# Surface Functionalization of Polyethylene by Silicon Nitride Laser Cladding
## 3 Experimental Results
### In Vitro Bacterial Testing  
A Microbial viability assay (WST) showed the amount of live bacteria on different sample surfaces (Figure 5). After 12 h of exposure, the Si\({}_{3}\)N\({}_{4}\)-cladded PE showed a lower amount of living bacteria as compared with both the negative (bulk PE) and the positive (Si\({}_{3}\)N\({}_{4}\)) control. After 24 h the number of bacteria on both the pristine and the laser-cladded PE samples increased, with the latter still showing better antibacterial performance. It should be noted that the positive Si\({}_{3}\)N\({}_{4}\) control, which showed a relatively higher amount of bacteria at 12 h, performed better at 24 h. This is in line with previous results on Si\({}_{3}\)N\({}_{4}\), where the \"activation\" of the surface antibacterial effects reflects the kinetics of released nitrogen species, in particular when the surfaces are polished [22]. At the end of the treatment (48 h) the presence of the bacteria on the Si\({}_{3}\)N\({}_{4}\)-cladded PE exhibits twice as high amounts of bacteria as compared to 24 h, but the values of Optical Density are still lower compared with the negative control.  
\begin{table}
\begin{tabular}{c c c c} \hline
**Bands Position (cm\({}^{-1}\))** & **Assignments** & **Bands Position (cm\({}^{-1}\))** & **Assignments** \\ \hline
184 & E\({}_{26}\) & 1062 & C-C stretching \\
204 & A\({}_{8}\) & 1081 & C-C stretching \\
227 & E\({}_{16}\) & 1129 & C-C stretching \\
448 & E\({}_{26}\) & 1170 & CH\({}_{2}\) rocking \\
613 & E\({}_{26}\) & 1271 & CH\({}_{2}\) twisting \\
727 & A\({}_{8}\) & 1295 & CH\({}_{2}\) twisting \\
857 & E\({}_{16}\) & 1304 & CH\({}_{2}\) wagging \\
922 & E\({}_{26}\) & 1369 & CH\({}_{2}\) bending \\
933 & A\({}_{8}\) & 1418 & CH\({}_{2}\) bending \\
1040 & E\({}_{26}\) & 1440 & CH\({}_{2}\) bending \\  & & 1460 & CH\({}_{2}\) rocking \\ \hline \end{tabular}
\end{table}
Table 1: Raman band assignments for Si\({}_{3}\)N\({}_{4}\) (left) and PE (right).  
Figure 5: Biological assay of _Staphylococcus epidermidis_ as a function of bacterial exposure time.  
Fluorescence microscopy analyses (Figures 6 and 7) showed the presence of dead and living bacteria on the different substrates. Three stains were used to identify the different bacterial states: CFDA green stain was used for live bacteria, PI red stain for dead bacteria, and DAPI blue stain for the nuclei of the bacteria. At all testing times, the PE substrate (Figure 6) showed higher amounts of live bacteria on its surface. The number of dead bacteria was very low as compared to the total number of bacteria present on the surface (expressed by the number of nuclei stained blue). A greater number of bacteria were present on the surface of the Si\({}_{3}\)N\({}_{4}\)-cladded PE (Figure 7, _cf._ blue stain), but most of them were dead (_cf._ red stain).  
Figure 6: Fluorescence micrographs of the PE substrate after bacteria testing. Green stain (CFDA) marked the live bacteria, the red stain (PI) the dead bacteria, and the blue stain (DAPI) the bacterial cells’ nuclei.  
Figure 7: Fluorescence micrographs of the Si\({}_{3}\)N\({}_{4}\)-coated PE substrate after bacteria testing. Green stain (CFDA) marked the live bacteria, the red stain (PI) the dead bacteria, and the blue stain (DAPI) the bacterial cells’ nuclei.