Prevotella intermedia

Strains of bacterial
cells, gram-positive facultative anaerobic bacteria (Streptococcus mutans ATCC 35668 and Actinomyces naeslundii ATCC 27039), gram-negative
facultative anaerobic bacteria (Escherichia coli ATCC 25922), and gram-negative obligate anaerobic bacteria (Porphyromonas gingivalis ATCC 63143627 and Prevotella intermedia ATCC 25611) were obtained from
the American Type Culture Collection (Manassas, VA, USA). The bacteria
were grown anaerobically using an Anaeropack system (Mitsubishi Gas
Chemical Company, Inc., Tokyo, Japan) and brain heart infusion broth
(Eiken Chemical, Co., Ltd., Tokyo, Japan) supplemented as follows:
no supplementation for E. coli; 1%
sucrose (FUJIFILM Wako Pure Chemical Corporation) and antibiotics;
0.1% gramicidin D and bacitracin (FUJIFILM Wako Pure Chemical Corporation)
for S. mutans; and 0.5% yeast extract,
0.0005% hemin (Sigma-Aldrich Co. LLC, St. Louis, MO, USA), and 0.0001%
menadione (Sigma-Aldrich Co. LLC) for P. gingivalis and P. intermedia. A. naeslundii was grown in Actinomyces broth (Becton
Dickinson and Company, Franklin Lakes, NJ, USA). For antibacterial
assessments, bacteria in log-phase growth were diluted to final concentrations
of approximately 1 × 10⁷ colony-forming units (CFU)
per mL.

Oral aerobes or facultative anaerobes (Streptococcus sanguis, Streptococcus oralis, and Peptostreptococcus anaerobius) and oral obligate anaerobes (Porphyromonas gingivalis, Prevotella intermedia, and Bacteroides forsythus) were purchased from American Type Culture Collection (Rockville, Maryland, USA) and cultivated according to the methods described earlier [8 , 9 (link)]. In brief, bacteria were grown in different media and the cultures were incubated for 48–72 h at 35–37°C. Purity was assessed by colony morphology and gram stain. Bacteria were harvested at the late log phase by centrifugation at 10,000 g for 15 min and then washed twice with 0.15 M sodium chloride. The bacteria were lysed using a sonicator, and after separation of the lysate, the protein concentration was measured and used for coating ELISA plates and antibody measurement.

Oral aerobes or facultative anaerobes (Streptococcus sanguis, Streptococcus oralis, and Peptostreptococcus anaerobius) and oral obligate anaerobes (Porphyromonas gingivalis, Prevotella intermedia, and Bacteroides forsythus) were purchased from American Type Culture Collection (Rockville, Maryland, USA) and cultivated according to the methods described earlier [8 , 9 (link)]. In brief, bacteria were grown in different media and the cultures were incubated for 48–72 h at 35–37°C. Purity was assessed by colony morphology and gram stain. Bacteria were harvested at the late log phase by centrifugation at 10,000 g for 15 min and then washed twice with 0.15 M sodium chloride. The bacteria were lysed using a sonicator, and after separation of the lysate, the protein concentration was measured and used for coating ELISA plates and antibody measurement.