Evaluation of different cryoprotective agents in maintenance of viability of Helicobacter pylori in stock culture media

ABSTRACT

Four different cryoprotective supplemented stock media were evaluated for maintaining better survival and recovery of H. pylori type strain NCTC 11637 at two different maintenance temperatures of -20°C and -80°C after one month preservation as frozen stocks. The spread plate colony count method was used to investigate the recovery rate of H. pylori from equally inoculated bacterial suspensions in differently prepared stock cultures. After the preservation of H. pylori for one month in different cryoprotectant-supplemented stock media, the recovery rates for -20°C obtained for stock cultures supplemented with dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), glycerol and glycerol+sucrose, as well as controls with and without human serum alone were 7.13, 6.97, 7.93, 7.99, 6.95 and 0.0 log CFU/ml, respectively. Maintenance of bacteria at -80°C gave statistically higher recovery rates compared to preservation at -20°C with the values of 8.55, 8.24, 8.59, 8.66, 8.01 and 0.0 log CFU/ml for these above mentioned stock cultures. The stock cultures supplemented with glycerol+sucrose and glycerol showed the highest recovery rates, 7.99 and 7.93 for -20°C vs. 8.66 and 8.59 for -80°C respectively, which were statistically different from the others. Our study revealed that H. pylori type strain NCTC 11637 could be better preserved at -80°C than -20°C. The best stock media which supported viability or culturability of bacteria were brain heart infusion broth (BHI)+glycerol+human serum and BHI+glycerol+sucrose+human serum, where the latter yielded the higher recovery rate.