*2.2. Oral Examination*

Two dentists visited the homes of the subjects. An oral examination was carried out with a penlight and a dental mirror. The definition and diagnosis of dental caries were based on the criteria of the World Health Organization [39].

### *2.3. Sample Collection*

As all subjects wore complete dentures, plaque samples were collected from the denture surface based on a method described in previous reports [40,41]. Briefly, the denture was brushed around the buccal surface with a toothbrush for 2 min, followed by immersion of the toothbrush with the attached plaque in sterilized, phosphate-buffered saline (PBS). Tongue coating samples were collected by using a mucosal brush (ERAC 510Lion, Tokyo, Japan). The tongue surface was brushed one way, from back to front, 5 times. Samples were treated in the same way as denture plaque samples. Samples were kept on ice before being transported to the Iwate Dental Association in an ice box with refrigerant, then were stored at −20 ◦C for further analysis.
