Exclusion criteria included the following: 1) Patients who did not meet inclusion requirements; 2) Patients who did not provide authorization for participation; 3) Patients who were younger than 16 years old; 4) Patients who were pregnant; 5) Patients who were diabetic; 6) Patients with a positive history of antibiotic use within the past month; 7) Patients whose tooth had been previously accessed or endodontically treated; 8) Teeth with root resorption, and 9) Immature/open apex, or a root canal in which patency of the apical foramen could not be established were all excluded from the study. Teeth with periodontal pockets deeper than 4 mm, or the presence of a periapical radiolucency more than 2 cm diameter also were excluded of the study.