Isolation and Identification of Anaerobic Gut Bacteria

Feces and large-intestine scraping were cultivated with selective medium on TSA Tryptic Soy Agar (Tryptic Soy Agar, DIFCO, Taiwan, China, cat no. 211043) supplemented with 5% sheep blood, 6.25 mg/μL rifampicin (rifampicin, Sigma-Aldrich, St. Louis, MO, USA, cat no. R3501), and 800 mg/L spectinomycin (Sigma-Aldrich, cat no. S9007), 25 mg/μL vancomycin (vancomycin, Sigma-Aldrich, cat no. V2002), 25 mg/L of colistin (colistin, Sigma-Aldrich, cat no. C1511) [32 (link)], and incubated for at least three days at 42 °C in jars with an anaerobic atmosphere. Anaerobic conditions were generated using a vacuum pump filled with a mixture of N₂ (80%), CO₂ (10%), and H₂ (10%) gases. To obtain pure colonies, several passages were performed until isolation. The isolates were stored in a freezer at −80 °C.
For differential diagnosis, at the end of the experiment, mucosal scrapings from the small intestine were cultivated on blood agar and MacConkey to evaluate the growth of enterotoxigenic Escherichia coli and scrapings from the large intestine mucosa were cultivated in Rappaport broth and Hectoein agar for Salmonella spp.