P. aeruginosa Biofilms Removal by APNTP Figure 7. CLSM images of the plasma treated biofilms. 3D rendered confocal laser scanning micrographs of 3-day P. aeruginosa biofilms, grown on polycarbonate coupons, exposed to the 20 kHz plasma jet for 0s (A and D), 60 s (B and E), and 240 s (C and F). Green colour indicates surviving cells whereas red colour indicates dead cells. Magnification power is 200x (a-c) and 600x (d–f). doi:10.1371/journal.pone.0044289.g007 dose which may render some of the biofilm cells non-culturable but still viable, which was also referred to in [39]. Live/Dead Staining and Confocal Microscopy Examination In addition, P. aeruginosa biofilms were stained with Live/Dead BacLight bacterial viability staining kit for examination by confocal laser scanning microscope. The viability kit consists of two dyes; green-fluorescent SYTO 9 which stains live bacteria, and red-fluorescent propidium iodide which only penetrates non- viable bacterial cells with damaged/perturbed cell membranes. The 3D rendered confocal images of the plasma exposed biofilms, presented in Figure 7 further supports the findings of the other experiments, namely the biofilm viable counting and XTT assays. While unexposed biofilm has been shown to be predominantly green (viable), the fraction of red colour (non-viable) increased upon increasing plasma exposure time such that the 240 s plasma exposed biofilm was almost entirely stained red; indicating that vast majority of biofilm cells were by then rendered non-viable. On the other hand, the maximum biofilm thickness observed in the biofilm areas examined by the confocal microscope was between 40 mm and 80 mm. After 240 s plasma exposure, cells in the whole biofilm thickness were affected which indicates the ability of the active plasma species to penetrate deeply into the biofilm and inactivate the cells within. Conclusions In this study a kHz-driven cold atmospheric pressure plasma jet has proven to be highly efficient in the in vitro inactivation of P. aeruginosa biofilms. A greater than 4-log (99.99%) reduction in the number of viable cells was achieved within 4 minutes of exposure to the plasma jet when operated at 20 kHz. While increasing the operation frequency to 40 kHz resulted in a complete eradication of the bacterial biofilm at 4 minutes. Survival curves, under both operating frequencies, exhibit a biphasic trend with a rapid decline in the number of surviving cells in the first 60 seconds followed by a slower rate of bacterial cell inactivation. Percentage cell reduction values obtained by XTT assay were, generally, in agreement with those obtained by colony count method with an exception at the early stage of exposure, this difference might suggest the presence of complex changes in cell physiology prior to their complete destruction. Confocal micros- copy examination, preceded by a differential staining with LIVE/ DEAD BacLight Bacterial Viability Kit, has supported the evidence of the bacterial inactivation effect exerted by the plasma jet through the whole thickness of P. aeruginosa biofilms. Plasma reactive species are highly reactive and relatively short-lived; although they have proven to exert an activity through full thickness of the bacterial biofilm, they are not expected to penetrate a host tissue or a thick substance. Therefore, the plasma application suggested by this study lies within the area of surface decontamination/sterilisation and not intended to cover biofilms protected by thick living tissues or growing in body cavities. On the other hand, the comparison between plasma-induced effects at the two frequencies is difficult since the gas temperature in the plasma plume increased with frequency. More detailed measurements are required in order to quantify specific plasma species for an improved understanding of the plasma environment. Although a comprehensive and systematic understanding of the mechanisms underlying biofilm eradication and bactericidal activity by atmospheric pressure non-thermal plasmas will prove necessary for the translation of this technology to clinical applications, atmospheric pressure non-thermal plasmas would appear to offer a promising, novel and highly efficient strategy for the control of microbial biofilms on inanimate surfaces, such as biomaterials employed in the manufacture of indwelling medical devices, as well as viable tissues. Author Contributions Conceived and designed the experiments: MYA QTA SPG WGG DO BFG. Performed the experiments: MYA QTA. Analyzed the data: MYA QTA GL WGG DO BFG. Contributed reagents/materials/analysis tools: MYA DO BFG. Wrote the paper: MYA QTA WGG DO BFG. PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e44289