Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

NOTE: A full description and catalog numbers for all supplies in this protocol can be found in the Table of Materials.

1. Preparation of M8T minimal media

1. Prepare 1 L of M8 minimal salts base (5x) by dissolving 64 g of Na₂HPO₄·7H₂O, 15 g of KH₂PO₄, and 2.5 g NaCl in 800 mL of ultrapure water (UPW, resistivity 18 MΩ/cm) in a 2 L bottle. pH to 7.6. Complete volume to 1 L with UPW. Autoclave for 45 min.
2. Prepare 500 mL of a glucose solution (20% w/v) by dissolving 100 g of glucose in 400 mL of UPW in a 1 L bottle. Complete solution to 500 mL with UPW. Autoclave for 45 min.
3. Prepare 500 mL of a tryptone solution (20% w/v) by dissolving 100 g of tryptone in 400 mL of UPW. Complete to 500 mL with UPW. Autoclave for 45 min.
4. Prepare 1 L of M8 + 10% tryptone (M8T) minimal media by adding 200 mL of 5x M8 minimal salts (1x final), 10 mL of 20% glucose (0.2% final), 1 mL of 1 M MgSO₄ (1 mM final), and 50 mL of 20% tryptone (1% final) to 600 mL of UPW. Complete to 1 L with UPW. Filter through a 0.2 μm sterile filter into a sterile 500 mL media storage bottle.

Day 1:

2. Preparation of bacterial overnight cultures

1. Inoculate 5 mL of M8T minimal media with a single colony of P. aeruginosa or S. aureus (including antibiotics when appropriate) and incubate overnight at 37 °C with aeration for no longer than 16 h.
   NOTE: The bacterial pathogens P. aeruginosa and S. aureus were used for this method, because they are commonly coisolated from chronic infections, and studying their interactions is important to understand how they contribute to patient outcomes during polymicrobial infections. Other bacterial species could be used depending on the focus of the study.

Day 2:

3. Subculture of bacterial strains

1. Subculture P. aeruginosa 1:500 and S. aureus 1:1000 in 5 mL of fresh M8T (include antibiotics when appropriate). Incubate with aeration at 37 °C until cultures reach mid-log phase (OD₆₀₀ = ~0.3 – 0.5).

4. Preparation of materials for pad molds

1. Prepare metal spatulas by heating the end of a flat, rounded laboratory spatula with a Bunsen burner until half of the flat end can be bent to a 90° angle. Heat the end of another flat, rounded laboratory spatula and slightly bend the last 10 mm to a 45 °C angle.
2. Cut off the four corners of the silicone molds so that the molds fit inside a 35 mm dish.
3. Sterilize the spatulas and a pair of tweezers by adding 70% ethanol and passing them through the flame of the Bunsen burner.
4. Clean the dish and silicone molds with 70% ethanol and dry them with lint-free wipes.

5. Preparation of agarose pads

NOTE: The pads are prepared with the M8T minimal media as the nutrient source for the bacteria used in this protocol. However, the nutrients used in the pads can be modified for different organisms.

1. Melt 2% low-melt agarose in 10 mL of M8T in a clean 50 mL Erlenmeyer flask. Microwave in short intervals (2-5 s) until the agarose is in solution in order to prevent the contents of the flask from boiling over. Once melted, let cool in a 50 °C water bath for at least 15 min.
2. Prepare molds by aligning the silicone cutout with the opening in the 35 mm dish and tap lightly with spatula to secure the silicone to the dish, and to remove all air bubbles between the mold and the dish.
3. Once cool, pipette 915 µL of molten agarose into the mold. Leave the lid ajar and let the pad dry at room temperature for 30 min.
4. Cover the dish with the lid and leave at room temperature for an additional 2 h.
5. Prepare humidity wipes by tightly rolling up a lint-free wipe. Place the rolled wipe inside a sterile Petri dish and evenly add 500 µL of sterile water across the wipe. Warm to 37 °C for 1 h.
6. Warm the pad and a sterile 35 mm dish to 37 °C for 1 h.

