Abstract
Pathogenic microorganisms are valuable biological resources, closely related to biosecurity, human health, environmental protection, and renewable energy. It is very important to properly preserve the microbial resources by methods to maintain the purity, viability, and integrity, and to avoid prolonged degradation. The present work aims to explore the cryopreservation technology of Nocardia farcinica (Gram-positive bacteria) and Yersinia pestis vaccine strains (Gram-negative bacteria). The effects of cryoprotectants (CPAs), freezing temperature, and freeze-thaw cycles on the two bacteria in the cryopreservation process were studied. The results showed that the addition of CPAs (glycerol, propylene glycol, sucrose, glucose, l-carnitine, l-proline, and skim milk) significantly enhanced the survival rates of the N. farcinica and Y. pestis vaccine strains. However, high concentrations of CPAs can produce biochemical toxicity in the two pathogens. The utilization of composite CPAs not only reduced the toxicity but also improved the survival rates of samples during cryopreservation. The optimal composite CPA for N. farcinica is 0.292 M sucrose, 0.62 M l-carnitine, and 2.82 M glycerol. The optimal composite CPA for Y. pestis is 0.62 M l-carnitine, 8.46 M glycerin, and 0.292 M sucrose. The results showed that the quality of the strains stored at -80°C and -196°C was better. For the case of freeze-thaw cycles, the two pathogens have different degrees of reduction, and the survival rate of Y. pestis decreased more than that of N. farcinica. The uniform distribution of bacteria in CPAs can form uniform nucleation sites in the solution system, which is beneficial to the cryopreservation of strains, as can be seen from the experimental results from a differential scanning calorimeter. This study may provide a reference for better preservation of precious natural biological resources of pathogenic microorganisms.
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