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

1. Coincubate bacterial strains

1. Choose two bacterial strains for single-cell bacterial competition assays. Here, two strains of V. fischeri are used: a target strain (ES11424) and an inhibitor strain (MJ1125) that is known to kill the target strain using the type VI secretion system on chromosome II (T6SS2)1, which is a contact-dependent killing mechanism.
2. Transform strains with stable plasmids encoding genes for different fluorescent proteins (e.g., GFP or RFP) to visually distinguish strain types on the microscope. Here, the inhibitor strain is tagged with a GFP-encoding plasmid (pVSV102), and the target strain is tagged with a dsRed-encoding plasmid (pVSV208).
3. Start with mid-log cultures for both strains, measure and record the optical density at 600 nm (OD₆₀₀) for all samples.
4. Normalize each sample to an OD₆₀₀ = 1.0, which corresponds to approximately 10⁹ CFU/mL for V. fischeri, by diluting the culture with LBS medium.
5. Mix the two competing strains together at a 1:1 ratio based on volume by adding 30 µL of each normalized strain to a labeled 1.5 mL tube. Vortex the mixed-strain culture for 1-2 s.
   NOTE: In some cases, it may be appropriate to mix cocultures in different ratios. For example, when one strain grows much faster than the other, it may be necessary to start the slower-growing strain at a numerical advantage in order to observe the competition. Optimization may also be required if OD₆₀₀ does not correspond to similar CFU/mL for both strains.
6. Repeat step 1.5 for each biological replicate and treatment. In the example shown here, this will result in a total of four mixed-strain tubes: two biological replicates with the wild-type inhibitor strain mixed with the target strain and two biological replicates with the type VI secretion system mutant strain mixed with the target strain.
7. To ensure competing cells are sufficiently dense for contact-dependent killing in the coincubation on the agar pad, concentrate each mixed culture 3-fold by centrifuging the mixed culture in a standard 1.5 mL centrifuge tube for 1 min at 21,130 x  g, discarding the supernatant, and resuspending each pellet in 20 µL LBS medium. Repeat for each sample.
   NOTE: Some bacterial cells are sensitive to damage by centrifugation at high rcf; in such cases, the mixed culture can be centrifuged for 3 min at 4600 x g. Additionally, when quantifying contact-dependent competition, it is important to ensure sufficient cell density on the slide to observe killing. In this article, "crowded" treatments, where killing is observed, had approximately 10 cells/20 µm².

2. Slide setup

1. When using an upright microscope, place a ~5 mm² agarose pad onto a standard 1 mm glass slide. Spot 2 µL of a mixed culture onto the agarose pad and place a #1.5 coverslip (25 mm²) over the spot. See Figure 1B.
2. When using an inverted microscope, spot 2 µL of a mixed culture onto the #1.5 coverslip bottom of a 35 mm Petri dish and place a ~5 mm² agarose pad over the coincubation spot. Place a 12 mm circular glass coverslip over the agarose pad. See Figure 1C for an example.
3. Repeat steps 2.1 or 2.2, depending on the microscope setup used, for the remaining three mixed cultures, resulting in four slides or dishes to be imaged.
4. Allow slides to sit on the benchtop for approximately 5 min before proceeding. This allows cells to settle on the agar pad and eliminate movement during the imaging process.

3. Fluorescence microscopy

1. Begin by focusing on cells using white light (phase contrast or DIC) to minimize the effects of photo-bleaching. Based on the average size of a single bacterial cell, use a 60x or 100x oil objective.
2. Adjust the exposure time and acquisition settings for each channel so that cells are visible in the appropriate channel with minimal background detection.
   NOTE: It is appropriate to use different exposure times for different channels, but the same exposure time should be used across all biological replicates and treatments for a given channel.
3. For each sample, select at least five fields of view (FOV) and acquire images in each appropriate channel using the acquisition settings (See examples in Figure 2). Save the XY points from each FOV so that the same FOV can be imaged during the final time point. Imaging the same FOV at each time point is necessary to determine the proportion of area occupied by target or inhibitor cells during the analysis steps.
   NOTE: In this example, the fluorescence of GFP is detected using a filter with an excitation wavelength of 467 – 498 nm and an emission filter of 513 – 556 nm and is false-colored green. The Fluorescence of dsRed is detected using a filter with an excitation wavelength of 542 – 582 nm and an emission filter of 603 – 678 nm and is false-colored magenta.
4. After 2 h, repeat the above step for each sample using the previously saved XY points (Figure 2).
   NOTE: The timing of subsequent images may need to be optimized for organisms with different growth rates or competitive mechanisms.