Method to Generate Chlorine Dioxide Gas In Situ for Sterilization of Automated Incubators

Pharmaceutical preclinical tests using cell cultures are nowadays commonly automated. Incubator microbial contaminations impact such tests. Chlorine dioxide (ClO₂) is widely used in aqueous solutions. However, a gaseous form, such as chlorine dioxide gas (gClO₂), can effectively access unreachable spaces, such as closed cell culture incubators. Steam sterilization requires a temperature rise to at least 121 °C, thus limiting the possibility of automation elements for sensors and actuators. gClO₂ sterilization is an ambient-temperature sterilization method. This article aims to demonstrate that gClO₂ generated from solid powder tablets is efficient for sterilizing incubators and can be automated. We selected (i) <i>Bacillus subtilis</i> strain, (ii) <i>Saccharomyces cerevisiae</i>, and (iii) T7 phages as representatives for (i) bacteria, (ii) fungi, and (iii) viruses for each domain to evaluate the sterilization efficiency. This study demonstrated that gClO₂ can be generated inside the incubator from a solid powder tablet without specific equipment and can effectively fight biological proxies in 15 min. After 30 sterilization cycles, the actuators and sensors mounted inside the incubator were still operating. Our proposed sterilization method seems to be generally applicable for automated in situ sterilization of incubators and medical robots.