such as "Introduction", "Conclusion"..etc
Although a number researchers have noted the phenomenon of microbial death 'when exposed to air ions', to the authors' best knowledge, none of the previous studies have made a rigorous attempt to separate out the various effects of electrical origin that may be responsible for cell death. For example, Kellogg et al.  and Digel et al.  made no attempt to distinguish effects arising from ozone or electric fields, from those caused by air ions themselves. By contrast Shargawi et al.  and Noyce and Hughes [7,8] did attempt to eliminate the effects of ozone, by performing a series of negative 'air' ion experiments in a pure nitrogen atmosphere. However this approach is inherently flawed, because although negative air ions readily form in air, they cannot form in pure nitrogen, and the mechanism is therefore believed to be electronic. One feature of negative coronas is that they can only be sustained in fluids which contain electronegative molecules, such as O2, H2O and CO2, since these gases have molecules which readily scavenge free electrons. Without electronegative molecules to capture free electrons, small negative cluster ions cannot form, with the result that a simple flow of electrons will occur in an ionized gas between the two electrodes and an arc will develop. Therefore any direct comparison between the action of negative ions in air and in nitrogen, such as that performed by Shargawi et al.  is somewhat erroneous, since both ozone and negative ions will form in air but not in nitrogen. Furthermore, irrespective of the nature of the atmosphere used, an electric field will always be present and this may have an impact on microbial viability. Given this, it is difficult to draw any firm conclusions about possible bactericidal mechanisms from much of the previous work.
Experiments such as described in this paper and those of previous researchers can produce microbial death by three, quite distinct, electrical phenomena:
• Electrodynamics – Ions, electrons and other ionising radiations
• Electrostatics – Electric charge/Electric field
• Electro-chemical effects – Ozone production
The experiments described in this paper have been designed to distinguish, for the first time, between these different effects. A mica plate was used to isolate the microorganisms from the ions and any ozone produced by the electric discharge. Similarly, an earthed wire mesh was used to prevent exposure of the microorganisms to both the ions and the electric field, allowing only the ozone to pass. In this way, it was possible to quantify with some accuracy the relative proportions of microbial mortality attributable to the different bactericidal mechanisms.
From the data presented in figures 2, 4 and 5 a clear consistent picture emerges. When exposed to 'negative ions' the principal bactericidal mechanism affecting most of the test species appears to be oxidation damage arising from exposure to ozone. This is clearly evident from Figure 5 which shows the reduction in the microbial population primarily due to the action of ozone. This finding echoes that of Shargawi et al. , who when working with Candida albicans found a strong correlation between cell death and the level of ozone present. The results achieved with the mica plate in place reinforce the opinion that ozone played an important role in the inactivation of most of the species tested. The results in Figure 4 indicate that for most of the species tested the negative ions and the electric field played only a minor role in cell death compared with the action of the ozone. Further evidence supporting this comes from the positive ion data presented in Figure 3. The mechanism by which positive ions are produced generates much less ozone than does its negative counterpart. This is because of the greater number of free electrons associated with negative coronas and the fact that the reactions which produce ozone are relatively low-energy. Consequently, when exposed to positive ions the test bacteria were also exposed to reduced levels of ozone compared with levels associated with negative air ionization, with the result that for most of the test species only a modest bactericidal action was observed. Interestingly, from the results in figures 2 and 5 it appears that some microorganisms (i.e. Serratia marcescens and Acinetobacter baumannii) are more susceptible to ozone alone compared with the combined action of ozone, negative ions and the electric field. However, the reasons for this phenomenon are unclear.
The behaviour of Mycobacterium parafortuitum is of particular interest. For this bacterium in the absence of ozone or negative ions (see Figure 4), a substantial reduction (i.e. 94.9% at 15 minutes) was achieved solely through the intervention of the electric field. This suggests that unlike the other bacteria, with this species the principal inactivation mechanism is electroporation. Corroboration of this comes from Figure 5 which shows that for this bacterium, the kill achieved through the action of ozone alone was actually less than that achieved by the electric field alone. Further evidence suggesting that ozone played only a relatively minor role in the inactivation of Mycobacterium parafortuitum comes from Figure 3, where it can be seen that under conditions of reduced ozone a large kill was still achieved. Of the bacteria tested this behavior appears unique to Mycobacterium parafortuitum. By comparison, the electric field appears to have played only a minor role in the inactivation of the other bacteria.
