P. aeruginosa Biofilms Removal by APNTP Figure 2. Bacterial growth inhibition zones. P. aeruginosa cell suspensions were spread over MHA plates (9 cm in diameter). The seeded plates were exposed to the 20 kHz plasma jet for (A) 0 s, (B) 120 s, and (C) 240 s and then incubated at 37uC for 24 hours. After incubation, photographs of agar plates, showing bacterial growth inhibition zones, were taken using a digital camera. doi:10.1371/journal.pone.0044289.g002 addition, a gas-only exposure (with the power supply switched off) was carried out as a control. senting that time point with the absorbance representing the untreated control samples. Cell Viability Determination After plasma exposure, the pegs were replaced in the wells of a 96-well Microtiter plate containing 200 ml PBS in each well and sonicated [17] for 10 minutes in order to dislodge the biofilm cells from the pegs and to re-suspend surviving bacteria in PBS to permit viable counting. Following sonication, the pegs were discarded and the resultant bacterial suspensions were used to determine the viability of surviving bacterial cells. After bacterial biofilm exposure to the plasma jet, the viability of surviving cells was quantitatively determined using two methods: standard colony count method and XTT viability assay. In the standard colony count method, the recovered bacterial suspen- sions were 10-fold serially diluted in a 96-well microtiter plate using sterile PBS, and three aliquots (20 ml each) from each well were spotted on the surface of MHA plate. After incubating the MHA plates at 37uC for 24 hours, the number of colonies originated from each spot was counted using a colony counter. The number of surviving cells was calculated as colony forming unit per peg (cfu/peg) and survival curves were constructed based on these values. Furthermore, percentage cell killing was calculated by comparing the number of surviving cells in each sample with the number of bacterial cells present in the untreated samples (zero exposure time) prepared under the same conditions. XTT viability assay (XTT based in vitro Toxicology Assay Kit, Sigma-Aldrich Company Ltd., Dorset, UK) was carried out as follows: an XTT stock solution was prepared by reconstituting a kit vial (containing 5 mg of XTT with 1% PMS) with 5 ml of PBS. 50 ml aliquots of the recovered bacterial suspensions (after plasma exposure) were transferred into the wells of a 96-well microtiter plate containing 50 ml of growth media (MHB), and then 20 ml of the XTT stock solution was added to each well. After incubating the microtiter plate at 37uC for 5 hours in an orbital incubator, the absorbance at 450 nm was measured, against blank controls (containing 50 ml PBS, 50 ml MHB, and 20 ml XTT solution), using a plate reader (BioTek EL808 Microplate Reader, BioTek UK, Bedfordshire, UK) in order to quantify XTT metabolic product, the intensity of which is proportional to the number of respiring cells. The percentage cell killing at each time point was calculated by comparing the absorbance of the samples repre- Confocal Scanning Laser Microscopy Following exposure of the biofilms grown on polycarbonate coupons to the plasma jet, biofilms were stained with LIVE/ DEAD BacLightBacterial Viability Kit (Molecular Probes, Eu- gene, OR, USA), and examined by confocal laser scanning microscope (Leica TCS SP2 Confocal Microscope, Leica Micro- systems, UK). Z-stacks of confocal images were rendered into 3D mode using Volocity software (PerkinElmer, UK). Results and Discussion Bacterial Growth Inhibition Zones In order to visually demonstrate the effect of the investigated atmospheric pressure plasma jet on the viability of bacterial cells, MH agar plates seeded with P. aeruginosa were exposed to the plasma plume and incubated overnight. As shown in Figure 2, the plasma-exposed plates showed significant bacterial inhibition zones which indicate the extent of bactericidal activity of the plasma jet, against planktonic P. aeruginosa spread over the surface of the agar. Although the maximum diameter of the plasma plume observed in the current jet is less than 5 mm, inhibition zones of several centimetres are obtained; which indicates that plasma-derived active species are not confined within the visible plasma plume, rather they are present in a larger volume around it. The observable inhibition zone diameter increased with increasing exposure time. This is most likely due to decreasing densities of the active bactericidal species as a function of distance from the centre of the visible plasma plume, as these species interact with components of ambient air. Therefore areas at the periphery of the inhibition zone would be expected to require longer exposure times in order to inactivate the bacteria present. Biofilm Eradication by 20 kHz Plasma and Its Survival Curve In biofilm eradication studies, the plasma jet under investiga- tion, operated at 20 kHz and 6 kV produced more than 4-log (99.99%) reduction in the number of viable cells of P. aeruginosa biofilm within 4 minutes of exposure, as shown in Figure 3. In an PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e44289