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Previous studies have demonstrated antimicrobial substantivity in root canal dentin up to 7 days after treatment with chlorhexidine. This in vitro study assessed the antimicrobial substantivity of chlorhexidine-treated bovine root dentin over a period of 21 days. Sixty standardized bovine root sections were randomly divided into three equal groups, and their canals immersed in one of the following solutions: (i) sterile saline; (ii) 2.5% NaOCl; or (iii) 0.2% chlorhexidine (CHX). Half the specimens in each group were treated with the solution for 5 min and the other half for 7 days. After solutions were removed, the specimens were incubated at 37°C in Brain Heart Infusion broth containing Enterococcus faecalis (ATCC 29212). A fresh inoculum was added to the broth every other day over a 21-day period. The canals were then enlarged with sterile burs, and the dentin shavings collected and cultured for the presence of …