Techniques for the growth of Porphyromonas gingivalis biofilms

We have described three systems used to grow P. gingivalis biofilms, two static systems and one flow-cell system. The first was a 96-well microtiter dish assay, based primarily on a protocol developed by Denis Mayrand and colleagues (57). This assay can be used as a first step in assessing biofilm formation by different strains of P. gingivalis. Furthermore, by modifying the CDM used in this assay by the addition of different carbon and energy sources, one can assess nutritional effects on biofilm formation. Although many studies on biofilm development use saliva as a preconditioning film or nutrient source, we propose that serum is more relevant for the study of P. gingivalis, because it resides in the subgingival crevice and is therefore more likely to be exposed to gingival crevicular fluid. It has been shown that P. gingivalis can grow on human serum diluted 1 : 10 in water that is supplemented with 0.01% hemin (57), and our preliminary studies indicate that P. gingivalis can make a biofilm when growing in CDM supplemented with 10% human serum (unpublished data). The development of our protocol using a CDM will be instrumental in future nutritional studies. The second system we describe is a 12-well assay that can be used to microscopically examine biofilms. Since it is not a flowing system, this system cannot be used to study the different stages of biofilm development over time, however it is a good method to inspect the biofilms visually at an early time point. Using this assay, we were able to obtain data showing that the three different wild-type strains used in this study varied greatly in their ability to form a biofilm. Most striking is our finding that strain W83 is unable to initiate biofilm formation. This strain is distinct from the other two strains. Unlike 33277 and 381, strain W83 is encapsulated and does not typically produce fimbriae. Since both of these structures affect the surface properties of the cell, and thus affect cell-cell and cell-surface interactions, we propose that this is likely to contribute to the inability of this strain to attach to the surface and initiate biofilm formation. We also describe the adaptation of a flow-cell system to grow biofilms of P. gingivalis anaerobically. The data presented are the first report of a fully developed P. gingivalis biofilm (constant flow of nutrients for 4 days). As described above, a 4-day-old P. gingivalis biofilm demonstrates characteristics that are typically associated with a mature biofilm, i.e. a heterogeneous architecture consisting of large macrocolonies surrounded by open areas - not a dense monolayer of cells on the surface. Also, the thickness of the biofilm was found to be approximately 200 μm, which is comparable to measurements obtained in studies on other model organisms. This indicates that although there are probably many differences in the nutritional and environmental parameters that regulate biofilm development by different bacteria, there are some characteristics (such as biofilm structure) that are conserved. It has been proposed that this architecture supports survival by facilitating the delivery of nutrients and the removal of end products (5), thus the development of a biofilm into this architecture may be a conserved characteristic. Further studies on biofilm development will likely enlighten us to other strategies that are used by bacteria to survive under different growth parameters. This flow-cell apparatus can be used to examine biofilm development under different growth conditions and to obtain biomass for chemical analyses or for the isolation of RNA or protein. The system is based on the principle that fresh medium is continuously supplied to a flow cell chamber. The walls of the chamber provide surfaces on which biofilm develops. One side of the chamber is a glass coverslip, and the chamber itself is small enough to be mounted on the stage of a microscope, so the biofilm can be imaged by either epifluorescent or confocal microscopy. In addition, each channel in the four-channel flow cell can be inoculated with a different strain by using a fine-gauge needle and syringe. The advantage of the four-channel system is that a single media reservoir and a single pump can be used to feed the four channels simultaneously, helping to maintain consistency from channel-to-channel and allowing the analysis of up to four different strains per experiment. Flow cells such as the one described here have been an instrumental tool in the study of biofilm morphology, chemistry, and physical parameters using a variety of model organisms (48, 49). Now that we have a system to grow P. gingivalis biofilms, we can begin to study the nutritional and environmental parameters that regulate development, as well as identify the genes that are expressed during different stages of biofilm growth. Since biofilm formation is essential to the persistence of this organism, such studies should lead to a greater understanding of how to control this opportunistic oral pathogen. © 2006 The Author.