Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response

Summary

We detail a simple method to produce high-resolution time-lapse movies of Pseudomonas aeruginosa swarms that respond to bacteriophage (phage) and antibiotic stress using a flatbed document scanner. This procedure is a fast and simple method for monitoring swarming dynamics and may be adapted to study the motility and growth of other bacterial species.

Abstract

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations of bacteria move over large distances in characteristic tendril-shaped communities over the course of hours. Swarming is sensitive to several factors including medium moisture, humidity, and nutrient content. In addition, the collective stress response, which is observed in P. aeruginosa that are stressed by antibiotics or bacteriophage (phage), repels swarms from approaching the area containing the stress. The methods described here address how to control the critical factors that affect swarming. We introduce a simple method to monitor swarming dynamics and the collective stress response with high temporal resolution using a flatbed document scanner, and describe how to compile and perform a quantitative analysis of swarms. This simple and cost-effective method provides precise and well-controlled quantification of swarming and may be extended to other types of plate-based growth assays and bacterial species.

Introduction

Swarming is a collective form of coordinated bacterial motility that increases antibiotic resistance and production of virulence factors in the host^1,2,3. This multicellular behavior occurs on semi-solid surfaces that resemble those of mucous layers covering epithelial membranes in the lungs^4,5. Biosurfactants are commonly produced by swarming populations to overcome the surface tension on surfaces and the production of these is regulated by complex cell-cell signaling systems, also known as quorum sensing^6,7,8. Many species of bacteria are capable of swarming, including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli^9,10,11,12. The swarming patterns created by bacteria are diverse and are affected by the physical and chemical properties of the surface layer including nutrient composition, porosity, and moisture^13,14. In addition to surface properties, growth temperature and ambient humidity affect several aspects of swarming dynamics, including swarming rate and patterns^12,13,14,15. The growth variables that affect swarming create challenges that impact experimental reproducibility and the ability to interpret results. Here, we describe a simple standardized method to monitor the dynamics of bacterial swarms through time-lapse imaging. The method describes how to control critical growth conditions that significantly affect the progression of swarming. Compared to traditional methods of swarm analysis, this time-lapse imaging method enables tracking the motility of multiple swarms concurrently during extended periods of time and with high resolution. These aspects improve the depth of data that can be gained from monitoring swarms and facilitate the identification of factors that affect swarming.

Swarming in P. aeruginosa is facilitated through the production and release of rhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids into the surrounding area^6,16. The introduction of stress from sub-lethal concentrations of antibiotics or infection by phage virus impacts the organization of swarms. In particular, these stresses induce P. aeruginosa to release the quorum sensing molecule 2-heptyl-3-hydroxy-4-quinolone, also known as the Pseudomonas quinolone signal (PQS)^17,18. In swarm assays that contain two populations of swarms, PQS produced by the stress-induced population repels untreated swarms from entering the area containing the stress (Figure 1). This collective stress response constitutes a danger communication signaling system that warns P. aeruginosa about nearby threats^18,19. The effects of stress on P. aeruginosa, the activation of the collective stress response, and the repulsion of swarms can be visualized using the time-lapse imaging method described here. The protocol described here explains how to: (1) prepare agar plates for swarming, (2) culture P. aeruginosa for two types of assays (traditional swarming assays or collective stress response assays) (Figure 1), (3) acquire time-lapse images, and (4) use ImageJ to compile and analyze the images.

Briefly, P. aeruginosa from an overnight culture is spotted in the middle of a swarming agar plate while P. aeruginosa that are infected with phage or treated with antibiotics are spotted at the satellite positions. The progression of P. aeruginosa swarming is monitored on a consumer document flatbed scanner that is placed in a humidity-regulated 37 °C incubator. The scanner is controlled by a software that automatically scans the plates at regular intervals over the swarm growth period, typically 16–20 h. This method yields concurrent time-lapse videos of up to six 10 cm swarming plates. The images are compiled into movies and the repulsion of swarms by stress-induced populations is quantified by using freely available ImageJ software. Special consideration is given to ensure consistency and reproducibility between different swarming experiments.

