actinomycetemcomitans JCM8577, A. actinomycetemcomitans SUNYaB67, A. actinomycetemcomitans SUNYaB75, Aggregatibacter naeslundii JCM8350, Prevotella loescheii JCM8530, Prevotella denticola JCM8525, Prevotella bivia JCM6331, Prevotella pallens JCM1140, Prevotella veroralis JCM6290, and Prevotella oralis ATCC
33322. STI571 Ethics statement All patients were treated in accordance with the Helsinki Declaration regarding the participation of human subjects in medical research. Ethics clearance for the study was obtained from the Ethics Committee of Kyushu Dental University Hospital (reference number 11–40). The parents of participants were fully informed about the study and signed informed consent forms. Study subjects and oral specimen sampling Twenty-one subjects ranging in age from 3 to 10 years and who had dental caries were included in the caries group (mean age ± S.D. = 7.86 ± 0.43 years; 11 males and 10 females). A healthy (completely caries-free) control group consisted of 24 subjects (ages 3 to 12 years; mean age ± S.D. = 7.29 ± 0.56 years; 13 males and 11 females). The carious dentin Selleck RG 7204 was
excavated from cavitated lesions. Before excavation of the carious dentin, the plaque on the surfaces of cavitated lesions was swiped. The dental plaque samples from healthy subjects were collected from the buccal or lingual surface of the second primary molar. Collected carious dentin and dental plaque were placed in 200 μl of PBS in a sterile 1.5-ml microcentrifuge tube. These samples were washed and placed in PBS solution adjusted to 1 mg per 100 μl. Saliva was collected
from both the caries and healthy control groups. Fifty microliters Ribociclib solubility dmso of saliva was washed with PBS and used for analysis. Bacterial counting from oral specimens on an agar plate Serially diluted carious dentin or dental plaque was plated on a Mitis-Salivarius agar plate (Becton Dickinson, Franklin Lakes, NJ) supplemented with 150 g/l sucrose and 200 U/l bacitracin for selection of mutans streptococci (MSB agar). Bacterial counting was performed using a magnifying loupe. Propidium monoazide treatment For only viable cell quantification, PMA (3-amino-8-azido-5-[3-(diethylmethylammonio)propyl]-6-phenyl dichloride; Wako Pure Chemical, Osaka, Japan) treatment was performed for bacterial cells prior to DNA extraction, as previously described [19]. Briefly, PMA was dissolved in 20% DMSO to produce a 25-mM stock solution. Following incubation with the dye for 5 min in the dark, similarly prepared cells were exposed for 5 min to a 500-W halogen light placed 15 cm above 500-μl samples in open microcentrifuge tubes on ice. The toxicity of PMA at 2.5–250 μM to S. mutans and S. sobrinus was analyzed at 37°C. In the present study, 25 μM PMA was employed for the analysis. All data presented are from triplicate independent cultures and/or biofilms.