Conical tube

Plates containing hundreds of synchronized day 1 adult worms were washed with 2 mL of sterile milli-Q water and transferred to Eppendorf conical tubes. After decantation, the supernatant containing water and bacteria was removed and 250 μL of a 5 % NP-40 solution was added. The worm suspension was heated at 75 °C for 10 minutes in a thermoshaker at 800 rpm. Samples were then vortexed and cooled at room temperature for 10 minutes. To eliminate the insoluble particles, tubes were centrifuged at 12000 × g for 5 minutes at 4 °C. The supernatant containing the lipids was then transferred to a new tube for triglyceride quantification. Triglyceride quantitation was performed using the LabTest kit according to the manufacturer’s instructions. Lipid extract (2 μL) or standard control (200 mg/dL) were added to 200 μL of detection buffer and the solution was heated at 37 °C for 10 minutes [62 (link)]. The product was quantified at 490 nm using a plate reader (EL808 Ultra Microplate Reader). Concentration of triglycerides was estimated according to the standard control and then normalized by protein levels, as measured by the BCA kit using the same extract.

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.

For each culture condition 20 mL samples were collected in 50 mL falcon tubes. For each condition, four sample aliquots were placed in the dark for 3 h and an additional 3 “light” sample aliquots were irradiated for 3 h. Post incubation, all samples were spun down at 4500 × g for 10 min at 4°C. The “light” and “dark” samples were then decanted of media and the cell pellets were transferred with 500 uL PBS to 1.5 mL Eppendorf conical tubes. The probes were then added to the samples by addition of 1 uL each of 60 mM stock Mal-RP and IAM-RP. Probed samples were incubated in the dark for 60 min at 37°C with 250 rpm shaking. Next, samples were spun down at 4500 × g for 10 min at 4°C to pellet cells, and the supernatant was discarded. Also collected include two 20 mL sample aliquots from each nutrient condition, which were pelleted to be used for no probe control samples and another two 20 mL sample aliquots were pelleted for global proteome analysis. All cell pellets were then frozen in liquid nitrogen and stored till further sample preparation steps. Preparation for LC-MS analysis of probe-labeled and global samples, and quantitative data analysis was performed as described previously (Sadler et al., 2014 (link)). Details of LC-MS analysis are provided in the Supplemental Information text, and was performed as described previously (Sadler et al., 2014 (link)).

Topsoil samples (10 g each) and water samples (approximately 50 mL each) were taken in November 2019 from three heavily polluted sites located at the historical oil field in Wietze (52° 39′ 0″ N, 09° 50′ 0″ E), Germany. In addition, two reference samples were taken from nearby unpolluted soils. Samples were placed into 50 mL Eppendorf conical tubes. The samples were transported to the laboratory on ice.

Household pairs were sampled every 2 weeks, for a total of 6 visits covering 10 weeks. At each visit, the participants provided a self-collected saliva sample (36 ) into an empty 25 mL Eppendorf conical tube and answered questions about their social activities, doctors’ visits, recent contact with children, and any respiratory symptoms they were experiencing or had experienced in the 2 weeks prior (Appendix). Study participants left their samples outside their front door for contactless-collection and transport back to the laboratory at room temperature.