pH-7) and vortexed for two minutes. After vortexing, the specimens were, as has been explained, exposed to three treatment regimens in study groups (MW-DW, DC-DW and MW-DC-DW) at different exposure times. For selective isolation of bacteria, *E. coli*, *S. aureus* and *S. mutans*

were cultured in Eosin Methylene Blue Agar (EMB Oxoid Basingstoke, Hampshire, UK) (Figure 3), Baird-Parker Agar with egg yolk tellurite (Fisher Scientific, Port Salvo, Portugal) (Figure 4) and Brain Heart Infusion (BHI) Broth (Difco, Detroit, MI, USA). The growth was monitored and CFU were counted.

**Figure 2.** (**A**) Acquisition of PMMA samples after bacterial incubation. (**B**) Incubation of PMMA samples for 24 h, 36 h, 48 h and 96 h.

**Figure 3.** Pure culture of *E. coli* on EMB agar.

**Figure 4.** Pure culture of *S. aureus* on Mannitol Salt agar.