Protocol

1. Preparing Swarming Agar Plates for P. aeruginosa Swarming Time-lapse Imaging

1. Prepare 1 L of 5x M8 minimum media in a glass bottle by adding 64 g of Na₂HPO₄•7H₂O, 15 g of KH₂PO₄, and 2.5 g of NaCl in 500 mL double-distilled water (ddH₂O). Adjust the final volume to 1 L with additional ddH₂O. Autoclave to sterilize and store liquid media at room temperature.
2. Prepare 100 mL of 1 M MgSO₄ (magnesium sulfate) in a glass bottle by adding 24.6 g of MgSO₄•7H₂O in 50 mL ddH₂O. Adjust the final volume to 100 mL with additional ddH₂O. Autoclave to sterilize. Store at room temperature.
3. Prepare 100 mL of 20% casamino acids in a glass bottle by adding 20 g of casamino acids in 50 mL ddH₂O. Adjust the final volume to 100 mL with additional ddH₂O. Autoclave to sterilize. Store at room temperature.
4. Prepare 100 mL of 20% glucose in a glass bottle by adding 20 g of glucose in 50 mL ddH₂O. Adjust the final volume to 100 mL with additional ddH₂O. Sterilize by filtration with 0.22 µm filter. Store at room temperature.
5. To make 10 swarming agar plates, add 1 g of agar in 100 mL of ddH₂O and adjust the final volume to 160 mL with additional ddH₂O in a 250 mL Erlenmeyer flask. Sterilize by autoclaving.
   1. Immediately after autoclaving, place the agar solution in a 55 °C water bath for 15 min.
   2. Remove the agar solution from the water bath and add 40 mL of 5x M8 minimum media, 200 µL of 1 M MgSO₄, 2 mL of 20% glucose, and 5 mL of 20% casamino acids¹⁵. Proceed to step 1.6 immediately after mixing.
      NOTE: The final concentrations are 0.5% agar, 1 mM MgSO₄, 0.2% glucose, and 0.5% casamino acids.
6. Using a 25 mL pipette for consistent volume, add 20 mL of the swarming agar solution per 10 cm diameter Petri dish.
   NOTE: A fixed volume of agar solution is important, as the volume affects the drying time and moisture content of the agar. Avoid bubbles when making the swarming agar plates.
7. Allow the agar to solidify by placing the swarming agar plates in a single stack with lids on for 1 h on the bench at room temperature. Turn on the dehumidifier to decrease relative humidity of the room to 40–50% 1 h prior to the next step.
8. Dry the swarming agar plates for an additional 30 min with the lids off in a laminar flow hood at 300 ft³/min with 40–50% relative humidity at room temperature. Dry the interior of the lids by placing them face up in the laminar flow hood. Store swarming agar plates at 4 °C for up to 24 h.
9. Prepare black 10 cm Petri dish lids for imaging by smoothing the inside of the lid with sandpaper. Put the lids inside a packaging box and place the packaging box under a chemical hood. Spray inside the lids using black spray paint. Allow the lids to dry.
   NOTE: Black lids may be re-used for additional experiments. It is important that the lids are painted so that they do not reflect light during scanning.

