Oral biofilms normally exist in dynamic equilibrium with host defenses and are important for preventing colonization by undesirable organisms (1
). However, changes in the composition and metabolic activities of biofilm communities that lead to increases in the proportion of pathogenic species can lead to oral diseases, including dental caries and periodontitis. Despite the importance of oral biofilms to health and disease, studies on the physiology and genetics of oral bacteria were primarily conducted using planktonic populations of bacteria. In recent years, the development of in vitro and in vivo biofilm methodologies to study sessile populations have demonstrated that there are many physiologic and molecular differences between planktonic and surface-bound bacteria, suggesting that the organisms can acquire a “biofilm phenotype” (2
Starting with the premise that “all models are wrong, but some are useful,” a quote attributed to the British statistician George Box, there are a variety of in vitro systems to study oral streptococci biofilms. These include simple and economical models in which bacteria are cultivated in batch systems using different surfaces such as glass, plastic, or hydroxyapatite (HA), the latter being used as a surrogate of tooth enamel. These systems can give reproducible results and can be scaled up to provide sufficient biomass for physiologic and genetic studies. In addition to batch and static systems, the use of shear force in continuous flow systems is considered ideal for the analysis of the dynamics of cell attachment to surfaces and the initial stages of biofilm development. Yet another commonly used system is the so-called constant depth film fermentor in which a scraper intermittently passes over the grown biofilms in wells to achieve constant biofilm depth. Batch and continuous feed systems can also be used to generate more complex multispecies biofilms of known composition, or microcosms can be formed from starter biofilm samples from the body and subsequent cultivation of biofilms in vitro. Because of the natural heterogeneity of these more complex biofilms, the interpretation of the behavior of these populations is very challenging.
Here, we focus on batch-culture systems that our laboratories have routinely used for studying the physiology of oral biofilms, with a particular emphasis on Streptococcus mutans biofilms. One of the advantages of the models presented in this chapter is that multiple biofilms can be formed simultaneously, which provides significant benefit in establishing reproducibility of the data and reducing variance. In addition, test agents can be applied and removed from the system instantaneously allowing a tightly controlled substance exposure time. Moreover, biofilms formed on glass slides or HA are amenable to confocal and electron microscopy and can yield a quantity of bacterial biomass that is sufficient for enzymatic assays. Finally, these model systems can be easily adapted for studies with non-streptococcal species.