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

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