Dismic 25ss

Equal volumes (0.1 mL each) of stationary-phase CD ATCC9689 and B. fragilis YCH46 or B. thetaiotaomicron VPI-5482 were inoculated into 10 mL fresh GAM broth. We monitored the visible changes of CD cells under co-cultivation with B. fragilis YCH46 or B. thetaiotaomicron VPI-5482. The co-culture was periodically sampled and spread on glass slides. After fixation with methanol, the slides were Gram stained using a staining kit (Favor G “Nissui”; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). Microscopic images were captured with a Leica ICC50 HD microscope. After 24-h incubation, the remaining cultures were centrifuged at 10,000× g for 15 min, and the supernatant was passed through a 0.22 µm filter (ADVANTEC, DISMIC-25SS) to remove bacterial cells. Aliquots of the filtrate were stocked at –80 °C until use for cytotoxicity assay described below.

The filtrate of culture supernatant of the selected bacteria was prepared to test the effect on CD toxin production. A single colony of each bacterial strain grown on GAM agar plates was inoculated into 10 mL GAM broth, and the media were anaerobically cultivated overnight at 37 °C. A part of the overnight culture (0.1 mL) was inoculated into 10 mL of freshly prepared GAM broth and incubated again for 24 h. The culture was then centrifuged at 10,000× g for 15 min, and the supernatant was passed through a 0.22 µm filter (ADVANTEC, DISMIC-25SS) to remove bacterial cells. Aliquots of the filtrate were stocked at −80 °C until use. Conditioned medium was prepared by mixing the filter sterilized culture supernatant with an equal volume of fresh GAM broth. Another type of conditioned medium that contained the polysaccharide fraction from the selected bacterial culture was prepared as described below (Section 4.10).