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The purpose of this study was to determine the effect of a separated rotary instrument on the time required for bacterial penetration of obturated root canalsTwenty-six extracted human mandibular premolars with single canals were used in this study. Group 1 consisted of teeth that contained a separated size 40 K3 rotary file, and were obturated with gutta-percha and AH-26 sealer to the level of the separated file. Group 2 consisted of teeth that were obturated similar to Group 1 but without a separated file. The negative control canals were obturated and had the entire root surface sealed with nail varnish. The positive controls were obturated without sealer. Enterococcus faecalis was placed in the access chamber daily, and penetration was determined when turbidity was noted in the culture broth.The results showed no significant difference between Groups 1 and 2. However, differences between the experimental groups and control groups were statistically significant.Under the conditions of the present study, the separated instrument itself does not play a significant role in the sealing ability of the obturation material.