6. Preparation of bacterial cells and inoculating pads

1. Measure the OD₆₀₀ of each subculture and dilute P. aeruginosa to an OD₆₀₀ = 0.03 and S. aureus to an OD₆₀₀ = 0.10 in M8T prewarmed to 37 °C. Mix P. aeruginosa and S. aureus in a 1:1 ratio and vortex.
   NOTE: If strains require antibiotics, the antibiotics can be added to the overnight and subculture, but should not be added when mixing species for imaging if it affects the other species in the coculture. Plasmid stability and the effects of antibiotics on all species in the coculture will need to be determined for each organism/plasmid being used.
2. Pipette 1 µL of coculture evenly across the bottom of a pre-warmed, sterile 35 mm glass coverslip dish.
3. Remove the silicone cutout from the mold using sterile tweezers.
4. Remove the pad from the dish using sterile spatulas.
   1. Slip the slightly bent spatula under the edge of the pad, while holding the mold upside down, to drop it out onto a sterile Petri plate lid.
      NOTE: Take care not to force the pad out or it will rip. Keep track of which side of the pad is the bottom.
5. Transfer the pad to the dish with the bacterial cells, bottom-side-down, by sliding the 90° angled spatula under the pad and placing it on top of the inoculated coverslip. Use the 90° angled spatula to make the pad flush against the coverslip and gently press out any air bubbles.
6. Remove excess moisture from moist wipes then place around the edge of the dish, making sure it does not touch the pad. The sample is now ready for imaging.

7. Setting up the microscope for live imaging

1. Warm environmental chamber to 37 °C at least 2 h prior to experimental set-up.
2. Turn on all components including microscope, computer, and light sources for brightfield and fluorescence imaging.
3. Open the imaging software and make sure that light sources are connected and running.
4. Perform Köhler illumination²⁷ to align all image planes.
   1. First, bring marked coverslip into focus with a 20x objective using a “dummy” dish. Make sure the condenser turret is set to the “open” position.
      NOTE: Köhler illumination should be performed for each unique objective/sample to properly align image planes. However, focus and alignment with the “dummy” dish on lower magnification expedites set-up on live samples at higher magnification to limit time between set-up and experiment start time.
   2. Focus the condenser.
      1. Close the field diaphragm.
         NOTE: An octagon-shaped aperture should appear. If the condenser is completely out of focus, the entire field of view (FOV) will appear dark.
      2. Rotate the condenser focusing knobs until the octagon edges are crisp.
         NOTE: The light intensity will increase as the image planes approach the correct alignment.
   3. Align the condenser.
      1. Center the field condenser by adjusting the aligning knobs.
         NOTE: The octagon should be centered to the middle of the FOV. The aligning knobs differ depending on the microscope. For example, some microscope condensers have knobs, while others have screws requiring a screw driver.
   4. Focus the lamp filament and adjust the condenser aperture.
      1. Place a phase telescope or Bertrand lens into the light path to observe the back focal plane of the objective.
         NOTE: There should be two concentric circles.
      2. Turn the Bertrand lens focus knob until the rings look crisp.
      3. Remove the Bertrand lens from the light path.
   5. Open the field diaphragm until the octagon is just outside of the FOV.
   6. Change to the 100x objective and slide the matching phase ring into place before adding a drop of immersion oil, and place the prepared sample dish on top.
   7. Perform Köhler illumination on the 100x objective with the bacterial sample dish.
5. Focus on the bacteria using the fine adjustment only. Once the bacteria in the FOV are focused through the eyepiece, switch the light path to the camera by pressing the camera button on the microscope.
6. Click the Phase option in the imaging software.
7. Adjust the percentage of DIA LED light emitted by selecting the light source in the software and either manually entering the desired percentage of light to be used or sliding the bar on the light percentage scale.
8. Adjust the DIA LED light exposure time by clicking on the light source and either manually entering the desired exposure time or selecting an exposure time from the provided drop-down menu.
   NOTE: The exposure time will vary depending on the camera being used.
9. If using fluorescence, adjust the camera settings in each corresponding channel (i.e., TxRed, GFP), by clicking on the fluorescent channel of interest.
   1. Set the percentage of fluorescence light emitted, then adjust the exposure time (as performed in steps 7.7 and 7.8 for DIA LED light).
   2. Alternatively, change the bit depth to adjust the dynamic range by selecting one of the other bit depth options in the visualization controls drop-down menu.
10. Choose the XY positions of interest by clicking on the XY option in the acquisition controls menu.
    1. Move the stage position with the joy stick, or by clicking and dragging the FOV on the screen, and click on the empty box to save the X and Y coordinates of a specific position.
       NOTE: Selection of no more than three different XY positions, as close together as possible, is recommended for time-lapse acquisition.
11. Turn on the perfect focus system (PFS) by either clicking the PFS box on the XY tab of the acquisition control menu or pressing the PFS button on the joy stick control panel.
12. Rotate the fine adjustment knobs to focus on the bacterial cells.
13. Click the PFS button for each XY position, once cells are in the desired plane of focus.
    NOTE: PFS compensates for drift in the Z-axis during time-lapse experiments. This is necessary to maintain focus of bacterial cells over time. Different manufactures have different compensation systems.
14. Choose the image acquisition conditions including acquisition interval and frequency for each channel (e.g., phase contrast and each fluorophore) by selecting from the options in the acquisition controls menu.
    NOTE: For the experiments presented here, phase contrast images are acquired at intervals every 5-10 s while fluorescence images in the GFP and TxRed channels are acquired every 20 min.
15. Begin imaging once the microscope and acquisition settings are set up.

