The purpose of this study is to understand the effect that 3 different types of root canal procedures have on bacteria.

Sample Size Calculation Sample size was calculated by STATISTICA v8.0 software (StatSoft, Tulsa, OK, USA), that demonstrated 23 teeth per group are needed to show a 5% difference in bacterial levels at 90% power. Considering possible losses, ninety adult subjects will be selected.

Case Selection and Eligibility Criteria Participants will be recruited at the Endodontic Clinic at the University of Iowa. The inclusion criteria will be patients 18 years or older, teeth with intact pulp chamber walls, no response to cold and electric pulp testing and apical periodontitis.

Teeth with vital pulp, incomplete root formation, extensive crown destruction, previous endodontic treatment or intervention (pulp debridement), acute/ chronic apical abscess, internal or external resorption, non-odontogenic facial pain, periodontal probing deeper than 4 mm, advanced untreated periodontal disease or recent periodontal surgery, teeth with mobility score > 2, fracture or visible crack, patients with diabetes or immune-compromised conditions, patients who received systemic antibiotics within the last three months, or taking corticosteroids, and pregnancy will be excluded.

Study Intervention Root canal samples will be taken under strict asepsis, as described by Rodrigues et al. (21). Operative field will be disinfected using 3% hydrogen peroxide and 3% NaOCl under rubber dam isolation. Once the access cavity is prepared, 10% sodium thiosulfate solution will be injected into the pulp chamber, and the sterility control (SC) will be taken from the access cavity walls' internal surfaces using sterile paper points. This sample will be transferred aseptically to a cryotube containing RNA later (Ambion, Austin, TX) and stored at -20°C.

The first sample (S1) will be taken as the baseline quantity of bacteria before chemomechanical preparation. One ml of 10% sodium thiosulfate solution will be injected into the canal, and a sterile paper point will be introduced up to that length for 1 minute, transferred in cryotubes containing RNA later, and stored at -20°C.

The root canal will be shaped and cleaned using rotary endodontic instruments up to size 35/.04, with periodic irrigation with 5 mL of 3% NaOCl between each instrumentation. After apical preparation, the canal will be dried with sterile paper points and then washed with 1 mL of 10% sodium thiosulfate for 1 minute to inactivate NaOCl. After that, a second sample (S2) will be collected and stored as described for S1.

Teeth specimens will be randomly assigned to three groups (n=30) according to the type of supplementary disinfection approaches used: (1) GWS, (2) LAI, and (3) UAI. These devices will be used as stated above in phase 1 of this study. After using supplementary disinfection approaches, a third sample (S3) will be collected from the root canal and stored as described above for S1 and S2.

Inclusion Criteria:

• Diagnosis of pulp necrosis with apical periodontitis (AP) confirmed by pulp tests, clinical and radiographic evidence.
• Tooth with no response to cold and electric pulp testing (done at screening).
• Single canalled premolars with intact pulp chamber walls.
• Patient must be 18 years of age or older

Exclusion Criteria:

• Teeth with vital pulp.
• Teeth with incomplete root formation (immature with apical periodontitis).
• Teeth with extensive crown destruction.
• Teeth with previous endodontic treatment or intervention (pulp debridement).
• Teeth with acute/ chronic apical abscess.
• Teeth with internal or external resorption.
• Teeth with non-odontogenic facial pain.
• Teeth with periodontal pockets deeper than 4 mm.
• Teeth with advanced untreated periodontal disease or recent periodontal surgery.
• Teeth with mobility score greater than 2.
• Teeth with a fracture or visible crack.
• Patients with diabetes or immune compromised conditions.
• Patients who received systemic antibiotics within the last 3 months.
• Patients taking corticosteroids.
• Patients who are pregnant.