Bacillus subtilis (natto) phage ONPA suspended in an SM buffer without a cryoprotectant is sensitive to freezing at −20°C to −40°C. Freezing with a cryoprotectant and/or at low temperatures (−80°C and −165°C) can dramatically improve the viability of ONPA. B. subtilis (natto) phage JNDMP, the other principal type of B. subtilis (natto) phage used in this study, was stable even without a cryoprotectant at −20°C. The observation of ONPA using electron microscopy, after freezing at −20°C and subsequent thawing, suggested that the phage tails contracted and the phage genome DNA remained in the heads. The contraction of the phage tails may have caused the inactivation of ONPA phage particles.