BACKGROUND: Periodontitis is caused by a highly complex consortium of bacteria that establishes as biofilms in subgingival pockets. It is a disease that occurs worldwide and its consequences are a major health concern. Investigations in situ are not possible and the bactieral community varies greatly between patients and even within different loci. Due to the high complexity of the consortium and the availability of samples, a clear definition of the pathogenic bacteria and their mechanisms of pathogenicity is still not available. In the current study we addressed the need of a defined model system by advancing our previously described subgingival biofilm model towards a bacterial composition that reflects the one observed in diseased sites of patients and analysed the structure of these biofilms. RESULTS: We further developed the growth media by systematic variation of key components resulting in improved stability and the firm establishment of spirochetes in the 10-species subgingival Zurich biofilm model. A high concentration of heat-inactivated human serum allowed the best proliferation of the used species. Therefore we further investigated these biofilms by analyzing their structure by confocal laser scanning microscopy following fluorescence in situ hybridisation. The species showed mutual interactions as expected from other studies. The abundances of all organisms present in this model were determined by microscopic counting following species-specific identification by both fluorescence in situ hybridisation and immunofluorescence. The newly integrated treponemes were the most abundant organisms. CONCLUSIONS: The improved model generates stable and reproducible biofilms that allow further investigations of interactions between individual species and of the effects of athmospheric or nutrional changes. It provides a basis for analyses of the EPS matrix. The composition of the biofilms indicates a high pathogenic potential, which could be used to examine interactions of these biofilms with host cells.