P. aeruginosa Biofilms Removal by APNTP Figure 1. The plasma jet used in this study. (A) Schematic diagram of the plasma jet. (B) Photograph of the plasma jet interacting with a biofilm sample. doi:10.1371/journal.pone.0044289.g001 streamer-like behaviour ([48–52], which is in good agreement with the model proposed by Lu and Laroussi [49]. Inhibition Zone Determination Inhibitory zones formed on P. aeruginosa lawns on solid agar cultures following exposure to the plasma plume were determined as described previously [43]. After exposure to the plasma, Mueller Hintonagar (MHA) plates which had been streaked with a phosphate buffered saline-diluted overnight culture of P. aeruginosa were incubated at 37uC for 24 hours in a static incubator and the diameter of zones of inhibition measured using Vernier callipers. Biofilm Growth For survival curves construction and XTT assay experiments, bacterial biofilms were grown on the peg lid of Calgary Biofilm Device (commercially available as the MBEC AssayTM for Physiology & Genetics (P & G), Innovotech Inc., Edmonton, Alberta, Canada). An overnight culture of PA01 was adjusted to an optical density (OD550) equivalent to 16107 cfu/ml. The standardized bacterial suspension was used to inoculate the Calgary Biofilm Device (with 150 ml in each well) which was then incubated at 37uC for 48 hours in a humidified compartment within an orbital incubator. The bacterial inoculum was replaced by fresh growth medium (i.e. MHB) after the first 24 hours of incubation. At the end of 48 hours of incubation, individual pegs were broken off the lid with sterile pliers and gently rinsed with 200 ml of PBS for 1 minute to get remove of any planktonic or loosely adhered bacteria before exposure to the plasma jet. For microscopic examination, bacterial biofilms were grown on polycarbonate coupons (10 mm diameter) in a continuous flow cell chamber (FC 271-AL-3610 Dual Channel Coupon Evaluation Flow Cell, BioSurface Technologies Corp., Bozeman, Montana, USA) for 3 days. Initially, fresh growth Mueller Hinton broth (MHB) was allowed to perfuse the flow cell at a rate of 0.1 ml/ minute for 24 hours, after which time the perfusion was arrested and 1 ml of a mid-logarithmic phase bacterial suspension was injected into the flow cell chamber and left for 1 hour (under static conditions) to allow for adherence of bacterial cells onto the coupon surface. Following cell adherence, the flow cell chamber was perfused with fresh MHB at a rate of 0.1 ml/minute for 3 days. At the end of the biofilm growth period, a final rinsing step was carried out by running 0.9% NaCl solution through the flow cell at a rate of 1.0 ml/minute for 10 minutes before removing the coupons bearing mature biofilm, using sterile surgical tweezers, and exposure to the plasma treatment. Treatment Conditions Individual pegs, bearing 48 hr PA01 biofilms, were exposed to the plume of the plasma jet for varying periods of time (0, 15, 30, 45, 60, 120, and 240 seconds) as described previously [43]. The treatment was repeated in triplicate at each time point. The distance between the end of the plasma tube and top of the peg was fixed to 15 mm. The atmospheric pressure plasma jet was operated at a frequency of either 20 or 40 kHz at a set voltage of 6 kV. For determining bacterial inhibition zones resulting from plasma exposure, the bacteria seeded agar plates were placed under the plasma plume at a distance of 25 mm away from the end of the plasma tube for the following time intervals: 0, 120, and 240 seconds. After plasma exposure, the plates were incubated and photographed as mentioned previously. Mature biofilms grown on polycarbonate coupons in the flow chamber described above were exposed to the 20 kHz plasma jet under the same operating conditions but at two time points (60 seconds and 240 seconds). In PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e44289