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Journal of Clinical Microbiology, November 2001, p. 3842-3850, Vol. 39, No. 11
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.11.3842-3850.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Helicobacter pylori Growth and Urease Detection in the Chemically Defined Medium Ham's F-12 Nutrient Mixture

Traci L. Testerman,* David J. McGee,dagger and Harry L. T. Mobley

University of Maryland School of Medicine, Department of Microbiology and Immunology, Baltimore, Maryland 21201

Received 24 April 2001/Returned for modification 13 July 2001/Accepted 9 August 2001

Obstacles continue to hinder in vitro studies of the gastric human pathogen Helicobacter pylori, including difficulty culturing the organism in the absence of serum or blood, rapid loss of viability following exponential growth due to autolysis, and the necessity for using high starting inocula. We demonstrate that H. pylori grows in the chemically defined broth medium Ham's F-12 nutrient mixture (F-12) in the absence of fetal bovine serum (FBS); this represents a breakthrough for studies in which serum components or proteins interfere with interpretation of results. Cultures can be continually passaged in fresh, FBS-free F-12 medium at an initial inoculum of only ~103 CFU/ml. All H. pylori strains (n = 21), including fresh clinical isolates, grew in serum-free F-12. H. pylori grew poorly in the related medium, F-10, unless additional zinc was supplied. Enhanced growth of H. pylori in F-12 broth was obtained by addition of bovine serum albumin (BSA) (1 mg/ml), beta -cyclodextrin (200 µg/ml), or cholesterol (50 µg/ml). H. pylori also grew in several simplified versions of F-12 broth lacking glucose and most vitamins but containing hypoxanthine, pyruvate, and all 20 amino acids. On F-12 medium solidified with agar, H. pylori only grew when BSA (98% pure; 1 mg/ml), cholesterol (50 µg/ml), beta -cyclodextrin (200 µg/ml), or FBS (2 to 4%) was added; addition of urea and phenol allowed colorimetric detection of urease activity. Thus, F-12 agar plus cholesterol or beta -cyclodextrin represents the first transparent chemically defined agar and the first urease indicator agar for H. pylori. Several lines of evidence suggested that BSA itself is not responsible for H. pylori growth enhancement in F-12 containing BSA or FBS. Taken together, these innovations represent significant advances in the cultivation and recovery of H. pylori using chemically defined media. Use of F-12 or its derivatives may lead to improved understanding of H. pylori metabolism, virulence factors, and transmission, and result in improved recovery and identification of H. pylori from clinical specimens.

* Corresponding author. Present address: Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL 36688. Phone: (251) 460-7447. Fax: (251) 460-7931. E-mail: ttesterm{at}jaguar1.usouthal.edu.

dagger Present address: Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL 36688.

Journal of Clinical Microbiology, November 2001, p. 3842-3850, Vol. 39, No. 11
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.11.3842-3850.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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