## 2 Materials and Methods
### Microbiological Assay  
The representative strains selected for this study were obtained from the American Collection of Type Cultures: Streptococcus mutans (ATCC 25175), Lactobacillus rhamnosus (ATCC 9595), Candida albicans (ATCC 90028), and Candida albicans (ATCC 10231) [32; 33; 31]. Zirconia crowns were used as the dental material, specifically two crowns of size BIL (second primary incisor size 1). The test samples were sterilized in a Vacuklav 24B+ autoclave (Melag, Germany) using the steam method, in accordance with European standard EN 13060, at a temperature of 121 \({}^{\circ}\)C.  
A quantitative assessment was performed to evaluate the adhesion capacity of the analyzed microbial strains and their ability to form a mixed biofilm on the selected dental material. Microbial suspensions were prepared from fresh cultures and adjusted to a  
Figure 1: NuSmile\({}^{\circledR}\) zirconia crowns, size BIL (Houston, USA), used in pediatric dentistry: (**A**) frontal view; (**B**) lateral view. McFarland standard of 0.5, corresponding to 1.5 \(\times\) 10\({}^{8}\) CFU/mL for bacterial strains and 1.5 \(\times\) 10\({}^{6}\) CFU/mL for fungal strains. Brain Heart Infusion Broth (BHI) supplemented with 5% sucrose (Biomaxima, Lublin, Poland) served as the growth medium. Two microbial mixtures were prepared by combining the strains in the following volumes: (i) 400 \(\upmu\)L of _Candida albicans_ ATCC 90028, 400 \(\upmu\)L of _Streptococcus mutans_ ATCC 25175, and 200 \(\upmu\)L of _Lactobacillus rhamns_ ATCC 9595; (ii) 400 \(\upmu\)L of _C. albicans_ ATCC 10231, 400 \(\upmu\)L of _S. mutans_ ATCC 25175, and 200 \(\upmu\)L of _L. rhamns_ ATCC 9595. Sterile dental crowns were then immersed into the prepared microbial suspensions. The samples were incubated for 24 h at 37 \({}^{\circ}\)C under aerobic conditions with elevated CO\({}_{2}\). Following incubation, the crowns were rinsed three times with sterile saline and subsequently vortexed for one minute in 1 mL of 0.5% saponin solution (Sigma-Aldrich, Poznan, Poland) to detach adherent microorganisms. The resulting suspensions were quantitatively cultured on BHI agar (Biomaxima, Lublin, Poland). After incubation, colony-forming units per milliliter (CFU/mL) were enumerated to determine the extent of microbial adhesion and biofilm formation. The CFU/mL value was determined using the following ratio:  
CFU/mL = average number of colonies \(\times\) inverse of dilution \(\times\) 10  
The test was carried out in 2 repetitions. For confocal microscopy analysis, a microbial suspension at a McFarland standard of 0.5 was prepared from fresh cultures of _Streptococcus mutans_, _Lactobacillus rhamns_, and _Candida albicans_ ATCC 90028. A 1 mL mixed suspension, composed of 400 \(\upmu\)L _C. albicans_, 400 \(\upmu\)L _S. mutans_, and 200 \(\upmu\)L _L. rhamns_, was incubated with the biomaterial for 24 h at 37 \({}^{\circ}\)C under aerobic conditions with elevated CO\({}_{2}\) levels. Following incubation, the biomaterial was rinsed with sterile saline and stained using the LIVE/DEAD BacLight Viability Kit (Invitrogen, Life Technologies Corporation, Oregon, USA). After a 30 min incubation period with the staining solution, the samples were washed and prepared for imaging. To immobilize the samples during microscopy, they were mounted on silicone paste and covered with a No. 1.5 coverslip. Initial imaging was conducted using a Zeiss Cell Observer SD confocal microscope. Further high-resolution imaging was performed using a Leica SP8 confocal microscope equipped with an HC Fluotar L 25\(\times\) water immersion objective (numerical aperture [NA] 0.95) and an HC PL Apo 10\(\times\) objective (NA 0.40). The confocal aperture was set to 1 Airy unit. The LIVE stain (Syto9) and DEAD stain (propidium iodide) were both excited using a 488 nm laser, with fluorescence emissions detected in the 493-539 nm range. Additionally, reflected light from a 561 nm laser was collected to visualize the coronal surface of the material.