Potassium tellurite solution

Aliquots of homogenized samples were decimally serially diluted in phosphate-buffered saline and subsequently volume of 25 µl was inoculated to Trypticase soy agar (TSA; pH 7.2 ± 0.1, Carl Roth GmbH and Co., Karlsruhe, Germany) containing 5% ram’s blood and Mitis Salivarius agar (MSA; pH 7.0 ± 0.2, Sigma Aldrich, Steinheim, Germany) with 1% potassium tellurite solution (Sigma Aldrich, Steinheim, Germany) according to Pieri et al.^{21 (link)}. Samples were cultured under aerobic and anaerobic (BBL GasPak Plus, Becton, Dickinson and Co., Maryland, USA) conditions at 37 °C. After 2 days of aerobic and after 3 days of anaerobic cultivation, individual solitary colonies with different morphological characteristics were selected and subcultured to obtain pure bacterial cultures. After 7 days of anaerobic cultivation, plates were again examined for detection of black-pigmented colonies of Porphyromonas. The pure bacterial colonies were transferred to Eppendorf tubes containing Brain Heart Infusion broth (BHI broth; pH 7.4 ± 0.2, HiMedia, Mumbai, India). Subsequently, glycerol (20% v/v) was equally (1:1) added and the isolates were stored at − 80 °C.

Upon arrival at ETC: PANC, samples were homogenized in a stomacher (Stomacher^® 400, Seward) for 3 min at 230 rpm. A 1 mL aliquot was collected and added to 9 mL of Baird Staphylococcus enrichment broth base (Sigma-Aldrich, St. Louis, MO) supplemented with 0.1 mL Potassium Tellurite solution (Sigma-Aldrich, St. Louis, MO), tubes were incubated aerobically at 37°C for 24 h. After incubation, samples were serially diluted in 9 mL of buffered peptone water (BPW, Sigma-Aldrich, St. Louis, MO), spread plated onto CHROMagar Staphylococcus agar plates (CHROMagar, Paris, France), and incubated aerobically at 37°C for 24 h. Based on colony morphology, up to 4 representative colonies were selected from each plate, streaked onto tryptic soy agar plates (TSA, Sigma-Aldrich, St. Louis, MO), and incubated at 37°C for 24 h. A single well-isolated colony was selected from each TSA plate, grown in 9 mL of tryptic soy broth (TSB, Sigma-Aldrich, St. Louis, MO) and incubated at 37°C for 24 h (overnight culture). A 1.5 mL aliquot of the overnight culture was used for gDNA extraction and a 500 μl aliquot was added to 50% glycerol and stored at −80°C for further testing.