Application of cryoprotectants for cryopreservation of pathogenic microorganisms (Yersinia pestis, Nocardia farcinia)
The importance of pathogenic microorganisms
Pathogenic microorganisms are of great value as biological resources for biosecurity, human health, environmental protection, and renewable energy. Proper preservation of these microbial resources is of paramount importance and requires methods that maintain purity, viability, and integrity while preventing continued degradation.
Investigation of cryopreservation of N. farcinica and Y. pestis
The objective of this study is to investigate cryopreservation technology for two species of bacteria: Nocardia farcinica (Gram-positive) and Yersinia pestis vaccine strains (Gram-negative). This research examines the effects of cryoprotectants (CPAs), freezing temperature, and freeze-thaw cycles on these bacteria during the cryopreservation process.
Effect of cryoprotectancts (CPAs) on bacterial survival
The results show that the addition of CPAs such as glycerol, propylene glycol, sucrose, glucose, L-carnitine, L-proline, and skim milk significantly increased the survival rates of inoculum strains of N. farcinica and Y. pestis. However, it should be noted that high concentrations of CPAs can produce biochemical toxicity in these two pathogens.
Mitigating biochemical toxicity: the role of composite CPAs
The use of composite CPAs not only reduces toxicity but also improves sample survival rates during cryopreservation. The optimal composite CPA for N. farcinica consists of 0.292 M sucrose, 0.62 M L-carnitine, and 2.82 M glycerol, while for Y. pestis it consists of 0.62 M L-carnitine, 8.46 M glycerol, and 0.292 M sucrose.
Optimal conditions: Temperature and freeze-thaw cycles
The results show that the quality of the strains stored at -80°C and -196°C is better. In the freeze-thaw cycles, the two pathogens show a different degree of reduction, with Y. pestis experiencing a greater decrease in survival compared to N. farcinica.
Optimization: uniform distribution of bacteria and the role of nucleation sites
The uniform distribution of bacteria in CPAs facilitates the formation of consistent nucleation sites in the solution system, thus favoring the cryopreservation of strains, as found in experiments using the Differential Scanning Calorimeter.