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

• Published in: Helicobacter (Cambridge, Mass.) Vol. 15; no. 4; pp. 295 - 302
• Main Authors: Joo, Jung-Soo, Park, Kyung-Chul, Song, Jae-Young, Kim, Dong-Hyun, Lee, Kyung-Ja, Kwon, Young-Cheol, Kim, Jung-Min, Kim, Kyung-Mi, Youn, Hee-Shang, Kang, Hyung-Lyun, Baik, Seung-Chul, Lee, Woo-Kon, Cho, Myung-Je, Rhee, Kwang-Ho
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.08.2010
• Subjects: Alcohol dehydrogenase; bacterial growth; Brucella; Catalase; Cell culture; Cell density; Culture Media - metabolism; Culture Techniques - methods; Cytotoxins; g-Glutamyltransferase; Growth curves; H. pylori; Helicobacter pylori; Helicobacter pylori - growth & development; Helicobacter pylori - metabolism; Liquid culture; Media (culture); Nuclease; reductase; Superoxide dismutase; Urease
• ISSN: 1083-4389; 1523-5378; 1523-5378
• DOI: 10.1111/j.1523-5378.2010.00767.x

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‐β‐cyclodextrin (200 μg/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 × 109 CFU/mL. γ‐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.