Histochemical tracing of bismuth in Helicobacter pylori after in vitro exposure to bismuth citrate

Background: Bismuth-containing drugs are widely used in the treatment of Helicobacter pylori associated peptic ulcer. The mechanism of action of bismuth salts is, however, not fully understood, and at present no histochemical techniques for the demonstration of bismuth in H. pylori are available. The aims were to present a histochemical method for the detection of bismuth in H. pylori and to demonstrate bismuth uptake in H. pylori after in vitro exposure to bismuth citrate. Methods: H. pylori cultures (the strain used in this study was CCUG 17874), were exposed to bismuth citrate at different concentrations (0, 4.6, 80, 200 μM) and for different lengths of time (0 min, 15 min, 1 h, 24 h, 48 h). The samples were fixed in glutaraldehyde, centrifuged, and exposed to autometallographic (AMG) development in order to detect bismuth histochemically. Results: A detailed protocol on the AMG bismuth technique on H. pylori exposed to bismuth in vitro is given. This method results in easily detectable AMG grains of silver enhanced bismuth particles at the electron microscopical level, and shows that bismuth accumulates in H. pylori, predominantly near the wall of the bacteria. Bismuth uptake is followed by bacterial degeneration. Conclusion: The present technique with its ability to trace bismuth constitutes a valuable tool in the efforts of clarifying the mechanism of action of bismuth on H. pylori, and supports the notion that bismuth has an antimicrobial activity in itself.