Development of an in situ sers based method for observation of biofilm formation

A biofilm is an assembly composed of microbial cells and extracellular polymeric substances, which provides and supports microorganisms for attach themselves onto a surface irreversibly and protect them from environmental stress conditions. Monitoring the in situ molecular changes during a biofilm formation can provide valuable insights in the fields including medicine, biology and related industrial processes. The conventional molecular and microscopy techniques are time consuming due to cumbersome sample preparation steps and destructive nature. With the aim of better understanding biofilm formation and possibility of detection, in this study, biofilm formation of clinically important microorganisms, Pseudomonas aeruginosa, Staphylococcus epidermidis and Candida albicans were monitored by utilizing surface-enhanced Raman scattering (SERS). Clinically relevant microorganisms threaten patient health often through biofilm formation on the surface of polymeric medical devices and implants. In this thesis work, the biofilm characteristics of the model microorganisms were identified on agar plates, 2D and 3D poly (methyl methacrylate) substrates and 3D glucose-gelatin scaffolds with the aim of understanding the influence of substrate type on biofilm formation process. The significant concentration changes on carbohydrates, lipids, proteins and genetic materials with increasing incubation time provided information about biofilm formation process. Moreover, the spectral data was attempted to confirm with confocal laser scanning microscopy and scanning electron microscopy analyses. The discrimination of microorganisms was also demonstrated from the SERS spectra using principle component analysis and linear discrimination analysis. A further step was taken in the way to clinical application of the approach by monitoring biofilm formation by using a multi-species sample. In summary, the results present a comprehensive evaluation of the applicability of SERS in clinically relevant biofilm formation with exciting outputs and opportunities.