Characterization and morphological methods for oral biofilm visualization: where are we nowadays?

The oral microbiome represents an essential component of the oral ecosystem whose symbiotic relationship contributes to health maintenance. The biofilm represents a state of living of microorganisms surrounding themselves with a complex and tridimensional organized polymeric support and defense matrix. The substrates where the oral biofilm adhere can suffer from damages due to the microbial community metabolisms. Therefore, microbial biofilm represents the main etiological factor of the two pathologies of dental interest with the highest incidence, such as carious pathology and periodontal pathology. The study, analysis, and understanding of the characteristics of the biofilm, starting from the macroscopic structure up to the microscopic architecture, appear essential. This review examined the morphological methods used through the years to identify species, adhesion mechanisms that contribute to biofilm formation and stability, and how the action of microbicidal molecules is effective against pathological biofilm. Microscopy is the primary technique for the morphological characterization of biofilm. Light microscopy, which includes the stereomicroscope and confocal laser microscopy (CLSM), allows the visualization of microbial communities in their natural state, providing valuable information on the spatial arrangement of different microorganisms within the biofilm and revealing microbial diversity in the biofilm matrix. The stereomicroscope provides a three-dimensional view of the sample, allowing detailed observation of the structure, thickness, morphology, and distribution of the various species in the biofilm while CLSM provides information on its three-dimensional architecture, microbial composition, and dynamic development. Electron microscopy, scanning (SEM) or transmission (TEM), allows the high-resolution investigation of the architecture of the biofilm, analyzing the bacterial population, the extracellular polymeric matrix (EPS), and the mechanisms of the physical and chemical forces that contribute to the adhesion of the biofilm to the substrates, on a nanometric scale. More advanced microscopic methodologies, such as scanning transmission electron microscopy (STEM), high-resolution transmission electron microscopy (HR-TEM), and correlative microscopy, have enabled the evaluation of antibacterial treatments, due to the potential to reveal the efficacy of different molecules in breaking down the biofilm. In conclusion, evidence based on scientific literature shows that established microscopic methods represent the most common tools used to characterize biofilm and its morphology in oral microbiology. Further protocols and studies on the application of advanced microscopic techniques are needed to obtain precise details on the microbiological and pathological aspects of oral biofilm.