Culturing and Maintaining Helicobacter pylori

Strains and plasmids used in this study are listed in Table 1. Unless noted otherwise, H. pylori strains were grown as previously described (Carpenter et al., 2015 (link)). Briefly, H. pylori stock cultures were maintained at −80°C in H. pylori freezing media [brain heart infusion broth (BD Biosciences) containing 10% fetal bovine serum (FBS) and 20% glycerol (EMD chemicals, Inc)]. Freezer stocks were plated on horse blood agar (HBA) plates comprised of 4% Columbia agar (Neogean Corporation), 5% defibrinated horse blood (HemoStat Laboratoris, Dixon, CA), 2 mg/mL β-cyclodextran (Sigma), and an antibiotic/antifungal cocktail [10 μg/ml vancomycin (Amresco), 5 μg/ml cefsulodin (Sigma), 2.5 U/ml polymyxin B (Sigma), 5 μg/ml trimethoprim (Sigma), and 8 μg/ml amphotericin B (Amresco)]. Following growth on HBA plates, H. pylori strains were grown in liquid culture. Liquid media consisted of brucella broth (Neogen Corporation) supplemented with 10% FBS (Gibco) and 10 ug/mL vancomycin. Where indicated, 25 μg/mL Kanamycin (Kan²⁵) was used to supplement the media. All cultures were grown at 37°C, in gas evacuation jars, under microaerobic conditions (5% O₂, 10% CO₂, and 85% N₂) generated with an Anoxomat gas evacuation and replacement system (Advanced Instruments, Inc.); in addition, liquid cultures were grown shaking at 100 rpm.