Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

1. Swarm Assay Media Preparation and Inoculation^4,5,7,8,11

1. Media Preparation
   NOTE: The medium composition described below is applicable to P. aeruginosa tendril formation studies. Please see media specifications on Table 1 for P. aeruginosa, B. subtilis, and M. xanthus surface motility assays.
   1. Mix 200 ml of FAB-minus (NH₄)₂SO₄ swarm medium (Materials Table), 0.9 g of Noble agar, and 0.2 g of Casamino acids (Table 1) by stirring with a magnetic stir bar. Use small volumes (100-300 ml) to improve consistency between experiments.
   2. Autoclave the 200 ml agar/media mixture using an exposure time of 22 min, exposure temperature of 121.1 °C, and a fast vent option. The autoclave settings will allow proper sterilization and agar melting, but prevent agar caramelization.
      NOTE: Noble agar is prone to caramelization; bacterial motility is altered on caramelized agar.
   3. Immediately after the sterilization cycle has finalized, close the cap of the media bottle to prevent water loss by evaporation. However, note that tight capping can cause a “vacuum sealing”-like effect on the bottle.
   4. Cool the media to 50 °C while stirring at room temperature (RT) and add 2 ml of sterile 1.2 M glucose. Alternatively, place the media in a 60 °C incubator or water bath until ready to use (up to 15 hr later), and proceed as indicated. To prevent the formation of bubbles in the media, mix thoroughly using the magnetic stir bar; bubbles on the surface of the agar will prevent even swarming.
      NOTE: For other assays, add at this step heat-sensitive components that cannot be autoclaved such as additional nutrients or dyes, as needed (e.g., addition of 8 µl Invitrogen Syto 63 dye per 100 ml melted agar to image M. xanthus as shown in Representative Results, below). Addition of some dyes may affect baseline swarming behavior, which should be checked against a non-dye control.
   5. In a laboratory hood, aliquot 7.5 ml of sterile media per 60 mm diameter polystyrene Petri dish and maintain the plates in a single layer (not stacked). For larger swarming surface, aliquot 25 ml of media per 100 mm diameter Petri dish. It is important to fill the dishes on an even horizontal surface. Use a bull’s eye level to check if the surface is leveled.
      NOTE: For P. aeruginosa assays, using a specific media volume per plate will improve consistency and reproducibility. For B. subtilis and M. xanthus assays, hand pouring yields results comparable to specific volume aliquots.
2. Plate curing
   1. For small plates (60 mm), allow the melted agar medium to cure (both set to semi-solid and dry excess liquid) in the hood uncovered (i.e., without lids) for 30 min. Larger plates (100 mm) require a longer curing time (see Discussion).
      NOTE: Alternatively, some assays may require plates to cure on the bench top overnight (20-24 hr) covered (i.e., lids on) in a single layer (Table 1). Swarming is sensitive to both excess and inadequate moisture. The humidity, airflow, and temperature of any given lab may necessitate variation to plate curing to promote optimal swarming of you bacterium.
   2. Inoculate plates immediately after the drying period is over. Do not store the plates for further use.
      1. Perform the “ink spread test” by spotting a test plate with 10 µl mixture of 0.50% (vol/vol) Higgins Waterproof Black India Ink and bacterial inoculum¹¹. If the ink/inoculum mixture spreads readily (i.e. does not retain droplet form) on the surface of the media, the media will need additional time to dry.
         NOTE: For species that are particularly sensitive to humidity (e.g., P. aeruginosa), perform a quick “ink spread test”¹¹ to determine if the plates are dry enough.
3. Swarm Assay Inoculation
   1. Inoculate 6 ml of broth culture media (see Table 1 for details) with an isolated colony from a fresh (< 5 days old if left at room temperature) Lysogeny Broth (LB) plate culture. Incubate broth cultures overnight (≤18 hr) at 30 °C or 37 °C with horizontal shaking (240 rpm).
   2. Inoculate swarm plates by spotting with 1-5 µl of overnight broth culture, or by “poking” the agar with a sterile tooth pick or wire inoculation needle.
      NOTE: We prefer the latter method because it decreases the likelihood of spattering the inoculum and prevents adding additional moisture to the swarm surface area.
4. Swarm Assay Incubation
   1. For general assay, incubate swarm assay plates at 30 °C or 37 °C (or even 42 °C for B. subtilis; Table 1)—this is bacterium specific. Invert the plates during incubation so that excess moisture condenses on the lid, not the agar.
      NOTE: Temperature can affect phenotype as well as kinetics. For P. aeruginosa swarms, incubation at 37 °C leads to faster growth and swarm expansion than incubation at 30 °C; however, the morphology of these swarms often differs with this change in temperature.
   2. For time-lapse imaging, incubate swarm plates at the appropriate temperature prior to transfer into the imaging station (see Table 1 for details).
      NOTE: This pre-imaging incubation allows swarms to start their development and become established before being moved to a new environment, which may or may not be optimal for swarming motility.

