A Novel Laboratorial Approach to Evaluate Bacterial Microleakage of Endodontic Sealers.

To avoid failure of endodontic treatment, it is important to properly seal the root canals after chemomechanical preparation in order to avoid microleakage. Typically, sealing involves the use of gutta-percha and sealing cement, the latter of which adheres to the dentin and fills the spaces between the gutta-percha and the dentin walls. Laboratory studies have been performed to detect microleakage of endodontic sealers based on various criteria, including microbiological ones; however, none guaranteed that the leakage occurred through the root canal space and not through other pathways. This article proposes a new laboratory assay to evaluate bacterial microleakage in endodontic sealers, ensuring visualization of the correct inoculum infiltration pathway. Furthermore, we present the main critical parameters and provide suggestions on how to manage them. These protocols allow the detection of bacterial microleakage in bovine teeth filled with endodontic sealer and inoculated with Enterococcus faecalis. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Bovine teeth selection and cleaning Basic Protocol 2: Bovine teeth inclusion Basic Protocol 3: Tooth standardization Basic Protocol 4: Set preparation Basic Protocol 5: Inoculum preparation Basic Protocol 6: Canal inoculation Basic Protocol 7: Set incubation.