Time laps images of <i>P. putida</i> KT2440 immobilized on PD islands printed on a PEGylated glass surface.

<p>The images are obtained for surfaces covered with medium between 0 and 160 minutes after changing the medium from LB to LB containing the inducer MB. MB induces the expression of GFP in the bacteria. The images are overlays of transmission light images and fluorescence images—both obtained using a Leica SP5 with a 20 × objective (N.A. = 0.7).</p

Right: Fluorescence micrograph of quantum dots deposited on a cleaned glass coverslip using μCP with PDMS stamps.

<p>Such images were used to study the reproducibility of the obtained patterns. Left: Distributions of observed diameters of the nine largest stamped islands compared to the mask hole diameters (blue triangles and corresponding blue linear regression line). Island diameters calculated from the area of each island as determined based on the ImageJ software and fluorescence micrographs of quantum dots. The red triangle indicate the most probable island diameter <i>d</i>_<i>m</i> and the red line is the linear regression obtained based on <i>d</i>_<i>m</i> obtained for the eight largest stamped islands.</p

Quantitative analysis of the number of bacteria immobilized onto each adhesive PD island of bacterial microarrays prepared on glass surfaces coated with PEG.

<p>The arrays were prepared using μCP with PDMS stamps obtained using a photolithography mask with <i>d</i>_<i>h</i> equal to 3.5 μm (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128162#pone.0128162.g001" target="_blank">Fig 1d</a>). <i>N</i>_<i>b</i> ≥ 1: one or more bacteria per island. <i>N</i>_<i>b</i> = 1: one bacterium per island.</p><p>Quantitative analysis of the number of bacteria immobilized onto each adhesive PD island of bacterial microarrays prepared on glass surfaces coated with PEG.</p

Images of glass surfaces and glass surfaces precoated with chemicals reducing bacterial adhesion, patterned with chemicals promoting bacterial adhesion, immersed in a solution containing bacteria and finally rinsed and covered with LB.

<p>Results obtained for the three chemicals potentially reducing bacterial adhesion (BSA, PVA or PEG) are shown. The substrates are patterned with one of three chemicals promoting bacterial adhesion (PLL, PEI or PD) using μCP with a PDMS stamp with 5 μm lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128162#pone.0128162.g001" target="_blank">Fig 1d</a>) and immersed in a solution containing bacteria. All scalebars are 10 μm. The combination of chemicals investigated in each experiment is indicated on the figure. The surfaces were rinsed in MilliQ water after the incubation with bacteria (<i>P. putida</i> KT2440) in order to remove weakly adhering bacteria. During imaging the surfaces were covered with LB in order to minimize stress induced in the attached bacteria. The images are obtained by using transmission light microscopy, and were captured on a Leica TCS SP5 with a 40 × objective (water, N.A. = 1.2).</p

Tapping mode AFM height topographs of PD printed on PEGylated glass.

<p>Tapping mode AFM height topographs of PD printed on PEGylated glass.</p

(a): The patterns on the photolithography masks used to produce PDMS stamps.

<p>The first pattern (left) consisted of 13 circular holes of diameter increasing from 0.8 μm to 4.4 μm on an opaque background. The mask contained four quadrants, each characterized by a vertical separation distance <i>d</i>₁ of 3, 4, 6 or 8 μm between the circular holes and a fixed horizontal distance <i>d</i>₂ between the center of each hole of 7.4, 8.4, 10.4 or 12.4 μm. The pattern on the second photolithography mask (right) consisted of circular holes with a diameter <i>d</i>_<i>h</i> of 3.5 μm with a separation distance <i>d</i>₃ between the circular holes equal to either 10 or 15 μm. (b), (c) and (d): SEM micrographs of gold coated PDMS stamps intended for patterning of glass surfaces by μCP. The stamps shown in (b) and (c) are produced using the photolithography masks schematically illustrated in 1(a). The stamp depicted in (d) was obtained using a photolithography mask with slits of width 5 μm interspaced by 5 μm.</p

Quantitative analysis of the number of <i>P. putida</i> KT2440 adhering onto each of the PD islands on the obtained microarrays.

<p>For each PD island size, both the fraction of the spots having one or more bacteria attached (<i>N</i>_<i>b</i> ≥ 1) as well as the fraction of the spots with only one bacterium attached (<i>N</i>_<i>b</i> = 1), were determined.</p

Fluorescence image reflecting the viability of <i>P. putida</i> KT2440 immobilized on arrays of PD islands on a PEGylated glass surface.

<p>Live bacteria are stained green, dead bacteria are stained red and the image is an overlay of both green and red fluorescent images. A single dead (red) bacteria is observed (white circle). The image is obtained for arrays covered in liquid using a Leica SP5 with a 10 × objective (N.A. = 0.4).</p