Anaerobic Cultivation of Oral Pathogens

Porphyromonas gingivalis ATCC 33277, Fusobacterium nucleatum CCUG 9126, Aggregatibacter actinomycetemcomitans ATCC 33384 and Streptococcus gordonii ATCC 35105 were acquired from the American Type Culture Collection (ATCC). The cultures were maintained in Horse Blood Agar (HBA) supplemented with 5 µg/mL of Hemin and 1.0 µg/mL of Vitamin K at 37 °C in an anaerobic chamber (5% CO₂, 10% H₂ and 85% N₂). For all the experiments, the bacteria were grown for 72 h in Tryptic Soy Broth (TSB) supplemented with 5 g/L of Yeast extract (YE), 5 µg/mL of hemin and 1.0 µg/mL of vitamin K at 37 °C [37 (link)] in an anaerobic chamber (5% CO₂, 10% H₂ and 85% N₂).
DMTU stock was prepared using 1% DMSO (Sigma Aldrich, St. Louis, MO., United states) as a solvent [33 (link)]. In all the experiments, growth media without DMTU served as the positive control while media with 1% DMSO was considered as a vehicle control. Growth medium without the culture served as a negative control. All experiments were performed in triplicates in three independent experiments.