Plan fluor 20x objective

The effects of bacteriophage on the growth and adherence of bacteria on the surface of hydrogels was assessed by weighing each hydrogel, then degrading it for 1 hr in 100 μl of 385 U/ml collagenase II in a 37°C water bath. This sample was diluted with 900 μl PBS, and 10 μl serial dilutions of this sample were plated on TSA and incubated overnight. Colonies in the highest concentration sample that produced distinct colonies were counted to derive the concentration of bacteria present on the original gel sample.
After biofilm formation, bacteria were stained with fluorescent dyes by flowing them over the biofilms at 5 μl/min for 20 min. Black and white images of the samples were acquired on a Nikon TE300 inverted microscope using a Plan Fluor 20X objective (Nikon) and subsequently pseudocolored in Photoshop CS6 (Adobe). The live/dead dye used was a 10 μl 1:1 mixture of 3.34 mM SYTO 9 in DMSO and 20 mM propidium iodide in DMSO (ThermoFisher, Waltham, MA) diluted in 990 μl of PBS. Live and dead images were taken at 485 and 535 nm respectively. FilmTracer SYPRO Ruby biofilm stain (ThermoFisher) was used for biofilm staining and images were taken at 535 nm.

The interactions between flowing bacteria and PEG coatings were studied using a custom-built flow chamber of dimensions 0.8 mm × 8 mm × 50 mm. The coated test surface of a microscope slide comprised one wall of the flow chamber, which was oriented vertically on an optical bench, with horizontal flow past that surface. This configuration eliminated gravitational forces normal to the surface. Videomicroscopy employed a Nikon Plan Fluor 20x objective with a numerical aperture of 0.5, producing a depth of field of approximately 3.5μm. Bacteria were flowed across the surface at a shear rate of 15 s⁻¹ for approximately 10 minutes. Data were recorded on DVDs and analyzed at a rate of 5 frames per second using FFmpeg software. Manual tracking employed FIJI is just ImageJ. In the analysis, all cells meeting the criteria for engagement, that were visible in the run, were analyzed. We found that, in a given run, the behavior of cells in the early minutes of the run was statistically identical to that of the cells towards the end of the run.

Aiptasia larvae were washed and diluted to a concentration of 300-500 larvae ml^-1 and were then incubated with 20 μg μl^-1 LPS (lipopolysaccharides from Escherichia coli O127:B8, Sigma-Aldrich), 1 μM XMD17-109 (0.1% DMSO {Dimethylsulfoxid, Carl-Roth}), 0.1 % DMSO, or without for 1 hour. Microalgae cultures were then added to a final concentration of 1 x 10⁵ microalgae ml^-1 of the respective microalgal types and incubated at 26 °C and exposed to a 12L:12D cycle. After a 24-hour infection, the larvae were fixed in 4% formaldehyde for 30 minutes, washed in PBS, and mounted in 100% glycerol for counting. Infection status was quantified in at least 100 larvae per infection using a Nikon Eclipse Ti epifluorescent microscope using a Nikon Plan Fluor 20x objective, utilizing microalgal autofluorescence. Data recording was performed in Microsoft Excel v16.16.6.