Phage Stability Across Conditions: Ensuring Accurate Use of Viral Surrogates in Antiviral Testing

Bacteriophages can serve as practical surrogates for human viruses in laboratory tests. They share key structural characteristics but are non-pathogenic and can be handled under lower biosafety conditions. This facilitates experiments on persistence, disinfection and materials-testing while reducing time, costs and logistical requirements. However, phage survival strongly depends on many factors including environmental conditions, such as temperature and relative humidity (RH). This study investigates the survival of bacteriophages Phi6, Qbeta and MS2 under different incubation conditions including storage on a surface, in liquid and in the fridge. Standardized ISO protocols for antimicrobial surface testing and extensions were applied. All phages demonstrated good survival in liquid, although stability was temperature-dependent. At refrigerator temperatures, phages remained stable for several months with only minor reduction in the initial titer. At 50 °C, the two non-enveloped phages survived for up to one week, whereas the enveloped phage Phi6 was inactivated within one day. On glass surfaces, Phi6 exhibited reduced stability and was detectable only up to one week at 25 °C and >90% RH. Qbeta and MS2 survived from several days at 50 °C to two to seven days at 37 °C, depending on RH, and remained detectable at 25 °C regardless of humidity. These differences highlight the importance of carefully selecting incubation conditions, as these directly affect phage stability. In particular, unsuitable conditions for antimicrobial testing may cause phage inactivation and thus lead to false-positive results. Therefore, it is essential to define the conditions under which each phage produces reliable results.