## 2 Materials and Methods
### Specim Preparation  
The sample size was obtained using Pass version 11 (NCSS Statistical software, Kaysville, UT, USA), employing one-way ANOVA with 99% confidence interval at 90% power, and with the means and standard deviation of viable cells in groups (DW = 7.47 \(\pm\) 0.68, DC = 4.82 \(\pm\) 0.73, DW + MW 1 min = 4.49 \(\pm\) 0.45, DC + MW 1 min = 2.64 \(\pm\) 0.59) from a previous study [21]. A sample size of ten per group was obtained using calculations (total 12 subgroups). A total of 128 specimens were fabricated (Table 1).  
In order to prepare the test specimens from PMMA acrylic resin, modelling wax (Yeti Dental, Berlin, Germany) was melted with help of a wax pot (Manfredi, Salerno, Italy) and poured into a three-part preformed metal mold with dimensions of 30 \(\times\) 30 \(\times\) 15 mm. Wax patterns were invested in a metallic denture flask filled with type III dental stone (Garrico Lab Stone, Rochester, NY, 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 dough stage according to the manufacturer's recommendations at a powder-to-liquid ratio of 2.3 (grams 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 plastic separating the two halves. Heat-cured PMMA was polymerized in a thermostatically controlled water bath (Manfredi--Acrydig 12, Salerno, Italy) and processed at 74 \({}^{\circ}\)C for two hours, followed by 100 \({}^{\circ}\)C for one hour (Figure 1). The amount of residual monomer for heat-cured PMMA (2 h cycle) ranged from 16 to 40 \(\times\) 10\({}^{-3}\)\(v/\)% [22]. However, the curing cycle protocol and storage conditions were standardized among all study specimens in order to standardize the residual monomer among samples.  
All samples were cooled at room temperature before being deflasked and immersed in distilled water at room temperature for 48 h in order to eliminate residual monomer. A metal bur (Denfac acrylic trimming burs) was then used to trim excess resin, and the finishing was performed using abrasive paper in a hand-held micromotor. All the specimens were autoclaved at 121 \({}^{\circ}\)C for 15 min. For reliability, a single operator prepared all the samples. All specimens' dimensions were checked repeatedly. Intra-operator reliability was assessed at a kappa score of 0.85.  
\begin{table}
\begin{tabular}{c c c c c} \hline
**Growth Time \& Exposure** & **Control CFU** & **MW-DC-DW** & **MW + DW** & **DC + DW** \\
**Time vs. Control** & & & & & \\ \hline
96 h & 1400 & MD (\(p\)-value) & MD (\(p\)-value) & MD (\(p\)-value) \\ \hline
1 min & & \(-\)429 (0.008 **) & \(-\)197 (0.008 **) & \(-\)94 (0.008 **) \\
2 min & & \(-\)447 (0.008 **) & \(-\)300 (0.008 **) & \(-\)98 (0.008 **) \\
3 min & & \(-\)1388 (0.007 **) & \(-\)1344 (0.008 **) & \(-\)98 (0.007 **) \\
4 min & & \(-\)1388 (0.008 **) & \(-\)1344 (0.008 **) & \(-\)200 (0.008 **) \\
5 min & & & \(-\)1384 (0.008 **) & \(-\)750 (0.008 **) \\ \hline \end{tabular}
**Significant at 1% using the Mann–Whitney test; MW: microwave; DW: distilled water; DC: sodium perborate tablet. Values are represented as the mean difference (\(p\)-value).  
\end{table}
Table 1: Pairwise mean CFU comparison for _C. albicans._