Sterile Tooth Sampling for Oral Microbiome Analysis

Samples were collected on teeth isolated with a rubber dam and using strict aseptic techniques. The tooth surface was cleaned with 30% hydrogen peroxide for one minute and NaOCl 3% for two minutes. The operation field (tooth, rubber dam, and clamp) was swabbed with 5% iodine tincture. To control the sterility of the operation field, samples of the disinfected tooth crown were taken with two foam pellets (Disposable Mini-Sponge Applicator; 3M ESPE, St. Paul, MN, USA) damped in thiosulfate solution (5%; surface control). One pellet was placed in fluid thioglycollate medium supplemented with agar (FTM). If growth occurred after seven days of aerobic incubation, the sample was excluded from the study. The other pellets were stored at −80 °C in Tris-EDTA (TE-buffer, Sigma, St. Louis, MO 63178, USA) for further DNA analysis.
For root canal samples, the access cavity was prepared with a sterile carbide bur, canals were gently filed with K-files (Dentsply Sirona Endodontics), and filled using a syringe containing sterile saline solution. The contents of the root canal were absorbed into sterile paper points and transferred to FTM. The paper points were moved to TE-buffer (1 mL) and ten-fold serial dilutions (0–10⁴) were cultured on fastidious anaerobic agar (FAA, Svenska LABFAB, #ACU-7531A) in an anaerobic atmosphere (5% CO₂, 10% H₂, 85% N₂, 37 °C) for one week. Colony-forming units (CFU) were counted, and colonies with different phenotypic patterns were selected from each plate for bacterial typing. Two colonies of each phenotypic pattern were collected. The remaining sample (800 µL) containing the paper points was stored at −80 °C for DNA extraction. The experimental workflow is presented in Figure 1. Up to 10 isolates with different phenotypic patterns were selected from each plate, amplified by PCR, and sequenced to identify bacterial species as previously described [13 (link)]. Thus, aliquots of the 16S rDNA PCR products were purified and sequenced with Sanger (Eurofins MWG Operon, Ebensburg, Germany), and sequences were compared with the eHOMD database (Expanded Human Oral Microbiome Database, Forsyth, (http://www.ehomd.org)).