A Simple Modified Method for Long-Term Glycerol-Based Cryopreservation of Leptospira Spp. in Liquid Nitrogen

A simple and reliable method for long-term preservation of Leptospira spp. is essential for maintaining reference strains and field isolates for diagnostic and research purposes. Although 10% glycerol is commonly used as a cryoprotectant for Leptospira preservation, the isolates in this study showed poor recovery, highlighting the need to optimize glycerol levels for cryopreservation in liquid nitrogen.

This study evaluated the storage of reference serovars Leptospira interrogans serovar Australis (pathogenic), Leptospira fainei serovar Hurstbridge (intermediate), and Leptospira biflexa serovar Patoc (nonpathogenic) with 10% and 15% glycerol and 28 additional reference serovars, and 11 field isolates using 15% glycerol in liquid nitrogen.

Cultures were grown for 7 days in Ellinghausen-McCullough-Johnson-Harris (EMJH) media supplemented with 10% rabbit serum and cryopreserved with 10% and 15% glycerol using a controlled-rate freezing protocol before storage at -196°C. Viability was assessed monthly for up to 15 months using dark-field microscopy and Petroff-Hausser chamber counts.

Both reference serovars (n = 31) and field isolates (n = 11) were successfully preserved using 15% glycerol and repeatedly recovered. Paired t-test showed significant differences (p < 0.01) between 10% and 15% glycerol, while Kruskal-Wallis analysis confirmed consistent cryoprotective efficiency of 15% glycerol across different Leptospira species. Microscopic agglutination test confirmed unchanged antigenic properties, while hamster model studies demonstrated retained virulence, evidenced by acute leptospirosis.

This optimized protocol supports reliable long-term cryopreservation of Leptospira spp. in liquid nitrogen without compromising viability, antigenicity, or virulence characteristics.