The procedure is essentially as previously described with some modifications (Kirsch et al., 2017 (link)). Suitable extracted human single-canal premolars were selected and stored in 0.1% thymol. Then the crowns of the teeth were separated from the roots and the roots were prepared with Pro Taper Gold F2 (Dentsply) under irrigation with sodium chloride (0.9%). Next, the roots were placed for 10 min in an ultrasonic bath with tryptic soy broth (TSB, Merck, Germany) to prepare the teeth for the bacterial incubation with E. faecalis. Then the roots were sterilized by autoclaving for 20 min in TSB and embedded with 3% agarose in 1.5 ml conical tubes (Eppendorf).
For inoculation of teeth root canals, E. faecalis was grown from a single colony for 16 h in TSB. Root canals of human premolars were then inoculated on two consecutive days with ≈1.5 × 108 CFU/ml (≈10–20 μl per root canal depending on the size of the canals). Teeth were incubated aerobically at 37°C for 3 weeks unless indicated otherwise. Medium was exchanged every day. Teeth were grinded using a 6775 Freezer/Mill® Cryogenic Grinder (SPEX®) with the following program: Precool 10 min, Run Time 1 min, Cool Time 1 min, Cycles 4, Impactor Rate 12. During the grinding procedure, teeth were constantly cooled with liquid nitrogen. The tooth powder was then transferred into 1.5 ml conical tubes.
For inoculation of teeth root canals, E. faecalis was grown from a single colony for 16 h in TSB. Root canals of human premolars were then inoculated on two consecutive days with ≈1.5 × 108 CFU/ml (≈10–20 μl per root canal depending on the size of the canals). Teeth were incubated aerobically at 37°C for 3 weeks unless indicated otherwise. Medium was exchanged every day. Teeth were grinded using a 6775 Freezer/Mill® Cryogenic Grinder (SPEX®) with the following program: Precool 10 min, Run Time 1 min, Cool Time 1 min, Cycles 4, Impactor Rate 12. During the grinding procedure, teeth were constantly cooled with liquid nitrogen. The tooth powder was then transferred into 1.5 ml conical tubes.