REPOSITORY > RESULTS

Abstract

The sterilization efficiency of oxygen plasma was studied. Plasma was created in pure oxygen with an
inductively coupled radiofrequency discharge. The discharge power was estimated to about 180 W. Plasma
parameters were measured with a double Langmuir probe and a catalytic probe. Plasma parameters
depended on pressure in the discharge chamber. The density of charged particles exhibited a well
pronounced maximum of 3.5×10[sup]16 m-3 at the pressure of 20 Pa. At lower pressure the density decreased
rapidly with decreasing pressure, while at higher pressure it decreased slowly with increasing pressure. At
75 Pa it was about 7×10[sup]15 m-3. The density of neutral oxygen atoms was increasing monotonously with
increasing pressure. At low pressure the increase was steep; while at pressure above 100 Pa the increase
was almost negligible. Most experiments were performed at 75 Pa, where the oxygen atom density was
3.5×10[sup]21 m-3 and the dissociation fraction of oxygen molecules was the highest. Some experiments were
also performed at 30 Pa and 150 Pa. Plasma was also characterized by optical emission spectroscopy to
detect the evolution of reactive products from plasma interaction with bacteria.
Three different types of bacteria were used: Escherichia coli, Bacillus stearothermophilus and
Staphylococcus aureus. Bacteria were typically deposited onto substrates in a monolayer. The sterilization
effects were studied by scanning electron microscopy (SEM), fluorescence microscopy (FM), plate count
technique (PCT), and fluorescence activated cell sorting (FACS). Samples were exposed to plasma for
different periods up to several minutes. The treatment time was chosen for each type of bacteria according
to the visual damage obtained at a preliminary investigation by SEM.
Bacteria were deposited onto well activated substrates made from different materials. Many materials
were heated in plasma due to the heterogeneous surface recombination of oxygen atoms on the surface. In
order to avoid the heating effects, the systematic experiments were performed using glass substrates which
minimized the thermal effects.
Results obtained by fluorescence microscopy were not found to be conclusive, which was explained by
the specifics of plasma sterilization. This effect was explained by the destruction of the bacterial DNA due
to plasma action. The PCT and FACS techniques gave sound results in most cases. The PCT was used to
determine the number of surviving bacteria and eventual sterility of the substrates, while the FACS allowed
for quantitative determination of the total number of bacteria (live and dead) on the surface of substrates
after plasma treatments.
The results of systematic measurements allowed for the calculation of the doses of plasma radicals
needed for the destruction of a certain number of bacteria. The sterility of substrates contaminated with
Escherichia coli and Staphylococcus aureus was obtained after receiving the dose of about 8×10[sup]25 m-2.
Whereas the sterility of substrates contaminated with Bacillus stearothermophilus dependened strongly on
the bacteria form – vegetative or spores. We also established that when the number of bacteria was higher
then a longer dose was required to obtain sterility.
The sterilization efficiency was found to be different for bacteria types, and depended primarily on the
dose of neutral O atoms on the surface, as well as on the influx of charged species (positive O ions). In the
case of Staphylococcus aureus, the efficiency of the process was achieved when we had a higher density of
ions (ni=3×10[sup]16m-3) and a moderate density of neutral atoms (nO=2×10[sup]21m-3). Whereas for Bacillus
stearothermophilus, the best efficiency was achieved by a balanced flux of positive ions and atoms on the
surface at densities of ni=1×10[sup]16m-3 and nO=3.5×10[sup]21m-3. Therefore, the density of different plasma species
and their influx to the surface gives different sterilization efficiencies depending on the bacteria type and
their structure form. Moreover, charging bacteria by plasma provided synergetic effects and accelerated the
bacteria inactivation process. Although the main reason for the destruction of cell wall was still chemical
etching by reactive neutrals, atom by atom. This conclusion enabled us to upgrade the inactivation model
presented by Moisan (Moisan et al., 2001), and proved Laroussi and Mendis’ theory that charged particles
play an important role in the plasma sterilization of bacteria.