5% yeast extract, 5 μg mL−1 of hemin and 1 μg mL−1 of vitamin K1 (BHI-HK). In some experiments, ferric citrate was also added to the medium. Porphyromonas gingivalis W83 (kindly supplied by Dr Koji Nakayama, Nagasaki University Graduate School of Biomedical Sciences) grown for 24 h was inoculated into the medium to give a final concentration of 106–108 cells mL−1 and incubated at 37 °C anaerobically (85% N2, 10% H2, and 5% CO2). At various time-points between 10 and 40 h, viable cells were enumerated by plating on blood EPZ015666 ic50 agar plates. Bacterial doubling times were established by dividing the time interval considered with the number of rounds of replication (n), which was
calculated as follows: n = ln(CFU2/CFU1)/ln2, where CFU1 and CFU2 are the numbers of CFU obtained at the beginning and end of the time interval, respectively (Chong et al., 2008). UV-visible spectroscopy using heme pigments from P. gingivalis cells was performed as described previously (Moon et al., 2011). Briefly, the bacterial Selleckchem Atezolizumab cells grown for 5 days on 5% sheep blood agar plates supplemented with DFO (0‒0.24 mM) were gently scraped, suspended in 500 μL of NaCl/Tris buffer (0.14 M NaCl/0.1 M Tris/HCl, pH 7.5) and sonicated on ice for 2 min. After centrifugation, UV-visible spectra of the supernatant buffer extract were recorded between 340 and 700 nm. The amount of hemin associated with P. gingivalis cells was measured
as described previously (Genco et al., 1994; Moon et al., 2011). Briefly, P. gingivalis cells were treated or untreated with 100 μM of CCCP for 60 min. After washing, the bacterial cells were suspended in BHI-HK and incubated anaerobically for 2 h with or without DFO. A 1.0-mL aliquot of each culture was centrifuged and the supernatant was assayed spectrophotometrically (OD400). Cell-associated hemin was calculated as the difference between the total amount of hemin added vs. the amount remaining in the
supernatant after 2-h incubation and normalized against the protein contents of that culture. Energy-driven active uptake of hemin was Carbohydrate calculated as difference between binding hemin of CCCP-untreated cells vs. CCCP-treated cells. Porphyromonas gingivalis cells (108 cells mL−1) were inoculated into BHI-HK with a twofold diluted series of H2O2 (0–0.8 mM) in the presence or absence of DFO. After 24-h incubation at 37 °C anaerobically the optical density at 600 nm was measured. The twofold serial dilutions of ampicillin, tetracycline and metronidazole (0–1.0 μg mL−1) were prepared in BHI-HK with or without DFO. In some experiments, ferric citrate was also added to the medium. Porphyromonas gingivalis cells (4–6 × 107 cells mL−1) were inoculated into the media. After 40-h incubation at 37 °C anaerobically OD600 was measured. After 24-h incubation, the viable cell numbers of P. gingivalis grown with DFO were statistically significantly lower than those grown without DFO when the inoculum size was 106 and 107 cells mL−1 (Table 1).