Maintenance of Helicobacter pylori Cultures in Agar Stabs

Helicobacter pylori (H. pylori) can be maintained on both solid [1-4] and liquid media [5-7] but maintaining viable bacteria for prolonged periods can be expensive or impractical. Growth of plate cultures of H. pylori under aerobic conditions has been reported [8], and we routinely grow cultures in standard tissue culture incubators set at either 5% or 10% CO2 (unpublished results). Most laboratories however must rely on the use of closed container systems complete with gas generating envelope technology to create the appropriate microaerobic environment. These envelope systems can constitute significant costs when performing frequent or multiple cultures. Additionally, one of the manufacturers of these systems has recently discontinued their product, resulting in limited options in the market place. Continuous culture of H. pylori requires frequent subculture of viable bacteria to fresh plates. Many laboratory and pathogenic bacterial strains can be kept viable on sealed plates and stored at 4 °C to arrest growth. H. pylori survival at 4 °C, however, varies significantly with the media being used and it typically dies rapidly within 2 days [3,9-12]. Optimal methods for storing plate cultures at 4 °C have achieved viability for up to 9 days [13]. Typically, however, H. pylori must be passaged every 3–5 days to prevent the culture from dying. Although liquid cultures of H. pylori can be maintained in standard tissue culture incubators under static conditions, long-term maintenance requires frequent passage and ultimately consumes media supplemented with fetal bovine serum. While many of these issues can be circumvented simply by recovering H. pylori from frozen stocks when needed, viability varies depending upon the freezing media being employed [5,14,15]. Additionally, recovery times can take up to 7 days, result in light growth, and occasionally fail outright (unpublished observations). The purpose of this study was to assess a mechanism for maintaining viable H. pylori cultures in the laboratory without having to perform frequent passaging. We now report that H. pylori strains grown in agar stabs can be stored for at least 8 weeks in agar stab cultures kept at 37 °C.