Use of fluorescent techniques for understanding the effect of non thermal plasma discharge on the E. coli cell

Summary form only given: It has been demonstrated that ambient air non-thermal plasma discharges are very effective in inactivating various microorganisms. Plasma is being considered as a potent sterilization technology. It is important to understand the biological effect of plasma on bacteria. Fluorescent techniques are often employed by cell biologists to detect cellular phenomenon. These phenomena include changes in pH, membrane permeation and certain cellular reactions. The LIVE/DEADreg BacLighttrade Bacterial Viability Kit (Molecular Probes, Eugene OR) consists of two stains that are used to detect live and dead bacteria in large bacterial populations. These stains are propidium iodide (PI) and SYTO 9. The SYTO 9 (Green) stain permeates the membranes of all bacteria, whereas the PI (red) stain only permeates the bacteria whose membrane has been irreversibly damaged. Hence the ratio of green to red fluorescence is considered the live/dead ratio. This kit has been used successfully to detect live bacteria in soil, milk and other environmental samples. It has also been used to determine the effectiveness of bactericidal treatments. E. coli bacteria were grown overnight and suspended in solution. The solution was treated with dielectric barrier discharge (DBD) plasma. The viable counts using plating techniques and colony forming unit (CFU) counting indicate ~2log reduction in the concentration of live bacteria. For the same treatment dose, the LIVE/DEADreg kit was added to the samples after they were plasma treated and then they were analyzed under a fluorescent microscope. Multiple fields of vision were analyzed and statistical analysis was performed on the samples. The results indicate that the number of bacteria fluorescing red was significantly lower than the expected number from viable counts. This indicates that membrane permeation is not the major cause of plasma induced inactivation of these bacteria. Further experiments are being performed to understand t- e effect of plasma treatment on the cellular respiration and other potential avenues for inactivation of the bacteria.