Bacterial swarm assays.
Swarming was studied for the strains listed in using plate assays containing 0.45% noble agar and FAB medium with 12 mM glucose (P. aeruginosa
) or 0.6% noble agar and LB with 0.5% glucose (S. enterica
serovar Typhimurium with P. aeruginosa
). Some plate assays contained dye mixtures that were added immediately prior to pouring the melted agar medium into plates. Approximately 6 h after pouring the plates, they were inoculated using a sterilized platinum wire with log-phase cells (optical density at 600 nm [OD600
] ≈ 0.7) grown in their respective media used for the swarm experiments. Swarm plates that were imaged only for their comparative endpoint swarm development (i.e., analysis of different P. aeruginosa
strains) were incubated at 30°C for 48 h prior to imaging.
Bacterial strains used in this study
Cell- and rhamnolipid-imaging dyes.
Swarm assays conducted with strains that were not fluorescent or bioluminescent contained Syto 24 (Invitrogen) at a concentration of 4 μl/100 ml to stain bacterial cells green.
Some plate assays were amended to include the lipid stain Nile red (20
) as a visual indicator of spatial rhamnolipid distribution. These rhamnolipid indicator plates included a 1-in-100 dilution of filter-sterile stock containing 1 mg ml−1
Nile red (MP Biomedicals) dissolved in 85% propylene glycol (prepared the day of use to limit photoinactivation). Plates were cured and incubated in the dark.
Imaging experiments were performed at room temperature (~23°C) on a Carestream multispectral FX (MSFX) (Carestream Health, Woodbridge, CT) image station equipped with a 16-bit charge-coupled device (CCD) sensor and 300-W light source. Up to four plates were placed on a tray inside the MSFX. Since the camera for the MSFX captures from beneath the imaging platen, the plates were inverted so that the agar would not obstruct the optical path. The lids of the petri dishes were placed open side up on top of their counterparts that held the inoculated agar. The lids of the petri dishes were filled with water, and another tray was used to enclose the petri dishes in order to maintain humidity throughout the experiment. Five types of images were collected as follows.
Green fluorescence: excitation, 480 ± 10 nm; emission, 535 ± 17.5 nm; acquisition time, 30.0 s; f-stop, 4.0; field of view (FOV), 190 mm; focal plane, 27.5 mm; binning, none. These settings were used to capture fluorescence from the green fluorescent protein expressed in cells.
Red fluorescence: excitation, 540 ± 10 nm; emission, 600 ± 17.5 nm was used to collect the data; duration, 60.0 s; f-stop, 4.0; FOV, 190 mm; focal plane, 27.5 mm; binning, 2 × 2. These settings were used to capture fluorescence from Nile red staining of rhamnolipid.
Luminescence I: excitation, off; emission, no filter; acquisition, 10 s; f-stop, 2.5; FOV, 190 mm; focal plane, 27.5 mm; binning, 16 × 16. This luminescent image served to turn off the excitation lamp during intervals between fluorescent images to prevent any excitation light interference with bacterial growth.
Luminescence II: excitation, off; emission, no filter; acquisition, 300 s; f-stop, 2.5; FOV, 190 mm; focal plane, 27.5 mm; binning, 8 × 8. These settings were used to capture emissions from bioluminescent bacteria.
Image preparation and analysis.
Carestream Multispectral Software was used to batch export each image as a 16-bit TIFF file. The images were taken and stacked according to category (GFP, Nile red, etc.) using ImageJ editing software. Image processing proceeded as follows: (i) intensity signals were inverted, (ii) the look-up table was inverted, and (iii) background was subtracted using a rolling-ball radius with a pixel radius equal to half of one dimension of the image size (e.g., 1,024 for a 2,048 × 2,048 image).
Only GFP and Nile red images were used for quantitative image analysis. Images in which the P. aeruginosa bacteria had reached the perimeter of the plate were omitted from further analysis by deleting them from the stack. The ICA (intensity correlation analysis) look-up table was applied to the stack to better visualize the borders of the bacterial colony. Next, the threshold utility (with “dark background” checked) was used to adjust the threshold to isolate the bacterial colony. The wand (tracing tool) was then used to automatically select the area superimposed over the bacteria from the threshold utility. After selecting the area with a region of interest (ROI), the threshold function was reduced to visually observe and confirm that the ROI had accurately captured the true bacterial area. This process must be performed for each image analyzed, as the bacterial colony will swarm and its characteristics will change. It is important to note that as the bacteria swarm, the threshold will change, i.e., increase, as the other characteristics change. Additionally, different thresholds are used among different data sets (e.g., GFP and Nile red). In order for the ROI to accurately represent the bacterial growth, it is recommended that the threshold be increased in small increments between consecutive images. For the time-lapse intervals used for these 2-to-3 day experiments, analyzing every 5th or 10th image tended to create the best data set.
The wand has now created an ROI that accurately captures the current bacterial colony. Using the “Measure” utility, one can acquire the data of the ROI (the bacterial colony). Various data can be gathered, including area, perimeters, and integrated density, to quantify these parameters of swarming.
Images of single cells were obtained using a Nikon A1 confocal microscope equipped with a 100× LU Plan Fluor objective with simultaneous excitation at 488 nm and 561 nm with emission capture using settings of 525 ± 50 nm and 595 ± 50 nm.
LC-MS analyses of rhamnolipid.
Supernatants from FAB-glucose-grown cultures were analyzed using liquid chromatography-mass spectrometry (LC-MS) (13
). Supernatants were prepared by harvesting cultures after 48 h of growth at 37°C by centrifugation at 7,500 rpm for 10 min and filtration through a 0.22-μm filter. Acetonitrile (960 μl) and an internal standard (40 μl) of 16-hydroxyhexadecanoic acid dissolved in acetonitrile (concentration, 1,000 ppm) were added to 1 ml of each sample. Liquid chromatography separation was achieved by using a Dionex Acclaim rapid-separation LC (RSLC) 120 C18
column (2.2-μm particle size, 120-Å pore size, 2.1 by 100 mm) with an eluent gradient of 50/50 (water/acetonitrile) to 10/90 (water/acetonitrile) containing 0.1% formic acid using a flow of 0.5 ml/min at 50°C. MS was performed on the samples by using a Bruker micrOTOF II in negative-ion mode. Chromatograms were extracted from the total ion chromatograph using pseudomolecular ion molecular masses.