Exp_20220305_1_MucinBeads_BT_detach

Detachment test of monoculture Bacteroides thetaiotaomicron VPI-5482 (DSM 2079T) in Wilkins-Chalgren Anaerobe Broth
Notes:
We first grew BT culture in a serum bottle containing 60 ml WC plus mucin beads, with a same inoculum size as described: 1 ml of OD600 0.1. After 24 h cultivation, 2 ml of the fermentation broth containing the suspended mucin beads were sampled to each 2-ml tube. 0.5 ml of phosphate buffer (PBS, pH 7.3) was added to each tube after washing with 1 ml PBS. The beads in PBS were transferred to the 12-well plates, with 1.5 ml sterile WC medium filled in each well. Control groups of fresh WC medium and WC medium plus new mucin beads were inoculated with BT at the same ratio as abovementioned. The experiment was performed in four replicate wells for each group. The plates were incubated at a constant stirring rate of 170 rpm at 37°C, with samples in liquid taken at 0 min, 30 min, 60 min, 90 min, 150 min and 240 min for live-dead staining followed by flow cytometry. live/dead cell staining followed by flow cytometry, using a benchtop CytoFLEX S flow cytometer (Beckman Coulter, Brea, USA). In addition to the instrumental calibration with CytoFLEX Daily QC Fluorospheres, 0.5 μm and 1 μm green-fluorescent beads (Thermo Fisher Scientific, USA) were applied to each batch of experiments as internal standards, to monitor the instrument stability and to make cytometric data from different batches more comparable.