Preparation of the specimens and inoculation of the teeth have been described previously [10 (link),34 (link)]. The crown of 78 human single-canal premolars was separated from the roots, and the roots were prepared with rotary nickel-titanium files Pro Taper Gold F1 and F2 (Dentsply, York, PA, USA) under irrigation with NaCl 0.9%. Along the roots, two external grooves were prepared longitudinally on opposing sides, in order to be able to bisect the roots. Next, the specimens were sonicated for 10 min in an ultrasonic bath in the presence of tryptic soy broth (TSB, Merck, Darmstadt, Germany), sterilized by autoclaving for 10 min, and embedded with 3% agarose in 1.5 mL conical tubes (Eppendorf, Hamburg, Germany).
The root canals of the teeth were then inoculated with E. faecalis. Therefore, a culture of E. faecalis was grown from a single colony for 16 h in TSB medium at 37 °C. The culture was afterwards diluted to ≈1.5 × 108 CFU/mL in fresh TSB medium. The root canals of the teeth were inoculated twice on two consecutive days with 10–20 µL of the diluted culture of E. faecalis (depending on the size of the root canal), and incubated for 6 weeks aerobically at 37 °C. The medium was exchanged every other day.
The root canals of the teeth were then inoculated with E. faecalis. Therefore, a culture of E. faecalis was grown from a single colony for 16 h in TSB medium at 37 °C. The culture was afterwards diluted to ≈1.5 × 108 CFU/mL in fresh TSB medium. The root canals of the teeth were inoculated twice on two consecutive days with 10–20 µL of the diluted culture of E. faecalis (depending on the size of the root canal), and incubated for 6 weeks aerobically at 37 °C. The medium was exchanged every other day.
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