Cultivation of Oral Pathogenic Bacteria

Streptococcus oralis (ATCC 9811; American Type Culture Collection, Manassas, VA, USA) was cultured overnight on Tryptone Soy Agar (TSA) plates at 37 °C. A few colonies were inoculated into Tryptone Soy Broth (TSB) (Oxoid Limited, Hamsphire, UK) supplemented with 10% yeast extract (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and 50 mM glucose (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) at 37 °C under constant shaking. Aggregatibacter actinomycetemcomitans (DSM 11123; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) or Porphyromonas gingivalis (DSM 20709; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured for 48 h on fastidious anaerobe agar (FAA) plates (LabM, Heywood, UK), supplemented with 5% sheep blood at 37 °C under anaerobic conditions (80% N₂, 10% H₂, 10% CO₂). A few colonies of A. actinomycetemcomitans or P. gingivalis were inoculated overnight into brain heart infusion medium (BHI; Oxoid, Wesl, Germany) supplemented with 10 μg/mL vitamin K (Roth, Karlsruhe, Germany) under anaerobic conditions [36 (link)]. Treponema denticola (DSM 14222; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) was cultured in new oral spirochete (NOS) medium at 37 °C for 72 h under static anaerobic conditions [37 (link)].