earlier short communication, we observed complete eradication of P. aeruginosa biofilms after 10 minutes of exposure using the same plasma jet operating at 20 kHz and 6 kV [43]. These results are superior to those reported for other atmospheric pressure plasma jets against various Gram negative bacterial biofilms; where less than a 2-log (99%) reduction was achieved in 4 minutes with Chromobacterium violaceum [39] and Neisseria gonorrhoeae [53] biofilms. In another study, only 40 fold reduction in the number of E. coli biofilm cells was achieved after 40 minutes of exposure to another setup of atmospheric pressure plasma [41], even though E. coli biofilms had been reported to be more susceptible, toward the plasma jet investigated in this study, than P. aeruginosa biofilms [43]. Treatment of fungal biofilms using three different plasma systems was also reported in [46]; where the most efficient system showed 5-log reduction after 10 minutes of plasma exposure, but complete biofilm eradication was not achieved with any of the three plasma systems [46]. On the other hand, activity of a radiofrequency plasma needle was evaluated against Streptococcus mutans biofilms [44]. Although this plasma needle was shown to have a good activity against biofilms, grown under certain conditions, no enumeration of surviving biofilm cells was carried out in this study. In another study, microwave-induced argon plasma has been shown to completely eradicate E. coli, S. epidermidis and MRSA biofilms after 20 seconds of exposure [45], but quantitative analysis of biofilm surviving cells was based on crystal violet (CV) assay. While CV assay is a good indicator of the total attached biomass, it is poorly suited to evaluate killing of biofilm cells [54,55]. In a recent study, oral biofilms formed in situ on titanium discs were removed by microwave-driven nonthermal plasma, however, ‘‘complete re- moval’’ was only achieved after another treatment with air/water spray followed by a second cycle of plasma treatment [47]. Although these three studies present valuable information on the use of atmospheric pressure plasma for biofilm eradication, no survival curves or log-reduction values were reported which makes it hard to compare the efficiencies of these plasma systems with that of the system being investigated in the current study. The survival curve presented in Figure 3 is characterised by a biphasic trend in which there are two bacterial inactivation phases; each with a distinct decimal reduction time (D-value, the time taken to reduce the bacterial population by 90% or a one log reduction). The first phase occurring during the initial 60 seconds of plasma exposure is characterised by a rapid reduction in the number of surviving cells with a D-value of 23.57 seconds. After 60 seconds of exposure, a second inactivation phase starts with a lower rate of bacterial destruction reflected by a higher D-value of 128.20 seconds. These data concur with similar biphasic survival curves reported for Chromobacterium violaceum [39] and Neisseria gonorrhoeae [53] biofilms, on exposure to different setups of atmospheric pressure plasma. The slower rate of biofilm reduction observed after 60 seconds of plasma exposure may result from the protection provided by the polymeric matrix surrounding cells in deep layers of the biofilm, or may be caused by the shielding effect of cellular debris produced from the plasma-lysed cells at the surface of biofilms, as previously proposed [39]. Effect of Plasma Frequency Variation on Its Anti-biofilm Activity As discussed above the kHz-driven plasma jet used in this study produces a pulsed plasma jet or ‘plasma bullets’ rather than a continuous plasma plume [48,49,56]. Thus a higher frequency results in more plasma pulses and effective plasma on-time; hence on time average a higher density of the plasma reactive species will be delivered [57]. It would therefore be expected that increasing P. aeruginosa Biofilms Removal by APNTP Figure 3. Survival curve of biofilm treated with 20 kHz plasma. 48-hour P. aeruginosa biofilms, grown on Calgary Biofilm Device, were exposed to the 20 kHz plasma jet for up to 4 minutes. The number of biofilm surviving cells in each sample was then calculated using colony count method and used to construct the log survival curve. (Each point represents the mean of 3 values 6 SE). doi:10.1371/journal.pone.0044289.g003 the frequency (in this case, by a factor of 2, from 20 kHz to 40 kHz) would yield improved plasma-mediated biofilm eradica- tion by increasing the effective plasma ‘dose’ delivered. Figure 4 demonstrates that 40 kHz plasma resulted in a significantly enhanced inactivation rate of P. aeruginosa biofilms, with complete biofilm eradication after only 4 minutes of plasma exposure. This would indicate that biofilm inactivation by atmospheric pressure plasma is ‘dose’ dependent, besides being time dependent. Through varying the plasma pulse repetition rate, at a constant applied voltage, we can increase the dissipated power into the plasma, while E/N remains unchanged. However, it is also important to consider the recombination chemistry of the plasma afterglow since the biofilm is exposed to the time averaged plasma. Species have varying lifetimes and some species densities will increase, while others will dramatically decrease in the plasma-off phase. Metastable molecular singlet delta oxygen (SDO) is an important ROS and known agent in numerous biochemical processes, it also has an extremely long radiative lifetime of more than 75 minutes in the gas phase. The absolute density of SDO, as measured through infra-red emission spectroscopy in the same plasma source, while in general increases with increasing frequency, between 20 kHz and 40 kHz remains constant at Figure 4. Survival curves of biofilms treated with 20 kHz vs. 40 kHz plasma. Comparison between log survival curves of P. aeruginosa biofilm cells, constructed as described above, upon exposure to both 20 kHz and 40 kHz plasma jet. (Each point represents the mean of 3 values 6 SE). doi:10.1371/journal.pone.0044289.g004 PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e44289