Effects of temperature, salinity and clay particles on inactivation and decay of cold-active marine Bacteriophage 9A

The effects of temperature, salinity and clay particles on the inactivation and decay of the cold-active Bacteriophage 9A, isolated from particle-rich Arctic seawater, were examined using a plating technique to evaluate infectivity (inactivation) and epifluorescence microscopy to measure phage concentrations (decay). Phage 9A was rapidly inactivated over a temperature test range of 25 to 55°C in marine broth (salinity of 36 psu), with half-lives ranging from 0.20). At the environmentally relevant temperature of -1°C, however, loss of infectivity in briny broth was 3 to 4 times greater than in marine or brackish broth. As commonly observed, viral decay determined microscopically often substantially underestimated loss of infectivity: at 30°C, loss of infectivity exceeded the viral decay rate by approximately 1000-fold, while at -1°C, microscopic counts did not detect any of the losses observed by plaque assay. Under conditions comparable to a winter sea-ice brine inclusion (-12°C and 161 psu), however, plating and microscopy were in substantive agreement, indicating relatively minor losses of 16 to 34 % losses over a 5 wk period. Illite, kaolinite or montmorillonite clays had no statistically significant effect on phage inactivation as a function of temperature or salinity, although rates tended to be slower in the presence of the clays. In general, our results emphasize the importance of working with cold-active phages under environmentally-relevant conditions of temperature and salinity. They also imply decay processes that involve viral proteins rather than nucleic acids; as a result, affected viruses may be recalcitrant to reactivation by known host-based repair mechanisms.