2. Macroscopic Imaging of Surface Motility Assays^7,8

1. For time-lapse imaging, after the pre-imaging incubation period place swarm assay plates on a clear imaging plate inside a commercial in vivo imaging station. Image up to six, 60 mm diameter or four, 100 mm diameter plates at a time. Since the camera captures images from beneath the imaging plane, invert the plates so that the optical path is not obstructed⁸. Alternatively, incubate at 30 °C or 37 °C (Table 1) for the duration of the experiment, and remove the plates to be imaged from the incubator at set time intervals.
2. Place the lids of the Petri dishes upright on top of the plate counterpart that holds the inoculated media. Fill the lids of the Petri dishes with water to prevent excessive drying during imaging, and enclose the entire set up using another clear tray to maintain humidity throughout the experiment.
3. Using Molecular Imaging (MI) software¹², run assay(s) at room temperature using the imaging settings described in Table 2. For time-lapse imaging, set up a protocol with the necessary steps and specifications.

3. Data Processing and Interpretation^7,8

1. Image Processing
   1. Use MI software to batch export images as 16-bit TIFF files: File > Export or Export Multiple > Select file(s) to export and export location.
   2. Use ImageJ to open a single image or import a time-lapse series:
      1. Open a single image: File > Open
      2. Import time-lapse image sequence: File > Import Sequence, and select “Sort names numerically”.
         1. For larger time-lapse files, select “Use virtual stack” in the “Import Sequence” window to stack the exported images into appropriate categories (i.e., GFP, RFP, etc.).
   3. If required, change images from 16-bit files to 8-bit files: Image > Type > 8-bit
      NOTE: Some ImageJ tools require 8-bit images.
   4. Determine if the intensity signal for an image or time-lapse sequence needs to be inverted. Place the cursor on a bright spot in the image (e.g., fluorescently labeled growth) and note the signal intensity “Value” from the ImageJ toolbar. Then, place the cursor in a dark spot outside the plate area and note the signal intensity. If the signal intensity for the dark spot is larger than the intensity for the bright spot, the image signal intensity needs to be inverted (follow substeps 1-2 below).
      1. Invert the intensity signals: Edit > Invert
      2. Invert the lookup table: Image > Lookup Tables > Invert LUT
   5. Subtract the background: Process > Subtract Background, and use a “Rolling ball radius” with a pixel radius that is one half of one image dimension (e.g., 1,000 pixels for a 2,000 x 2,000 pixel image).
   6. Artificially color an image or time-lapse sequence: Image > Lookup Tables, and select the appropriate color from the list options.
   7. For movies with two or more channels, merge and balance the colors prior to saving as a movie (Image Processing, Step 8). To merge images together, open all image stacks in ImageJ, then select Image > Color > Merge Channels, and assign each stack to a color channel.
   8. Save time-lapse sequence as AVI or QuickTime Movie: File > Save as, and choose desired format and specifications.
2. Data Analysis
   1. Acquiring Bacterial Surface Growth Area to Quantify Expansion Rate
      1. Open image(s) in ImageJ
      2. To calculate the diameter of the plate in pixels, draw a line across the center of an assay plate with the “Straight” tool from the and measure its length: Analyze > Measure
      3. The default measurement unit in ImageJ is the pixel. Obtain a conversion factor by dividing the diameter of the assay plate (e.g., 60 for a 60 mm plate) by the pixel length obtained in the previous step.
      4. Change the unit of measurement from pixel to mm: Image > Properties
         1. Change the “Unit of Length” to “mm”, and the “Pixel Width”, “Pixel Height”, and “Voxel Depth” to the conversion factor calculated in the previous step. Select the “Global” box to maintain this conversion factor across multiple images.
            NOTE: If ImageJ is closed and reopened, or the field of view of an image is changed (i.e., one image is zoomed in more than another), the conversion factor must be recalculated. Alternatively, perform all analyses in pixels and then converted to mm.
      5. For every frame, trace and measure the swarm area using the “Freehand Selections” tool in the toolbar to trace the outline of the swarm and measure the area using: Analyze > Measure. This will generate a measurements log that can be saved for further analysis in Microsoft Excel or similar programs: File > Save As
   2. Acquiring Bacterial Surface Growth Intensity to Quantify Surface Growth Rate
      1. Once the background is subtracted (Image Processing, Step 5), use the last frame of the sequence to determine the maximum area of swarm (Data Analysis, Step 1).
      2. Use the “Oval” selection tool from the toolbar to draw a box around the bacterial surface growth.
      3. Set the mean intensity measurement of pixels in the box using: Analyze > Set Measurements, and select "Mean gray value".
      4. To obtain intensity signal measurements for each frame in the time-lapse sequence, while on the first frame of the sequence go to: Analyze > Measure. This will generate a measurements log that can be saved for further analysis in Microsoft Excel or similar programs: File > Save As
   3. Alternative to the previous section (Data Analysis, Step 2). Use the ImageJ Macros plugin to setup and run a Macros surface growth intensity measurement script.
      1. Setup an automated measurement script to analyze multiple frames simultaneously: Plugin > New > Macro, and paste the provided script (below) into the box and save as an ImageJ Macros text file: File > Save, and save to the ImageJ application folder under “macros”.
         numberOfFrames = N
         for(i=0; i< numberOfFrames; i++){
         run('Measure');
         run('Next Slice [>]');
         }
         NOTE: Here the variable “N” is for an undefined number of frames.
      2. Edit the “numberOfFrames” in the Macros plugin for each experiment to reflect the number of frames in the image sequence prior to running the script. Use: Plugin > Macros > Edit, and type in the correct number of frames in the sequence and save (File > Save).
      3. Follow substeps 1-3 in Data Analysis-Step 2, and while on the first frame of the sequence run the Macros plugin: Plugin > Macros > Run. This will generate a measurements log that can be saved for further analysis in Microsoft Excel or similar programs: File > Save As