The effects of cryopreservation conditions on viability of escherichia and staphylococcus genus

Abstract

Maintaining the collections of microorganisms requires the long-term conservation of strains in viable state without changes in their biological properties. Cryopreservation is considered as one of the most effective means of long-term storage of microorganisms. The use of cryoprotective compounds ensures new possibilities to protect microbial cells from cold shock and alterations at freezing procedure. Specification of the preservation methods adapted for a specific type of microorganisms, that would provide high viability and stability of biological properties, is an actual task of modern microbiology. Objective. This study aimed to investigate the effect of cooling regiments, composition of conservation medium, and low temperature storage on the viability of different types of microorganisms. Methods. The study was conducted on 5 strains of E.coli and 5 strains of Staphylococcus spp. Cryoprotective mediums comprising 1% glucose or 10 % glycerol were used for deep freezing. Two cooling modes were used: mode 1 - direct immersion of the samples in liquid nitrogen; mode 2 - cooling with programmable freezer from 20 to −70 ° C with a speed of 10 ° C / min, followed by a temperature stop at −70 ° For 10 min and further immersion. The samples were thawed in a water bath at a temperature of +37 ° C for 120 seconds. Viability of tested strains was tested by the Koch method after 3 and 6 month of storage in liquid nitrogen. Statistical analysis was performed with nonparametric methods using MX Excel 2007 and STATISTICA 6.0 software. Results. Significant strain-specific differences in survival rates were established. At both cooling modes adding of the glycerol as a cryoprotectant provided significantly higher viability of bacterial cells then adding of glucose. The average number of viable cells after freezing was 8.1 × 10⁸CFU / ml vs 1.3 × 10⁸CFU / ml for glycerol and glucose respectively (p <0,05). There was no significant difference in viability of strains after deep frozen storage for 3 and 6 month. All strains had similar viability rates when glycerol was used as a cryoprotectant. Staphylococci were less vulnerable to freezing stress at both cooling modes when medium with glucose was used. Conclusion. High efficiency of one step deep freezing with 10 % glycerol as a method for preserving of Staphylococcus and Escherichia collection strains has been demonstrated. The viability of bacteria during cryopreservation was influenced by the composition of the preserving medium, the cooling mode and the species and stain-specific morpho-functional features. Glycerine was found to be the optimal cryoprotectant at both single-stage and two-stage cooling mode. The one stage freezing was much more preferable when a cryoprotective medium with glucose was used. Duration of the sample storage in liquid nitrogen did not affect the number of viable cells.