*2.1. Sampling*

This study was approved by the Institutional Review Board of the School of Dentistry, Kyung Hee University (KHD-IRB-1509-5). Ten children were recruited as study subjects. Six of the children had black stained teeth while four children maintained good oral hygiene and had no black stained teeth. Oral examinations were performed on all the children, and decayed-missing-filled teeth (DMFT) indices were estimated. The BS scale was estimated using the Shourie method [10]. The criteria for classifying BS were as follows: 1; no line, 2; incomplete coalescence of pigmented spots and 3; continuous line formed by pigmented spots. After classification, subjects with parental consent were instructed not to brush their teeth for 24 h before sampling. Sampling was performed in the morning from the subjects, who were instructed to skip breakfast. Sterilized dental explorers were used to collect supragingival plaque on the buccal and lingual surfaces of all teeth. The samples were collected in 1 mL of sterile phosphate-buffered saline and stored at −80 ◦C after liquid nitrogen treatment.
