Emission Spectroscopy and Atomic Force Microscopy Studies of Plasma Assisted Biofilm Inactivation

Summary form only given. The removal and disposal of biofilms is an expensive, time-consuming effort required in many industries (medical, and water purification, to name a few). Biofilms are microbial communities that coalesce in an exopolysaccharide matrix which enables them to adhere to different surfaces and make them more resistant to conventional sterilization techniques than free, mobile bacteria. Gas discharge plasmas are a novel approach to inactivating biofilms. They contain a mixture of charged particles, chemically reactive species, and UV radiation, all of which are well-established sterilization agents against free microorganisms. Four day-old single-species biofilms were grown in 96-well polystyrene microplates using Chromobacterium violaceum, a gram negative bacterium. The gas discharge plasma was produced with an Atomflo 250 plasma reactor (Surfx Technologies) in which discharge occurs in gas flowing through two narrowly spaced, perforated electrodes (one at ground, one at 100 MHz RF). A mixture of helium and a secondary gas, nitrogen, was used. Bacterial biofilms were exposed to it for various time periods. The spectroscopy data were obtained using an Ocean Optics HR-2000 spectrometer with 0.5 nm resolution. The microscopy data were obtained using a Quesant atomic force microscope (AFM) in air in intermittent contact mode. Our data show that after a 10-minute plasma treatment, almost 100% of the viable cells are removed. The kinetics show a rapid initial decline in CFU/mL followed by a much slower subsequent decline. Spectroscopy data show the presence of two different chemically reactive oxygen species: NO and OH. These reactive species interact with biomolecules in the bacterial cell membranes and walls. The microscopy data show that non culturable cells are still intact at short exposure times. The microscopy data also suggest that cells may undergo cell wall damage after longer plasma exposure periods. These results indicate the potentia- l of plasma as an alternative way for biofilm inactivation.