Microbiological samples were collected of supragingival and subgingival plaque, saliva and the tongue. Pooled supragingival plaque samples were taken with the aid of curettes at the four single-rooted teeth with the deepest initial pocket in each quadrant. Before sampling, the sites were isolated from saliva with the aid of cotton rolls and then dried with compressed air. All supragingival plaque from these sites was dispersed. Samples were dispersed using a vortex mixer and immediately frozen at -20°C until analysis. On the same teeth pooled subgingival plaque samples were collected per site. After 10 s, the samples were transferred to a sterile Eppendorf tube, as described Saliva samples were obtained by collecting unstimulated saliva in a sterile cup. Finally the biofilm of the tongue was collected with a cotton swab. These were wiped 10 times over the tongue starting from the tongue dorsum.