Sample Preparation and Imaging of Fluorescently Labeled Bacteria

Begin with a large glass slide with smaller glass slides placed on either edge in a double-layer.

Pour molten agarose into the space and cover it with another large glass slide.

Chill the setup to solidify the agarose.

Remove the glass slides and cut the agarose into small pads.

Place the agarose pads inside an adhesive frame attached to a slide.

Take a culture of pathogenic bacteria, genetically modified to express a fluorescently tagged secretion system essential for host infection.

Use a dense bacterial patch and suspend it in water.

Vortex to disperse the bacteria uniformly.

Apply the suspension on the agarose pad to immobilize the bacteria.

Seal with a coverslip to maintain hydration.

Under brightfield illumination, select a field of cells for imaging.

Optimize microscope settings to reduce photobleaching and increase signal sensitivity.

Switch to the fluorescence channel to detect the fluorescently tagged bacteria.

Start by making agarose pads for live cell imaging. Prepare about 30 milliliters of 1% low melt agarose solution in water and microwave it in a glass flask for about 90 seconds, swirling it occasionally until the agarose is completely dissolved.

Place two 22 by 22 by 0.15 millimeters glass slides on the edge of a 25 by 75 by 1.1 millimeter glass slide, one on top of the other. Then, stack two more of the smaller slides on the other edge. Pipette about 1 milliliter of the molten agarose into the center slide between the two upper glass slides and place another large slide on top of the molten agarose, making sure to avoid formation of air bubbles.

Cool the slides at four degrees Celsius for 15 minutes, then use a scalpel or razor blade to gently cut the pad into small squares. Fix a double-sided adhesive frame on a 25 by 75 by 1.1 millimeter glass slide and place several pads on the slide. Dissolve a heavy patch of Legionella pneumophila in 1 milliliter of double-distilled water.

Then vortex, and pipette 2 to 3 microliters of the dilution onto the pads. Gently place a 58 by 24 by 0.15 millimeters cover slip over the adhesive frame. Select cells for imaging using bright field lighting.

Then, in the capture window of the microscope's software, adjust the ND to 180 and the binning to 2 by 2. Use the 488 nanometer channel to expose the sample for 500 to 1000 milliseconds and validate the specificity of the fluorescence by imaging untagged Legionella pneumophila with the same parameters.