Effects of Cell Density on Efficacy of Atmospheric Plasma Inactivation of Spores of Bacillus Subtilis

Abstract

Summary form only given. Atmospheric pressure glow discharges have in the past decade been shown to be capable of inactivating a wide range of micro-organisms including vegetative bacteria and bacterial spores. This promises a generic plasma-based sterilization and sanitation procedure with application in both the healthcare and food industries. It also encourages an in-depth and systematic study of how plasma species may impact on cell viability and how different cellular conditions may influence inactivation efficacy. One of the greatest knowledge gaps surrounds the issue of whether the resistance of spores can be affected either by conditions prevailing at the time of sporulation, or by the environment in which the spores are subsequently exposed to the plasma. Also hitherto neglected is the effect of spore density on atmospheric plasma inactivation. Without a clear understanding of these issues, it will be difficult to arrive at robust procedures that can be used to achieve the desired levels of sterility. In this work, we report on the kinetics of inactivation of Bacillus subtilis spores supported on membrane filters. Using a 20 kHz low-power atmospheric glow discharge, Bacillus subtilis spores of different initial cell densities were exposed to plasma treatment over different periods of time up to 10 minutes. It is shown that Bacillus subtilis of higher cell density were more resistant to plasma treatment. We also varied the physiological state of the spores by desiccation, and exposed the desiccated spores to plasma treatment to establish changes to inactivation kinetics. Fluorescence microscopy and scanning emission microscopy are also used to support the above studies and to enable new insights to be gained into plasma injuries of Bacillus subtilis. Finally plasma chemistry is studied using time-resolved emission spectroscopy to understand the make-up of plasma species to which Bacillus subtilis spores are exposed. The synergistic interplay of these studies is then used to attempt a coherent picture of the injuries inflicted by atmospheric glow discharges