Fig 1.
Plasma-treated P. aeruginosa biofilms stained with Calcofluor White.
Biofilms were treated with plasma for 0, 3, and 30 min prior to staining. Images were acquired with a 355/433 nm excitation/emission filter at 400 X and processed with the Leica Application Suite software.
Fig 2.
Plasma-treated P. aeruginosa biofilms stained with SYTO9 and Calcofluor White.
Biofilms were treated with plasma for 0, 3, and 30 min prior to staining. Column A depicts images corresponding to SYTO9 staining and visualized with the 480/500 nm excitation/emission filter. Column B shows images of the same samples subjected to Calcofluor White staining and obtained with a 355/433 nm excitation/emission filter. Column C depicts superimposed images from the previous two panels. 400 x images were acquired with the Leica Application Suite software.
Fig 3.
Image binarization of plasma-treated P. aeruginosa biofilms stained with SYTO9 and Calcofluor White.
Biofilms were treated with plasma for 0, 3, and 30 min prior to fluorescent staining with Calcofluor White for the biofilm matrix (blue) and SYTO9 for the bacterial cells (green). 400 X images were acquired with the Leica Application Suite software. Column B corresponds to image digitalization to black and white and column C shows the further binarization with ImageJ software (right panel).
Table 1.
Ratio between the biofilm matrix area and the sessile cells area obtained from binary images of plasma-treated biofilms.
Fig 4.
SEM images of plasma-treated P. aeruginosa biofilms.
Biofilms were treated with plasma for 0, 3, and 30 min (from top to bottom). Magnification: 5000X and 10000X for images on the left and right panels, respectively.
Fig 5.
P. aeruginosa biofilms grown in continuous culture on stainless-steel coupons in AB synthetic medium were treated with DNase and exposed to plasma generated in moistened air. Results are the average of four independent experiments. Each experiment was performed in duplicates. Error bars represent the standard error of the mean.
Fig 6.
A/T FT-IR absorption spectra of P. aeruginosa biofilms.
(A) Three average spectra obtained from three independent experiments corresponding to P. aeruginosa biofilms with no plasma treatment (control samples). Spectral windows (W1 to W4) associated to functional groups in biomolecules are indicated. (B-D) second derivatives at the different spectral windows: (B) W1 spectral windows associated to lipids, (3,000–2,800 cm-1), (C) W2 region assigned to protein absorptions (1,800–1,500 cm-1), (D) W3 the mixed region (1,500–1,200 cm-1), and (E) W4 assigned to carbohydrates absorption bands (1,200–900 cm-1).
Table 2.
Spectral windows associated to functional groups in biomolecules and band assignment.
Fig 7.
Plasma effect on whole P. aeruginosa biofilms.
Cluster analysis obtained with 2nd derivatives as input data using scaling to first range for spectral distance calculation in the entire IR spectrum: (3,000–2,800 cm-1), (1,800–1,550 cm-1), (1,500–1,250 cm-1), (1,200–900 cm-1). Dendrogram was constructed using Ward´s algorithm. The cut-off value of D = 6 is indicated with the dotted line.
Fig 8.
Plasma effect on protein conformation.
(A) Vector-normalized second-derivatives of the average spectra of plasma-treated biofilms (blue, 3 min; red, 30 min; and black, control) in the amide I and II regions. (B) Cluster analysis obtained with 2nd derivatives as input data using scaling to first range for spectral distance calculation in the spectral ranges: (1,669–1,697 cm-1), (1,633–1,661 cm-1). Dendrogram was constructed with Ward´s algorithm. The cut-off value of D = 6 is indicated with the dotted line.
Table 3.
Semi-quantitative analysis of primary lipid oxidation products and final oxidation products.
Fig 9.
Plasma effect on biofilm carbohydrates.
(A, B) Vector-normalized second-derivatives of the average spectra in the C-H stretching vibration region (W1 3,000–2,800 cm-1) (A), and in the carbohydrate region (W4 1,200–900 cm-1) (B). Black, blue, and red lines correspond to biofilms treated with plasma for 0, 3, and 30 min, respectively. (C) Cluster analysis obtained with 2nd derivatives as input data using scaling to first range for spectral distance calculation in the spectral ranges: 2,880–2,870 cm-1 and 1,200–900 cm-1. The dendrogram was constructed with Ward´s algorithm. The cut-off value of D = 6 is indicated with the dotted line.
Fig 10.
Cluster analysis obtained with the 2nd derivatives as input data using scaling to first range for spectral distance calculation in the spectral ranges 1,500–1,200 cm-1. Dendrogram was constructed with Ward´s algorithm. The cut-off value of D = 6 is indicated with the dotted line.