Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level

Measure and record the optical density at 600 nanometers for all samples. Normalize each sample to an optical density of 1 by diluting the culture with LBS medium.

Mix the two competing strains at an equal ratio by adding 30 microliters of each normalized strain to a labeled 1.5-milliliter tube. Vortex the mixed strain culture for 1 to 2 seconds. Mix the competing strains for each biological replicate and treatment to generate a total of 4 mixed-strain tubes.

Concentrate each mixed culture three-fold by centrifuging, to ensure sufficiently dense competing cells for contact-dependent killing during co-incubation. Discard the supernatant. Resuspend each pellet and 20 microliters of LBS medium, and perform concentration procedures for each sample.

If imaging on an inverted microscope, begin by spotting two microliters of a mixed culture onto the #1.5 coverslip bottom of a 35-millimeter Petri dish and place the agarose pad over the co-incubation spot. Place a 12-millimeter circular glass coverslip over the agarose pad. Repeat spotting and placing of coverslip for the remaining 3 mixed cultures, which will result in 4 dishes to be imaged.

Before proceeding ahead, let the slides sit on the benchtop for about 5 minutes to allow the cells to settle on the agar pad, to avoid movement during the imaging process.

Begin by focusing on cells using DIC to minimize photobleaching effects. Based on the average size of a single bacterial cell, use a 100x oil objective. Adjust the exposure time and acquisition settings for each channel with minimal background detection. Select at least 5 fields of view, and acquire images in each appropriate channel.