Collectively the data in figures 2, 4 and 5 suggests that for most of the bacterial species tested, negative air ions play only a relatively minor role in the bactericidal process. Microbial inactivation appears to occur mainly through a combination of ozone damage and damage caused by the electric field, with very little contribution from the negative ions themselves. Although not conclusive, Figure 3 suggests that the positive ions also have only a very limited affect on the bacteria studied. Indeed, the similarity between figures 3 and 4 is striking. Thus, a very interesting picture emerges. It would appear that some bactericidal action results from all the electrical phenomena tested. The disinfectant properties of ozone are well known and this mechanism seems to be responsible for the majority of the kills. However, it also appears that both the electric field and air ions have a contributory role.
Although it might be argued that the magnitude of the applied field is not high in relative terms, the complete dynamics of the time development of the electric field in a spherical dielectric shell representing the cellular membrane can be obtained using an analytical solution of the Ohmic conduction problem. Indeed, it has been found that the field in the membrane can reach a maximum value two orders of magnitude higher than the original Laplacian electrical field . The plasma membrane of a cell serves the vital function of partitioning the contents of the cytoplasm from the external environment. These membranes are largely composed of amphiphilic lipids which self-assemble into highly insulating structures and thus present a large energy barrier to trans-membrane ionic transport. However, the lipid matrix can be disrupted by a strong external electric field leading to an increase in trans-membrane conductivity and diffusive permeability. These effects are the result of formation of aqueous pores in the membrane, which alter the electrical potential across the membrane and may ultimately lead to cell lysis.
Electroporation is a mechanical method used to introduce polar molecules into a host cell through the cell membrane. In this procedure, a large electric pulse temporarily disturbs the phospholipid bilayer, allowing molecules like DNA to pass into the cell. Electroporation is also the basic mechanism of tissue injury in high-voltage electric shock. If the strength of electrical field and duration of exposure to it are properly chosen, the pores formed by the electrical pulse reseal after a short period of time, during which the extracellular compounds have a chance to get inside the cell. However, excessive exposure of live cells to electrical fields can also cause apoptosis. Indeed, such harsh treatments have been used for killing tumour cells.
Care has been taken in the experimental protocols described in this paper to distinguish between the potential biocidal effects of the ions and the possibility of cell death induced by the electric field alone. Onset of cell lysis generally takes place at electric field strengths of around 100 V/mm or equivalently 100 kV/m. The ionising electrodes in our experiments are located 25 mm from the agar surface. At a potential of 10 kV this results in an 'estimated' field strength of 400 kV/m, which suggests that cell death due to electroporation may indeed have occurred. (It should be noted that because of the large field gradient at the electrode tip, this simple computation over-estimates the field strength at the agar surface. However, for the purposes of this study this estimate is a useful enough indicator of the field strength at the agar surface.) To quantify the effect of the electric field, a mica barrier was inserted between the ion source and the agar surface. The mica stopped the ions and ozone but had little effect on the electric field. In this way it was possible to measure the background effect of the electric field in isolation. From Figure 4 it can be seen that in addition to the dramatic effect on Mycobacterium parafortuitum, the electric field alone appears to have resulted in a modest kill in most of the other microbial species tested.
Collectively, the findings of the study indicate that negative air ions have little bactericidal effect on the species studied, despite being generated in close proximity to the microorganisms. This confirms the observations of Kerr et al. , who in a study on an intensive care unit (ICU), found the action of negative air ions to be associated with; (a) increased environmental isolates of Acinetobacter spp, and (b) a marked decrease in numbers of Acinetobacter infections/colonizations. Clearly, if the negative ions had had any bactericidal effect, then Kerr et al. would have observed a decrease in environmental isolates. This suggests that in the ICU study the reduction in observed Acinetobacter infections/colonizations was mainly due to physical effects rather than any bactericidal phenomena.
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