Dispersed bacteria versus biofilm.

<p>Confocal laser scanning images with 400x magnification of <i>G</i>. <i>vaginalis</i> biofilm in 2 vaginal slides (A and B) in a superimposed image: vaginal epithelial cells DAPI in blue and <i>G</i>. <i>vaginalis</i> specific PNA-probe Gard162 with Alexa Fluor 647 in red. A: vaginal sample with dispersed bacteria; B: vaginal sample with bacteria in biofilm.</p

Superimposed image of polymicrobial biofilm of <i>A</i>. <i>vaginae</i> and <i>G</i>. <i>vaginalis</i>.

<p>Montage of confocal laser scanning images with 400x magnification of polymicrobial biofilm in 6 vaginal samples (A-F) in a superimposed image: vaginal epithelial cells DAPI in blue, <i>G</i>. <i>vaginalis</i> specific PNA-probe Gard162 with Alexa Fluor 647 in red and <i>A</i>. <i>vaginae</i> specific PNA-probe AtoITM1 with Alexa Fluor 488 in green. For clarity we omitted the BacUni-1 plane; the bacteria that are not bound by the specific probes are visible in DAPI blue.</p

Polymicrobial biofilm of <i>A</i>. <i>vaginae</i> and <i>G</i>. <i>vaginalis</i> in different panes.

<p>Confocal laser scanning image with 400 x magnification of polymicrobial biofilm in different panes, A: vaginal epithelial cells DAPI in blue, B: all bacteria, BacUni-1 PNA-probe with Alexa Fluor 555 in yellow, C: <i>A</i>. <i>vaginae</i> specific PNA-probe AtoITM1 with Alexa Fluor 488 in green, D: <i>G</i>. <i>vaginalis</i> specific PNA-probe Gard162 with Alexa Fluor 647 in red (superimposed image can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136658#pone.0136658.g003" target="_blank">Fig 3A</a>).</p

Specificity testing in duplicate of PNA probes using cultured bacteria.

<p>¹(+) Presence of hybridization</p><p>²(-) Absence of hybridization.</p><p>The signal was considered positive if it had a positive counterpart in the DAPI stain and displayed a positive signal simultaneously with the broad-range probe. The signal was considered negative if no signal was seen with the species-specific probe.</p

<i>G</i>. <i>vaginalis</i> biofilm.

<p>Montage of confocal laser scanning images with 400x magnification of <i>G</i>. <i>vaginalis</i> biofilm, negative for <i>A</i>. <i>vaginae</i>, in 4 vaginal samples (A-D) in a superimposed image: vaginal epithelial cells DAPI in blue and <i>G</i>. <i>vaginalis</i> specific PNA-probe Gard162 with Alexa Fluor 647 in red. For clarity we omitted the BacUni-1 plane; the bacteria that did not hybridize with Gard162 are visible in DAPI blue.</p