Favor g nissui

IZ6 grows best when cultivated in R2A Broth “DAIGO” (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) at 25 °C. This study examined temperature-induced morphological changes. Cell morphology of IZ6 was observed by microscopy (Olympus BX-50F4, Olympus Optical Co. Ltd., Tokyo, Japan) with a Gram-staining kit (Favor G “Nissui”; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) after cultivation at 25 °C for 2 weeks, 25 °C for 2 weeks followed by 4 °C for 3 weeks, and 15 °C for 3 weeks. Culture fluid was filtered through a 0.22-μm filter (Millex-GV Syringe Filter Unit SLGV033RS; Merck Millipore, Tokyo, Japan), and the cells in the filtrate were also observed. Cell volume was calculated with the formula described in [15 (link)].

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.