Biopreservation and biobanking · 2026
A Simple Modified Method for Long-Term Glycerol-Based Cryopreservation of Leptospira Spp. in Liquid Nitrogen.
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Ponnidurai Malini, Pal Archana, Kirubakaran Vinod Kumar, Krishnamurthy Abhilasha, Venkatappa Bharath, Yeluvaguli Prassanna Arun, Prashant Bokade Prajakta, Munagala Sree Vidhya, Lakshmipathy Archudhan, H B Chethan Kumar, Mohandoss Nagalingam, Vinayagamurthy Balamurugan
Аннотация
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.
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.