Evaluation of Various Strategies for Isolation and Culturing of Helicobacter pylori

The effects of transport media, transportation time, culture media, blood from different sources, and effectiveness of a low cost atmosphere generation system (AGS) on isolation and culturing of H. pylori was studied. Biopsy samples from 56 dyspeptic patients were transported for culturing in sterile normal saline and Brain Heart infusion (BHI) broth. Both normal saline and BHI broth were effective transport media. Samples were plated on Columbia blood agar plates supplemented with 5% sheep blood and Dent antibiotic supplement (CBA-D) for primary isolation and determining the effect of transportation time. A low cost envelope system was compared with standard Campygen gas packs and anaerobic jar system for the production of microaerophilic environment. Isolates were subcultured on CBA and BHI agar plates having different concentrations of blood from various sources. When the samples were cultured within 2 hours of sampling, both transport media gave similar isolation rate of 87.5% (49/56). Isolation rate decreased to 75%, 62.4% and 46.4% in case of biopsy specimens transported in normal saline. Whereas, for specimen transported in BHI broth it was 76.8%, 71.4% and 53.6%, when culturing was delayed upto 4, 6 and 8 hours, respectively. CBA and BHI agar were comparable for subculturing with 100% and 94% efficiency. Thirty six (73.5%) out of 49 isolates were successfully subcultured using atmosphere generated by low-cost envelope system as compared to that produced by Campygen gas pack with anaerobic jar. When subcultured on CBA supplemented with 5% human or sheep blood, growth was observed for all isolates.. However, on 3% human or sheep blood concentration in the medium, 75.5% and 71.4% growth was observed, respectively. Isolation rate was higher when biopsy samples were cultured within 2 hours of sampling. CBA-D was found to be the best selective medium for H. pylori isolation. Microaerophilic environment could be created using low-cost systems but needs further studies.