Gaspak ez gas generating pouch systems

An H. pylori strain (HP99) was isolated from the gastric mucosa obtained from a Korean patient with duodenal ulcer at Seoul National University [17 (link)]. HP99 was kindly provided by Dr. HC Jung (Seoul National University College of Medicine, Seoul, Korea). These bacteria were inoculated onto chocolate agar plates at 37°C under microaerophilic conditions using GasPak EZ Gas Generating Pouch Systems (BD Biosciences, San Jose, CA, USA). Prior to stimulation, H. pylori was harvested and then resuspended in antibiotic-free cell culture medium. H. pylori was added to the cultured cells at a bacterium : cell ratio of 500 : 1 in a 1-mL volume.

Each sample (100 mL) was pretreated at 75 ± 5 °C for 15 ± 1 min in a water bath. After filtration, the membrane filter (47 mm Ø and 0.45-µm pore size; Millipore, Milan, Italy) was placed on Triptosio Solfito Cicloserina Agar (Biolife Italiana srl, Milan, Italy) plates, and incubated at 44 °C for 24 h in an anaerobic atmosphere generated by GasPak EZ Gas Generating Pouch Systems (BD Diagnostics, UK). Black colonies were considered to be spores of sulfite-reducing clostridia. The results were reported as CFU/100 mL [59 ].