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The oral microbiota, which is known to include at least 600 different bacterial species, is found on the teeth and mucosal surfaces as multi-species communities or biofilms. The oral surfaces are covered with a pellicle of proteins absorbed from saliva, and biofilm formation is initiated when primary colonizers, which express surface adhesins that bind to specific salivary components, attach to the oral tissues. Further development then proceeds through co-aggregation of additional species. Over time, the composition of oral biofilms, which varies between different sites throughout the oral cavity, is determined by a combination of environmental factors such as the properties of the underlying surface, nutrient availability and oxygen levels, and bacterial interactions within the community. A complex equilibrium between biofilm communities and the host is responsible for the maintenance of a healthy biofilm phenotype (eubiosis). In the face of sustained environmental perturbation, however, biofilm homeostasis can break down giving rise to dysbiosis, which is associated with the development of oral diseases such as caries and periodontitis.In vitro models have an important part to play in increasing our understanding of the complex processes involved in biofilm development in oral health and disease, and the requirements for experimental system, microbial complexity, and analysis techniques will necessarily vary depending on the question posed. In this chapter we describe some current and well-established methods used in our laboratory for studying oral bacteria in biofilm models which can be adapted to suit the needs of individual users., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Secreted and transmembrane mucins are important components of innate defence at the body's mucosal surfaces. The secreted mucins are large, polymeric glycoproteins, which are largely responsible for the gel-like properties of mucus secretions. The cell-tethered mucins, however, are monomeric but are typically composed of two subunits, a larger extracellular subunit which is heavily glycosylated while the smaller more sparsely glycosylated subunit has a short extracellular region, a single-pass transmembrane domain, and a cytoplasmic tail. These two families of mucins represent high-molecular-weight glycoproteins containing serine and threonine-rich domains that are the attachment sites for large numbers of O-glycans. The high-M ( r ) and high sugar content have been exploited for the separation of mucins from the majority of components in mucus secretions. In this chapter, we describe current and well-established methods (caesium chloride density-gradient centrifugation, gel-filtration and anion-exchange chromatography, and agarose gel electrophoresis) for the extraction and purification of gel-forming and cell-surface mucins which can subsequently be used for a variety of structural and functional studies.