## 2 Materials and Methods
### Antimicrobial Studies
#### 2.5.3 Microbial Adhesion and Mixed Biofilm Formation on Nanopowder  
The tooth surface is susceptible to bacterial adhesion, colonisation, and biofilm maturation. A biofilm is defined as an organised cluster of bacteria and fungi. It is a system of numerous synergistic and antagonistic interactions that generate microbial interdependencies and provide it with resistance to external agents. The objective of this study was to investigate the in vitro adhesion capacities of _S. mutans_, _C. albicans_ and _L. rhamn_. _S. mutans_ was selected for investigation in this study due to its well-established association with dental caries and periodontitis [51]. _C. albicans_ is frequently isolated alongside _S. mutans_ from various lesions in children with severe ECC (early childhood caries) infections. _L. rhamn_ plays a role in the formation of deep enamel caries and in the development of dental materials. In the present study, a three-species _Streptococcus-Candida-Lactobacillus_ biofilm was created on Hap and FAp discs in order to reproduce conditions more similar to those found in the oral cavity. The susceptibility of biomaterial surfaces to adhesion in a multispecies environment differs significantly from that observed in a single-species setting [52]. The combination of _S. oralis_ and _C. albicans_ results in the formation of more robust biofilms than would be observed with either microorganism alone. An increase in the invasiveness of _C. albicans_ and the promotion of bacterial biomass proliferation in the presence of _S. mutans_ were observed [53; 54].  
In this scientific work, a suspension of 0.5 McFarland (1.5 \(\times\) 10\({}^{8}\) CFU/mL for bacteria) and (1.5 \(\times\) 10\({}^{6}\) CFU/mL for fungi) density was prepared from fresh cultures of the analysed strains in BHI Broth liquid medium with 5% sucrose (Biomaxima, Lublin, Poland). Subsequently, 100 \(\upmu\)L of _C. albicans_, 100 \(\upmu\)L of _S. mutans_ and 50 \(\upmu\)L of _S. mutans_ were transferred to 750 \(\upmu\)L of liquid BHI medium (Biomaxima, Lublin, Poland) with 5% sucrose. A 1-mL aliquot of the microbial suspension was introduced into the biomaterial, which was then incubated at 37 \({}^{\circ}\)C with elevated CO\({}_{2}\) for 2, 4, 8, and 24 h. Subsequently, the biomaterial was washed three times in NaCl and shaken for one minute in 1 mL of 0.5% saponin solution (Sigma-Aldrich, Saint Louis, MI, USA). A 100 \(\upmu\)L aliquot of the suspension was diluted to 900 \(\upmu\)L NaCl in geometric progression and seeded 50 \(\upmu\)L onto BHI agar (Biomaxima, Lublin, Poland). The incubation was performed at 37 \({}^{\circ}\)C for 24 h under elevated CO\({}_{2}\). The colony-forming units per millilitre (CFU/mL) value was calculated according to the formula: CFU/mL = average colony count \(\times\) reciprocal of dilution \(\times\) 10. The study was performed in triplicate.  
The effect of time and material on strain abundance and susceptibility of biomaterials to bacterial adhesion was assessed using the Relative Treatment Effect (RTE) with the aid of the R-package \"nparLD.\" RTE is a measure of effect size based on the probability of the observation from a given subgroup to be larger than a random observation from other subgroups [55]. This method was chosen due to the very small sample size and, therefore,the inability to reliably check the normality of the distribution of variables, sphericity, and homogeneity of variance, necessary in the analysis of variance for repeated measurements.