Optimized Protocol for Collecting Root Canal Biofilms for in Vitro Studies

Endodontic retreatment is often necessitated by several factors, including the persistence of microorganisms in the root canal system (RCS). Their complex organization in biofilms increases their pathogenic potential, necessitating new disinfection strategies. This study aimed to standardize a new in vitro protocol for collecting biofilm from the RCS. Thirty-four bovine incisors were used in the study, divided into two experimental groups with two collection steps each: (a) biofilm collection protocol and (b) absorbent paper points protocol. Twelve specimens from each group were selected for counting colony-forming units (CFUs), while eight specimens were prepared for scanning electron microscopy (SEM). Two additional specimens served as sterilization controls to ensure that experiments were free of contamination. The coronal region was removed and standardized at 15 mm. After preparation with ProTaper up to F5, the apical foramen was sealed with composite resin, and the roots were stabilized with acrylic resin in 1.5-mL Eppendorf tubes. The specimens were sterilized and inoculated with Enterococcus faecalis NTCT 775 every 24 h for 21 days. After this period, each group underwent biofilm collection protocols, and CFU and scanning electron microscopy (SEM) data were analyzed. The Shapiro–Wilk test was performed to assess the normality of log-transformed data, and the results indicated a normal distribution for all groups, allowing parametric testing. The Levene test was used to evaluate the equality of variances. The proposed biofilm collection method yielded significantly higher CFU counts compared with the absorbent paper points method, particularly when analyzed on a log₁₀ scale. An independent samples t-test confirmed a statistically significant difference between the two methods (p < 0.0001). The proposed protocol achieved an efficiency rate of 95.85 % ± 1.15 %, whereas the absorbent paper points protocol yielded a lower efficiency of 5.46 % ± 1.37 %. Therefore, the biofilm collection protocol proposed in this study proved to be more effective for biofilm removal from the RCS.