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 millim