Microbial survival during repeated freeze–thaw cycles: species-dependent responses rather than glycerol concentration effects

Freezing is widely used for microbial preservation; however, it promotes ice crystal formation, which may compromise cellular integrity. Given the variability among cryopreservation protocols used in laboratories and reported in the literature, this study evaluated microbial responses to successive freeze-thaw cycles using different glycerol concentrations in Luria-Bertani (LB) broth and two storage temperatures. Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Saccharomyces cerevisiae were selected as representatives of a Gram-negative bacterium, a Gram-positive bacterium, a spore-forming bacterium, and a yeast, respectively. Microorganisms were cryopreserved at -20 °C or -70 °C with 10%, 20%, 35%, or 50% glycerol and evaluated over nine successive freeze-thaw cycles. Viability was assessed by loop inoculation recovery and total viable cell counts determined by surface plating. Repeated freeze-thaw cycles significantly affected microbial viability, with responses varying among the evaluated species. E. coli and S. aureus remained stable throughout storage, whereas B. subtilis showed a progressive decline and S. cerevisiae exhibited variable responses. Storage at -70 °C showed a trend toward greater cell viability, although the overall effect of temperature was not significant. Glycerol concentration did not significantly affect microbial survival. A positive correlation was observed between loop inoculation recovery and microbial load, indicating that higher numbers of viable cells increased the likelihood of successful recovery. These findings demonstrate that microbial survival during cryopreservation depends primarily on species-specific characteristics rather than glycerol concentration, highlighting the importance of tailoring preservation protocols to different microorganisms.