Samples preparation

Five roots containing bacteria were used as positive controls to assess the bacterial viability throughout the experiment. The remaining fifty-two roots were randomly divided into four groups (n = 13) corresponding to the file used for instrumentation. Root canal instrumentation was accomplished according to the manufacturer’s instructions using rotary motion generated by a torque-controlled electric motor (seongseo ro, Daegu, Korea) with a 16:1 gear reduction contra-angle handpiece. During instrumentation, the samples were immersed in a warm water bath at 37 ± 1 °C [25, 26].

In group A, XPS (#30/0.04) was performed at 800 rpm and 1 Ncm. It was applied with gentle and slow up-and-down strokes reaching the working length. In group B, HEDM (#25/ ~) was adjusted at 500 rpm and 2.5 Ncm in up and down movement. In group C, OC (#25/0.06) was operated at 300 rpm and torque 2.5 Ncm in pecking motion till reaching the working length. In group FO (#25/0.06) was operated at 500 rpm and torque 2.6 Ncm in up and down movement. Each file was discarded after shaping four canals.

After each of the four up-and-down movements, all root canals were irrigated with 3 mL 2.5% sodium hypochlorite for 1 min by a 30-gauge irrigation needle. Finally, 5 mL of 17% ethylenediaminetetraacetic acid (EDTA) were used as a final rinse for 3 min. Next, 2 mL distilled water was used.

After instrumentation, the second bacterial samples (S2) were collected from the root canals using three sterilized #20 paper points, as described previously [27]. Aliquots (0.1 mL aliquots) were cultured on M-Enterococcus agar plates and incubated at 37 °C for 48 h. Colonies were counted and transferred to actual counts according to previously recorded dilution factors. Roots from each group were fixed, split longitudinally using a chisel and mallet, and dehydrated by immersion in ethanol (50, 80, 90, 96, and 100%). Each root canal lumen was topographically evaluated in the coronal, middle, and apical segments using SEM [Jeol JSM-6510L.V, Jeol, Tokyo, Japan] at 3000X, 6000X, and 12000X magnifications.

The data were statistically analyzed using the Kruskal–Wallis test, followed by the Mann–Whitney U test, to compare the efficiency of the used files to reduce the bacterial count (P < .05). The data was analyzed using SPSS (statistical package for social science, version 25).