Influencing factors on the preservation of lytic bacteriophage VP3

• Scientific question : Long-term and stable storage of bacteriophage is important for its use in scientific and industrial areas. Lytic bacteriophage VP3 is one of the typing phages of Vibrio cholerae O1 biotype El Tor. So far, there is no description of the storage method of bacteriophage VP3. • Evidence before this study : Many external factors are considered to be influential to the storage of bacteriophages. First, temperature determines phage activity during storage. Second, a solution with stable and neutral pH and low ionic content is generally preferable for most phages. Also, cryoprotectants, which are essential to mitigate cold-induced damage, are usually added during the long-term storage of phage. Moreover, some phages are found to be affected by alternate freeze–thaw cycles. It should be noted that there does not exist a universal preservation method that fits every phage. • New findings : In this study, we explored different external factors, including temperature (4 °C, −20 °C, −80 °C and −196 °C in liquid nitrogen), solution (LB medium and SM buffer), cryoprotectant (DMSO and glycerol), 20-week cooling/freezing or thirteen cycles of alternate cooling/freezing and thawing, on the stability of phage VP3 during storage. We found that, for bacteriophage VP3, the best preservation method is to store the phage stocks in LB medium at −80 °C or −196 °C, without adding cryoprotectant. Frequent cooling/freezing and thawing cycles are not recommended for phage VP3. • Significance of this study : Our results provide insights into the storage condition of phage VP3, which is helpful to understand the external influencing factors on phage storage and to improve the adoption of phages in the future. Long-term and stable preservation of bacteriophages is of crucial importance. Although many efforts have been made in the past decades to explore the influence of external factors on bacteriophage preservation, there is still little understanding, and a systematic description is lacking. In this study, we explored the influence of different factors on the preservation of lytic bacteriophage VP3, one of the typing bacteriophages of Vibrio cholerae O1 biotype El Tor, and attempted to optimize its preservation. We examined external factors, including temperature, solution, and cryoprotectant, in stable cooling/freezing conditions or alternate cooling/freezing and thawing. We found that whether in Luria-Bertani (LB) medium or SM buffer, in terms of 20-week stable cooling or freezing, −20 °C was the most damaging while 4 °C, −80 °C, and −196 °C were protective. Thirteen cycles of alternate cooling/freezing and thawing caused a loss in the survival rates of bacteriophages. The addition of cryoprotectant, glycerol (30%, w/v) or dimethyl sulfoxide (DMSO, 10%, w/v) significantly improved the survival rates of bacteriophages preserved at −20 °C. However, at 4 °C, −80 °C, and −196 °C, the cryoprotectant effect was only slightly positive or even harmful. In summary, for bacteriophage VP3, the best preservation method is to directly preserve the bacteriophage stocks in LB medium at −80 °C or −196 °C instead of storing them in SM buffer or adding cryoprotectant. Our results provided insights into the external influencing factors on bacteriophage VP3 during preservation at low temperature and can be applied to the optimization of bacteriophage preservation in the future.