Table 1. D-values of 20 kHz and 40 kHz plasma jets against P. aeruginosa biofilm cells. Operating Frequency Phase 1 20 kHz 40 kHz D-Value (sec) 23.57 15.97 doi:10.1371/journal.pone.0044289.t001 Phase 2 128.20 69.79 261014 cm-3 [32]. Since the measured gas temperature in the plasma increases with pulse repetition rate we might expect a reduction in ozone density; the dependence of ozone density on gas temperature is well known. With increasing gas temperature, the ozone production rate decreases and its destruction rate increases, both leading to a lower ozone density [58]. From this we can conclude that the mechanisms responsible for enhanced biofilm inactivation with pulse repetition rate are complex and it probably not solely an individual species responsible. The survival curve for the 40 kHz plasma also exhibits a biphasic behaviour similar to the 20 kHz plasma but with lower D-values of 15.97 seconds and 69.79 seconds for the first and the second inactivation phases, respectively, compared to 23.57 and 128.20 seconds for 20 kHz (Table 1). A noteworthy point related to the D-values is that doubling the operating frequency from 20 kHz to 40 kHz resulted in reducing the D-value of the second phase by almost half. Unfortunately, doubling the plasma operating frequency increased not only the dose of active species delivered by the plasma but also the gas temperature of plasma discharge. While the 20 kHz plasma plume exhibited a gas temperature, measured as described by [59], around 39uC, at 40 kHz the gas temperature of the plasma jet was about 57uC. Although measuring the ‘‘local biofilm temperature’’ would be an interesting way of studying its effect in the eradication process, the determination of this temperature is not practically feasible because of the micro nature of bacterial biofilms. Nevertheless, since the biofilm is exposed to the plasma in an open ambient environment and under a continuous plasma gas flow, it is less likely that local temperature of the exposed biofilm will significantly exceed that of the plasma gas temperature. However, the higher temperature, observed at 40 kHz, may limit potential applications of this discharge when operating at such higher frequencies, especially for heat sensitive materials and viable tissues, but further modifications of this discharge setup, that may reduce its gas temperature, could overcome this issue. Cell Viability Determination by XTT Assay The XTT assay has been frequently used for the quantification of bacteria and bacterial biofilms [42,60] where it detects the presence of cells that are viable and metabolically active [42] regardless of being culturable. The viability of P. aeruginosa biofilms treated with a 20 kHz plasma discharge was evaluated using this method. After the samples had been prepared, as previously described, absorbance at 450 nm was measured to quantify the presence of orange-coloured formazan product which results from XTT reduction by metabolically active cells, to estimate the number of surviving cells. Absorbance values (at 450 nm) measured after exposing P. aeruginosa biofilms to the plasma jet for different time intervals are presented in Figure 5. The values continuously drop with increasing the time of plasma exposure to reach almost nil after 60 seconds which indicates the continuous P. aeruginosa Biofilms Removal by APNTP Figure 5. Absorbance of XTT-assay product. 48-hour P. aeruginosa biofilms, grown on Calgary Biofilm Device, were exposed to the 20 kHz plasma jet for up to 4 minutes. After plasma exposure, bacterial cells were dislodged off the pegs into PBS buffer by sonication. 50 ml aliquots of the recovered bacterial suspensions were then mixed with 50 ml of MHB and 20 ml of XTT stock solution and incubated at 37uC for 5 hours. After incubation, the absorbance at 450 nm was measured to quantify XTT metabolic product, the intensity of which is proportional to the number of viable (respiring) cells. (Each point represents the mean of 3 values 6 SE). doi:10.1371/journal.pone.0044289.g005 Figure 6. Percentage cell reduction curves based on colony count method vs. XTT assay. Percentage cell reduction curves of P. aeruginosa biofilm cells upon exposure to the 20 kHz plasma jet. The dotted line is based on the standard colony count method whereas the solid line is based on the XTT assay. (Each point represents the mean of 3 values 6 SE). doi:10.1371/journal.pone.0044289.g006 reduction in the number of surviving cells upon plasma exposure. This supports the evidence for the inactivation effect exhibited by the plasma against the bacterial biofilm. Percentage cell reduction values calculated based on XTT assay show good correlation with those obtained by the colony count method (Figure 6). One major difference between results of the two methods lies in the 15- seconds exposure point; the colony count method indicates that 85% cell killing was achieved at this point, while the value was about 36% from the XTT assay results. This finding may indicate that plasma exposure for this period of time provides a sub-lethal PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e44289