Initial Concentration of Streptococcus pneumoniae Cells as One of the Factors Influencing Cryopreservation Efficiency

The Streptococcus pneumoniae bacteria are among the main respiratory disease agents worldwide. Nowadays one can secure about 90 pneumococcal serotypes with different prevalence and medical significance according to the structure of capsular polysaccharides, thereby complicating serological diagnosis and prevention of pneumococcal infections. Therefore, the tasks of the integrity of S. pneumoniae clinical isolates, obtained in different geographical areas, are topical for designing diagnostic and specific prophylaxis agents. Freeze-drying and freezing down to low temperatures (cryopreservation) are currently considered to be the most efficient methods of storage. Numerous research papers on microorganism cryopreservation reported towards the following factors affecting microbial cell integrity: the structure and functional characteristics stipulated by taxonomic position of a microorganism, freeze-thawing regimens, preservation medium composition, culture conditions (growth medium composition, aeration, cultivation temperature), as well as the growth phase of a batch culture. However, in some cases it is not advisable to include the widely used cryoprotective substances into the preservation medium. Therefore the search for conditions of microorganism cryopreservation in the media free from these substances is now in progress. Concentrations of microbial cells per unit of volume were suggested to have a significant effect on the final cryopreservation result. However, this problem is still poorly understood. The findings obtained by different authors are contradictory. We assumed the viability of microbial cells with a high initial concentration as might be affected by intracellular substances released from cells during cooling and by a change in crystallization processes. This paper deals with the study of the S. pneumoniae cell viability after freezing in the samples with different initial cell concentrations, as well as those frozen in cryolysates, obtained via threefold freezing down to -196˚C of bacteria Enterococcus faecalis, Staphylococcus aureus, Escherichia coli B and Klebsiella pneumoniae. During S. pneumoniae bacteria freezing the initial cell concentration was first shown to cause a significant effect on their viability. Under initial concentration of 1011 CFU/ml the maximum cell survival after freezing was observed, regardless of the preservation medium composition, and when reducing the initial cell concentration down to 1010-109 CFU/ml the viability indices decreased significantly. The composition of preservation medium was established to affect the indices of cell viability as well, the highest results were achieved when freezing cell suspension in meat-peptone broth, and the lowest ones were obtained during bacteria suspending in physiological saline. The findings on S. pneumoniae freezing in cryolysates of other bacterial species testify to the fact that the different intracellular substances, released from bacterial cells at cooling stage, have a cryoprotective effect.