Exp_20220618_1_RI_mucin_sugars_plates

Mucin sugar test for Roseburia intestinalis L1-82 (DSM 14610T) in Wilkins-Chalgren Anaerobe Broth
Notes:
The degradation products of mucin including Neu5Ac, N-acetylgalactosamine, N-acetylglucosamine, fucose, mannose and galactose were tested for their effects on the growth of RI. The tests for each species were performed by using 24-well plates (Sarstedt, Germany) filled with two types of media: the standard WC and WC without carbon sources glucose and pyruvate. Each medium was split into eight aliquots, each of which supplemented with either one of those mucin sugars in 1 mM or a mixture of all six sugars together in 1 mM or without any sugar added as the control. We first inoculated 0.5 ml preculture with OD600 0.1 to 20 ml of each aliquot, and then transferred 1.2 ml to each well in three replicates. The plates of RI were incubated at a constant stirring rate of 120 rpm at 37°C for 24 h or 48 h, respectively, with samples taken every 12 h for pH and live-dead staining followed by flow cytometry measurements. 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.