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. 2024 Nov 20;13(11):1024.
doi: 10.3390/pathogens13111024.

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

Cédric Schicklin  1 , Georg Rauter  1 , Philippe Claude Cattin  2 , Manuela Eugster  1   3   4 , Olivier Braissant  5
Affiliations

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

Cédric Schicklin et al. Pathogens. .

Erratum in

Abstract

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) Bacillus subtilis strain, (ii) Saccharomyces cerevisiae, 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.

Keywords: bacteria; chlorine dioxide; decontamination; fungi; viruses.

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Conflict of interest statement

The authors declare that this study was partially funded by F. Hoffmann-La Roche Ltd. However, the funding organization had no role in the design of the study, data collection and analysis, decision to publish, or preparation of the manuscript. The authors affirm that the integrity of the research and the objectivity of the findings were maintained throughout the study according to the University of Basel code of conduct (https://www.unibas.ch/dam/jcr:1019dd24-ed06-4c5a-ade2-52e8db41115d/Code%20of%20Conduct_2020_EN.pdf (accessed on 12 November 2024)). All results and interpretations presented in this publication are solely those of the authors and have not been influenced by the funding source.

Figures

Figure 1
Figure 1
Schematic of the sterilization activation. (a) The initial load before activation was visualized (blue: water; yellow: ClO2 tablet). (b) The rocking station tilts in motion and pours the water on the tablet to generate the chlorine dioxide gas. (c) After activation, water with by-products remains.
Figure 2
Figure 2
This figure shows the experimental protocol for the sterilization of B. subtilis. The incubation time, temperature, and culture medium differed for S. cerevisiae (Section 2.4.2. Biological Indicator Sterilization) and T7 (Section 2.4.2. Biological Indicator Sterilization). The hourglass represents the passing of time.
Figure 3
Figure 3
(a) Before activation, the sterilization plate was loaded with water (17 mL) (1) and a fraction of a powder tab (here 2 g) (2) and was placed inside the incubator on the rocking station (3). (b) After activation of chlorine dioxide powder with water, gaseous chlorine dioxide (gClO2) was produced.
Figure 4
Figure 4
Bacterial spores, fungi, and viruses’ colony-forming units (CFU)/mL with SD (when applicable) for different chlorine dioxide gas (gClO2) concentrations. SD: standard deviation. ND: not determined. *: p-value < 0.05 between 0 PPM and assessed ClO2 concentration.
Figure 5
Figure 5
Photo of the human colon cells cultivated post-sterilization under the microscope.
See this image and copyright information in PMC

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