*2.1. Specimen Preparation*

Sample size was calculated using Pass version 11 (NCSS Statistical software, Kaysville, UT, USA), employing one-way ANOVA with 99% confidence interval, 99% power of the test, and with means and standard deviation of *C. Albicans* viable cells in groups: DW = 7.47 × 105, DC = 4.82 × 105, DW with MW 1 min = 4.49 × <sup>10</sup>5, DC with MW 1 min = 2.64 × 105, DC with MW 2 min = 0.00, DW with MW 3 min = 0.00, DC with M 3 min =0.00 [16]. Sample size was calculated with at least 10 specimens per sub-group (total 12 subgroups). However, a total of 168 specimens were fabricated (*n* = 14).

Test specimens (*n* = 168) of PMMA acrylic resin denture base were fabricated with the help of modelling wax (Yeti Dental GmbH, Engen, Germany) melted in a wax pot (Manfredi, San secondo di Pinerolo TO, Italy) and poured into a three-part preformed metal mold (30 × 30 × 15 mm). Wax patterns were impressed in a metallic denture flask filled with type III dental stone (Garrico Lab Stone, Heber Springs, AR, USA) to produce PMMA samples. De-waxing was performed using boiling water for 6 min. Heat polymerized PMMA acrylic resin was mixed and packed at a doughy stage according to manufacturer recommendations at a powder: liquid ratio, 2.3 g of polymer powder to 1 mL of liquid monomer (heat-cured acrylic provided by MR Dental, Plymouth, UK). A Hydraulic press was used for packing the denture base resin with a sheet of separating plastic between the two halves. Heat cure PMMA was polymerized in a thermostatically controlled water bath (Manfredi–Acrydig 12) and processed for 74 ◦C for two hours followed by 100 ◦C for one hour (Figure 1).

All specimens were allowed to cool at room temperature before de-flasking and were immersed in distilled water at room temperature for 48 h for residual monomer elimination. The excess resin was trimmed with a metal bur (Denfac Acrylic trimming burs). All PMMA specimens were wet-polished with abrasive paper (# 1200, water resistant) and buffed (Dialap ML150P; Maruto, Tokyo, Japan). Final finishing was performed with an aluminabased abrasive (particle size: 0.3 μm). Post-finishing, all specimens were autoclaved at 121 ◦C for 15 min. For the purpose of reliability, a single operator prepared all the specimens.

#### *2.2. Study Groups*

All specimens were divided into three main groups based on disinfection techniques, which are as follows.

Group MW-DW: microwave (MW) radiation was used to disinfect the specimens contaminated with a mixture of three isolates immersed in distilled water in a glass beaker. The glass container was placed in the microwave oven and the specimens were sterilized at 450 W. Based on duration of MW radiation, specimens were divided into MW-DW1 (1 min), MW-DW2 (2 min) and MW-DW3 (3 min). Different specimens in each subgroup were assessed at 24 h, 36 h, 48 h and 96 h, respectively.

Group DC-DW: specimens were immersed in distilled water with a denture cleaning tablet (sodium perborate) (DC) (Fittydent international, GmbH, Wien, Austria) added to it. Based on the duration of immersion in DC, the specimens were divided into DC-DW1 (1 min), DC-DW2 (2 min) and DC-DW3 (3 min). Different specimens in each subgroup were assessed at 24 h, 36 h, 48 h and 96 h, respectively.

**Figure 1.** PMMA sample fabrication. (**A**) PMMA packing in plaster molds and (**B**) Finished PMMA samples. (**C**) Dimensions of each PMMA sample.

Group MW-DC-DW: the glass beaker containing 200 mL of distilled water in which a denture cleaning tablet was dissolved for five minutes was placed in the microwave oven and irradiated at 450 W. Based on duration of MW radiation, specimens were divided into MW-DC-DW1 (1 min), MW-DC-DW2 (2 min) and MW-DC-DW3 (3 min). The temperature of the solution was kept between 65 ◦C to 71 ◦C with ±2 ◦C. Different specimens in each subgroup were assessed at 24 h, 36 h, 48 h and 96 h, respectively. With five specimens in each subgroup, a total of 180 specimens in each disinfection group were employed.

Positive Control Group: in this group the acrylic resin specimens were immersed in glass beaker (250 mL size) containing 200 mL of distilled water at room temperature. The glass container was placed in the center of the microwave oven chamber (Samsung 2450 MHz, 800 W) but was not irradiated.

Negative Control Group: the purpose of this group was to establish the disinfection of the specimens and accuracy of the test. For each bacterium, sterilized specimens were placed in a container with sterilized water.
