The present randomized controlled clinical trial will be conducted in the Department of Dentistry, AIIMS Guwahati, from January 2026 to July 2027 to compare the efficacy of various irrigant activation techniques on intracanal microbial reduction in single-rooted teeth. Fifty one patients aged 18–65 years requiring root canal treatment in single-rooted teeth with single canals were selected based on specific inclusion and exclusion criteria. Inclusion criteria comprised symptomatic teeth requiring root canal therapy, teeth with healthy periodontium, no anatomical variations, and presence of sinus tract. Exclusion criteria included previously treated, fractured, or resorbed teeth, pregnant or lactating women, and teeth with incomplete root formation or open apices. Eligible patients fulfilling the criteria were informed about the study, and written informed consent was obtained prior to participation.

The patients will be randomly divided into three groups of seventeen each using computer-generated randomization with sealed opaque envelopes to ensure allocation concealment. Group 1 will undergo conventional needle irrigation using 6 mL of 3% sodium hypochlorite (NaOCl) for 90 seconds. Group 2 will recieve ultrasonic activation with three 30-second cycles using 6 mL of 3% NaOCl. Group 3 will undergo diode laser-activated irrigation following the same protocol. All procedures will be carried out under rubber dam isolation. Access cavities will be prepared using Endo Access burs after proper disinfection, and canal patency will be established with a #10 K-file. The first microbial sample (S1) will be collected using a sterile #15 paper point placed 1 mm short of the apex for one minute and transferred to thioglycolate and brain heart infusion (BHI) broths. Thereafter, in the next appointment, biomechanical preparation will be performed with ProTaper Next rotary system. The irrigation protocol used in this study is standardized for all groups, differing only in the method of irrigant activation. During biomechanical preparation, canals will be irrigated with 3% sodium hypochlorite (NaOCl) between each file. After instrumentation, canals will flushed with 3 mL saline, followed by activation of 6 mL of 3% NaOCl for 90 seconds according to group allocation. After activation, canals will be rinsed with 3 mL saline, followed by 1 mL of 17% EDTA for 1 minute to remove the smear layer and a final saline rinse. As the last step, 2% chlorhexidine will be used as the final irrigant to provide residual antimicrobial action. This will be followed by collection of the second microbial sample (S2) .

In the microbiology laboratory, samples from thioglycolate and BHI broths will be cultured on Brucella blood agar and blood agar, respectively. Brucella plates were incubated anaerobically for 72 hours, and blood agar plates will be incubated aerobically for 48 hours to identify aerobic bacteria and E. faecalis. Colony-forming units (CFU) will be counted, and mean CFU values will be calculated to assess the percentage reduction in aerobic, anaerobic, and E. faecalis counts before and after irrigation activation, allowing comparative evaluation among the three groups.