(93b) Mechanisms of Supercritical Carbon Dioxide Sterilization of Bacterial Spores

Sterilization of implants and medical devices (such as endoscopes) is essential to prevent clinical infection. Commercial sterilization using the steam autoclave, ethylene oxide, or irradiation has serious limitations when sterilizing heat-sensitive polymers or substrates with complex internal structures. To meet the clinical needs associated with these situations we evaluated the viability of using SCCO₂ as a sterilant. Experimental results verified that with approximately 200 ppm of H₂O₂ (equivalent to 5 microliters of 30% H₂O₂), SCCO₂ has successfully sterilized all three commercial sterilization indicators. However, the addition of 1 mL of de-ionized water was not as effective as 5 microliters of H₂O₂.

The TEM images of SCCO₂/H₂O₂ treated B. atrophaeus spores revealed visible damage of spore exosporium and spore coat. BacLight fluorescence assay showed higher degree of penetration of fluorescent dyes with SCCO₂/H₂O₂ treated spores, comparing with the staining pattern of untreated spores. The deeper penetration of fluorescent dyes indicates compromised spore permeability barrier. DPA analysis showed higher amount of DPA leakage after SCCO₂/H₂O₂ treatment compared to the trace amount of DPA release from untreated and pure SCCO₂ treatment. This was an indication of perforation of the spore coat. Based on these studies, we infer that disruption and perforation of the outer layers of spore structures are the cause of spore deactivation.