Efficacy assessment of different cryoprotectants for preserving the viability of Enterobacterales strains at - 20 °C.

The preservation of microorganisms is pivotal in microbiological practice. Currently, cryopreservation is assumed to be an effective and inexpensive approach for the storage of microorganisms, including bacteria. The key point of cryopreservation is optimal cryoprotectant selection. In the present study, different cryoprotectant compositions were tested for long-term storage of 15 Enterobacterales bacterial strains at - 20 °C. The survival rates of the bacterial strains were evaluated in four different cryoprotectant solutions containing 70% glycerin only (cryoprotectants 1 and 4), 10% dimethyl sulfoxide (DMSO) with 70% glycerin (cryoprotectant 2), and 10% DMSO (cryoprotectant 3). In addition, cryoprotectants 1 and 2 contained peptone and yeast extract as nutritional supplements. The general survival rates of the bacterial strains were evaluated after 12 months of storage. After 12 months, the survival rates of the different cryoprotectants were as follows: cryoprotectant 1-88.87%; cryoprotectant 2-84.85%; cryoprotectant 3-83.50%; and cryoprotectant 4-44.81%. Thus, the composition of cryoprotectant 1 (70% glycerin with nutrient supplements) was optimal for preserving 15 tested strains of the order Enterobacterales. Despite these findings, the biochemical properties of the tested strains changed after cryopreservation for 12 months in the presence of 1 or 3 cryoprotectants. Alterations in the biochemical profile could be related to changes in environmental conditions and cold adaptation. We assume that the composition of cryoprotectant 1 can be optimal for storing the order Enterobacterales at - 20 °C. However, further investigations are needed to elucidate the problem of cryopreservation and to support our assumption.