Gaspak eztm pouch systems

All isolates (disease-associated, n = 83; HCs, n = 50) used in this study were isolated from faecal samples of patients with CD (n = 30), UC (n = 30), IC (n = 23), and HCs (n = 50) during the study of culturomics to isolate previously uncultivated human gut microbiota associated with IBD or IC (unpublished). Briefly, fresh faecal samples obtained from KNUH were immediately placed in an anaerobic gas pouch (BD GasPak EZ^TM Pouch Systems; BD; Franklin Lakes, NJ, USA) and transferred to the laboratory’s anaerobic gas chamber (BACTRON anaerobic chamber; Sheldon Manufacturing, Inc.; Cornelius, OR, USA). One gram of each stool sample was enriched in 9 mL of defibrinated sheep blood and incubated at 37 °C for 2 days under aerobic and anaerobic conditions. Serial dilutions up to 10⁻⁸ were made in phosphate buffered saline (PBS), and 100 µL of each enriched faecal sample (10⁻⁵–10⁻⁹) was plated on 34 uniquely defined agar plates (Table S1, see Supplementary Materials) and incubated at 37 °C for 3–7 days under both conditions (aerobic and anaerobic). Each distinct colony on agar plate was selected and streaked onto a new agar plate until a pure culture was obtained. For long-term preservation, pure colonies were stored in 50% glycerol stock at −80 °C.

Fresh fecal sample collected from a healthy Korean individual at Kyungpook National University Hospital (KNUH) was placed in an anaerobic gas pouch (BD GasPak EZTM Pouch Systems, BD, NJ, United States) and transported to an anaerobic gas chamber in the laboratory. To enrich the fecal samples, 1 g of feces was mixed with 9 mL of defibrinated sheep blood and incubated under aerobic and anaerobic conditions at 37°C for 2 days. The remaining samples were stored at −70°C for further analysis. Serial dilutions were made up to 10⁹ in phosphate-buffered saline, and 100 μL of each enriched fecal sample (10⁵–10⁹) was plated on brain heart infusion agar (BHA) with 5% of defibrinated sheep blood and incubated at 37°C for 3–5 days. Pure colonies were obtained by selecting and streaking each colony on a new blood agar plate until a pure colony was obtained. The purified colonies were stored at −70°C in a 50% glycerol stock for long-term preservation and have been deposited in different culture collection centers such as Korean Collection for Type Cultures (KCTC), China General Microbiological Culture Collection (CGMCC), and Thailand Bioresource Research Center (TBRC).