A thin-layer liquid culture technique for the growth of Helicobacter pylori.
OBJECTIVE
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 microg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD(600) with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD(600) contained 1.3 +/- 0.1 x 10(9 )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.
Joo
Jung-Soo
JS
Department of Microbiology, Gyeongsang National University School of Medicine, Jinju, Gyeong-Nam 660-751, Korea.
Park
Kyung-Chul
KC
Song
Jae-Young
JY
Kim
Dong-Hyun
DH
Lee
Kyung-Ja
KJ
Kwon
Young-Cheol
YC
Kim
Jung-Min
JM
Kim
Kyung-Mi
KM
Youn
Hee-Shang
HS
Kang
Hyung-Lyun
HL
Baik
Seung-Chul
SC
Lee
Woo-Kon
WK
Cho
Myung-Je
MJ
Rhee
Kwang-Ho
KH
D003470:Culture Media / Q000378:metabolism; D046508:Culture Techniques / Q000379:methods*; D016480:Helicobacter pylori / Q000254:growth & development* / Q000378:metabolism
D003470:Culture Media