8. Optional: Modifications for live/dead imaging

1. Melt 2% agarose in 10 mL of M8T and let cool in 50 °C water bath for at least 15 min.
2. Add 1 mM of propidium iodide to the molten agarose.
3. Once cooled, pipette 915 μL of agarose in the prepared mold.
4. Leave the lid ajar and let pad dry at room temperature for 30 min. Protect from light.
5. Cover the dish with the lid and leave at room temperature for an additional 2 h.
6. Prepare humidity wipes.
   1. Roll a lint-free paper wipe up tightly.
   2. Place the wipe in a sterile Petri dish and add 500 µL of sterile water to the wipe.
   3. Incubate at 37 °C for 1 h.
7. Incubate the pad and a sterile 35 mm dish at 37 °C for 1 h.
8. Proceed to inoculate the dish as indicated in section 6: Preparation of bacterial cells and inoculating pads.

9. Data analysis

1. Identification of cells
   1. Open image file in the imaging software and crop the file to only include the frames to be used for tracking, zoomed in to the cells of interest, and only in the phase contrast channel.
      NOTE: The cropped file can be saved as a new file in which tracking data can be stored without interfering with the original file.
   2. Select the option to choose regions of interest (ROI) in the analysis controls menu and define ROIs by tracing either individual bacterial cells or clusters of cells in the first frame to be used for analysis.
      NOTE: The ROIs can either be defined manually or as binaries.
      1. Manually define ROI
         1. Trace the perimeter of each individual bacterial cell or clusters of cells to manually define the ROIs.
            NOTE: In the analysis software, P. aeruginosa, or other rod-shaped cells, can be manually traced by selecting the Ellipse ROI and drawing an ellipse adjusted to the size of the bacterial cell. Alternatively, the Polygon ROI option can be selected to trace non-traditional-shaped ROIs, such as clusters of cells.
      2. Identify binary ROIs
         1. Click the Binary to ROI option in order to define binary ROIs.
            NOTE: Objects are defined in a binary layer based on separation of the darker-pixelated bacterial cells from the lighter-pixelated background in the phase contrast channel.
         2. To threshold the cells, select the Threshold option in the analysis controls menu. Select the channel of interest and slide the bars in the fluorescence histogram to adjust the threshold interval values.
            NOTE: Binary object identification for ROIs can also be defined in fluorescent images by thresholding the bacterial cells. Thresholding establishes which fluorescence intensities are considered objects and what fluorescence intensities constitute the background.
2. Cell tracking
   1. To manually track ROIs, select the next frame in the imaging sequence and adjust the position of the ROIs by clicking and dragging each ROI to align with the new position of the original bacterial cell.
      NOTE: If the bacterial cells have not changed position, the ROIs do not need to be moved.
   2. Repeat in all sequential frames for which cells are to be tracked.
   3. As cells divide, define the daughter cells as new ROIs, as described in step 9.1, and begin tracking the newly divided cells.
      NOTE: If cells are identified as binaries, use the Track Binaries function to automatically track ROIs in selected frames.
   4. Once cells have been tracked through all selected frames, export the data to be analyzed.
   5. Open the data spreadsheet and identify the measurements required for analysis (i.e., object speed, acceleration, or path length).
      NOTE: In the case of measuring directedness, the Path Length and Line Length measurements are required. The Line Length is a measurement of the Euclidian distance, or the straight distance from the track origin to the edge of the S. aureus colony. The Path Length is a measurement of the accumulated distance, or the sum of track segments from all frames. Directedness can be calculated as a ratio of the Euclidian distance, D_(E), (Line Length), over the accumulated distance, D_(A), (Path Length).
3. Fluorescence quantification
   1. Define ROIs for fluorescent bacterial cells as described in step 9.1.
   2. Repeat tracing or movement of ROIs for bacterial cells or clusters in the remaining fluorescent frames.
   3. Export the generated table to a spreadsheet file for analysis of fluorescent intensity.
   4. In the data spreadsheet, look for the column with “Mean Intensity” which represents the average fluorescence intensity for the traced bacterial cell(s) ROI.
   5. Graph the Mean Intensity values to look at the changes in fluorescence over time.
      NOTE: The changes in fluorescence over time represent the fluctuation of gene expression for the fluorescently labeled gene of interest.