Sterilization of bacterial spores using supercritical carbon dioxide

Sterilization of Bacterial Spores Using Supercritical Carbon Dioxide Jinn Zhang Sterilization of medical instruments and devices is required to prevent infections. Current technologies have drawbacks for certain applications. Supercritical carbon de (SCCO2) has been shown to have a strong biocidal effect against microorganisms. Sterilization requires the complete killing, of all microorganisms including bacterial spores; however, reports on killing spores with SCCO2 are incomplete and unsatisfactory.;In this work, we first investigate the sporicidal capability of pure SCCO2 and SCCO2 with various additives. It was shown that pure CO2 was not effective against spores at 4000 psi and 80°C. The addition of deionized water enhanced the killing effects, but it still does not meet the 6 log reduction required by the Food and Drug Administration.;The most significant discovery of this work is that after 4-hour exposure at 4000 psi, addition of trace levels of aqueous hydrogen peroxide, into SCCO2 can completely kilt 6 log B. pumilus at 60°C and B. atrophaeus at 40°C. Furthermore, it was shown that B. pumilus has higher resistance to SCCO 2+H2O2 treatment than B. atrophaeus and B. anthracis.;In order to reveal the interactions between the spores and SCCO 2+H2O2, we further investigated the deactivation mechanisms of bacterial spores. Transmission Electron Microscopy (TEM) imaging of the spores stained with ruthenium red revealed physical damage to the exosporium and possibly the spore coats, indicating possible breakdown of the Outer permeability barrier. In DNA staining experiments, it was shown that not only the outer permeability barrier was compromised, leading to the penetration of DNA stains into the core. The breakdown of the permeability barrier was further confirmed through the analysis of dipicolinic acid (DPA) released from the spore core. Phase contrast microscopy showed that germination is not involved in the sterilization mechanism. Based on these observations, it is proposed that SCCO2 relaxes or damages the permeability barrier, allowing the uptake of H 2O2 and oxidation of the spore inner membrane, Much causes the spore death.;Finally, we predicted the solubility of aqueous H2O2 in the SCCO2 using the Peng-Robinson-Soave-Vera (PRSV) equation of state with the Panagiotopoulos and Reid mixing rules. This model should be useful in analyzing a SCCO2+H2O2 based sterilization process.