A Thin-Layer Liquid Culture Technique for the Growth of Helicobacter pylori

Abstract

Background and Aims: Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin-layer liquid culture technique for the growth of H. pylori. Methods: A thin-layer liquid culture system was established by adding liquid media to a 90-mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Results: Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI-1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum-free RPMI-1640 supported the growth of H. pylori when supplemented with dimethyl-beta-cyclodextrin (200 mu g/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD600 with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD600 contained 1.3 +/- 0.1 x 109 CFU/mL. gamma-Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin-layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Conclusions: Thin-layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.