## 3 Surface Characteristics of Zirconia Surface  
Scanning electron microscopy (SEM)  
Scanning electron microscope (Scanning Electron Microscope, Model JSM-6390, JEOL, Inc., Tokyo, Japan) was used for the quantative analysis of the surface morphology of different zirconia groups of samples. The samples were gold-sputtered, and the analyzing procedures were carried out at 150\(\times\) magnification. SEM images were taken of representative zirconia samples of each subgroup in three spots. Sample regions with pronounced surface changes are shown.  
Biofilm formation assay  
The microbial biofilms of each bacterial/fungal species were formed on surfaces on different zirconia samples. The following strains were used: _Staphylococcus aureus_ ATCC 11632, _Streptococcus sanquinis_ ATCC 10556 and _Candida albicans_ ATCC 10231. Each strain was formed on 16 samples (4 control--untreated material (F1, F2, C1 and C2) and 12 samples treated with ultrasonic scaling (treatment 60 s) and a brush (treatment 60 s) (F1a/b, F2a/b, C1a/b and C2a/b). Following procedure [22], with some modifications, the samples (dimensions 4 \(\times\) 4 \(\times\) 2 mm) were placed in 200 \(\mu\)L of medium at 37 \({}^{\circ}\)C for 24 h. For _S. aureus_ and _S. sanguinis_ biofilm formation, triple soya broth with 2% glucose (Torlak, Belgrade, Serbia) was used, and for the _C. albicans_ biofilm, yeast extract lepton dextrose (YPD) medium (HiMedia, Thane West, Thane, Maharashtra, India) was used.  
Since only one surface of the samples was treated with ultrasonic scaling or brushing, after incubation, the biofilm was removed mechanically (with alcohol) from all other surfaces. Then, the samples were washed with sterile phosphate-buffered saline (PBS) and placed in sterile plastic tubes containing 500 \(\mu\)L of sterile PBS. Each tube was treated in an ultrasonic bath (40 kHz for 10 min) in order to remove the biofilm from the desired surface. The dilutions were seeded in plate counting agar (PCA) (Neogen, Heywood, UK) and incubated at 37 \({}^{\circ}\)C for 24 h. After 24 h, the colonies were counted using a microprocessor colony counter (Supertek, Digital Colony counter, model No. LT-37, Panchkula, Haryana, India), and the results were presented as colony-forming units (CFU)/plate [23].  
\begin{table}
\begin{tabular}{c c c} \hline \hline  & **Samples Name** & **Samples Code** \\ \hline  & \multicolumn{1}{c}{Polished} & F1 \\ \cline{3-3}  & \multicolumn{1}{c}{Polished, treated with ultrasonic scaling} & F1a \\ \cline{3-3}  & \multicolumn{1}{c}{Polished, treated with brushing} & F1b \\ \cline{3-3}  & zirconia & Glazed & F2 \\ \cline{3-3}  & \multicolumn{1}{c}{Glazed, treated with ultrasonic scaling} & F2a \\ \cline{3-3}  & \multicolumn{1}{c}{Glazed, treated with brushing} & F2b \\ \hline  & \multicolumn{1}{c}{Polished} & C1 \\ \cline{3-3}  & \multicolumn{1}{c}{Polished, treated with ultrasonic scaling} & C1a \\ \cline{3-3}  & \multicolumn{1}{c}{Polished, treated with brushing} & C1b \\ \cline{3-3}  & zirconia & Glazed & C2 \\ \cline{3-3}  & \multicolumn{1}{c}{Glazed, treated with ultrasonic scaling} & C2a \\ \cline{3-3}  & \multicolumn{1}{c}{Glazed, treated with brushing} & C2b \\ \hline \hline \end{tabular}
\end{table}
Table 1: Samples used in the study.  
Data were analysed and presented by a Microsoft program (Excel Spreadsheet, Software Microsoft 365, Excel 2010). The average values and the standard deviations were calculated as colony-forming units (CFU). Statistical analysis was performed by the Student's \(t\)-test, with * = \(p\)\(\leq\) 0.05. The results of the treated samples were compared with the results of the untreated control samples from the same materials.