Evaluation of techniques for isolation, subcultivation, and preservation of Helicobacter pylori

Abstract

With sheep blood agar (SBA), Belo Horizonte medium, and Brussels campylobacter charcoal agar, 104 strains of Helicobacter pylori were detected in 309 gastric biopsies. Each medium revealed only 69 to 71% of the strains. Ten strains grew solely on SBA, and four strains each grew on Belo Horizonte medium and Brussels campylobacter charcoal agar. Subculturing of 50 fresh H. pylori isolates on SBA revealed a progressive reduction in growth with increasing passage. Thirty strains stopped growing between passages two and seven. Four strains survived more than 20 passages. The preservation of fresh H. pylori isolates at -193 and -70 degrees C and by lyophilization was compared by use of 10% porcine mucin solution, fetal calf serum, and a commercial cryopreservative fluid. Of 30 strains, 77 to 90% could be recultivated on SBA after preservation at -70 degrees C in all three storage media. The data indicate that for the primary isolation of H. pylori, not only one selective medium but several selective media with different antibiotic supplements plus at least one nonselective medium should be used to yield the highest culture rates. Frequent subculturing of H. pylori on SBA selects strains which may not be representative of clinical isolates. Storage of fresh H. pylori isolates at -70 degrees C in 10% mucin solution is a simple and effective preservation procedure.