2. Growth of P. aeruginosa and Plating Conditions

1. Prepare 400 mL of lysogeny broth (LB) by adding 10 g of LB-Miller powder mix into 400 mL ddH₂O. For 2% LB-agar Petri dishes, add an additional 8 g of agar. Autoclave to sterilize.
2. Pour 20 mL of molten LB-agar medium into 10 cm diameter Petri dishes and allow them to solidify at room temperature overnight. Store liquid media at room temperature and agar plates at 4 °C.
3. Streak P. aeruginosa on an LB-agar Petri dish from a frozen stock stored at -80 °C using sterile loops or wooden sticks. Incubate the Petri dish upside-down overnight at 37 °C. Store LB-agar plate at 4 °C for up to 1 week.
4. Pick a single colony from the Petri dish with a sterile loop or wooden stick, inoculate it into 2 mL LB medium, and incubate the culture to saturation overnight (16–18 h) at 37 °C in a roller drum set at 100 rpm.
5. Pipet 5 µL of overnight culture from step 2.4 using a P20 pipet and spot at the center of the swarming agar plate by approaching the pipet tip at an angle (10–45°) 2.5 cm above the spotting area, pipetting down to the first stop, and touching the agar with only the liquid drop (Figure 1B).
   1. Avoid touching the agar with the pipet tip as it damages the agar. Use a template in order to position the spot consistently across different swarming agar plates (Supplementary Figure S1).
   2. For traditional swarming assays, use only the center spot and skip to step 2.8. For collective stress response assays continue to step 2.6 (for phage infection) or step 2.7 (for antibiotic stress).
6. For phage infection, mix 30 µL of overnight culture of P. aeruginosa from step 2.4 with 6 µL of 1 x 10¹² pfu/mL phage DMS3vir²⁰. Proceed immediately to the next step.
   1. Pipet 6 µL of the P. aeruginosa-phage mixture from step 2.6 using a P20 pipet and spot at 6 equidistant satellite positions on a 2.8 cm radius concentric circle that is centered at the Petri dish by approaching the pipet tip at an angle (10 to 45°) 2.5 cm above the spotting area, pipetting down to the first stop, and touching the agar with only the liquid drop (Figure 1C).
   2. Avoid touching the agar with the pipet tip as it damages the agar. Use a plating template for consistency (Supplementary Figure S1). Proceed to step 2.8.
7. For antibiotic treatments, mix 30 µL overnight culture P. aeruginosa from step 2.4 with 6 µL of 3 mg/mL gentamycin, 10 µL of 100 mg/mL kanamycin, or 7.5 µL of 100 mg/mL fosfomycin. Proceed immediately to the next step.
   1. Pipet 6 µL of antibiotic treated P. aeruginosa from step 2.7 using a P20 pipette and spot at 6 equidistant satellite positions on a 2.8 cm radius concentric circle about the center of the dish by approaching the pipet tip at an angle (10 to 45°) 2.5 cm above the spotting area, pipetting down to the first stop, and touching the agar with only the liquid drop (Figure 1D).
   2. Avoid touching the agar with the pipet tip as it damages the agar. Use a plating template for consistency (Supplementary Figure S1). Proceed to step 2.8.
8. Replace the clear Petri dish lids with black lids made in step 1.9 (Figure 2A).
9. Place the swarming agar plates on a scanner in an incubator set at 37 °C with a 10 L water bath to maintain humidity at 75% (Figure 1E, Figure 2B).
   CAUTION: Do not disturb spotted cells on the swarming agar plates. Keep plates facing up at all times.

3. Image Acquisition with Scanner

1. Decrease the ambient lighting of the Petri dishes by attaching black matte fabric to a rack 40–60 cm above the flatbed document scanner. Secure it using zip ties (Figure 2B).
2. The scanner will be controlled using a scanning software and an automatic scripting software.
   1. In the scanning software, select Home Mode (Figure 3A). Capture images in color by selecting Color under Image Type. To set the image quality, select Other under Destination and adjust the Resolution to 300 dpi. Keep the standard size for the images by selecting Original for Target Size. Leave all options under Image Adjustments unchecked for standard image quality.
      NOTE: Target Size is set to Original by default. To select other options for Target Size, click on Preview first.
3. Set the saving path of images by clicking on the folder icon to the right of Scan to open File Save Settings (Figure 3A).
   1. Select the folder destination for saving images by selecting Other under Location and click on Browse. Choose a folder to save the images.
   2. Name the images in the Prefix text box. Set Start Number 001 to begin naming sequence for the images. Set the file format to JPEG by choosing JPEG (*.jpg) for Type under Image Format and click on Options to adjust for Details. Set the image format quality by adjusting Compression Level to 16, Encoding to Standard, and check Embed ICC Profile. Click OK to close the window (Figure 3B).
   3. Leave the first option unchecked ("Overwrite any files with the same name") and check the 3 next options ("Show this dialog box before next scan", "Open image folder after scanning", and "Show Add Page dialog after scanning"). Click OK to close the window
   4. Check the image quality by clicking on Preview. The preview window appears, and the Scan icon becomes functional (Figure 3C).
4. Use the scripting software to automate the image acquisition. The provided script clicks on Scan in the Scan window and OK in the File Save Settings window at 30 min intervals.
   1. Import the script by clicking on Task | Import and select both Single_scan.tsk and Idle_scanning.tsk (TSK files provided as Supplemental Files 1 and 2). See Figure 3D.
      NOTE: Single_scan.tsk clicks on the Scan button in the Scan window and OK in the File Save Settings window. Idle_scanning.tsk activates Single_scan.tsk every 30 min. One may change the scan frequency by changing the activation of Idle_scanning.tsk.
   2. Enable automatic scanning at 30 min intervals by selecting both Idle scanning (imported) and Single scan (imported), right clicking on Idle scanning (imported), and left clicking on Enabled (Figure 3D, Supplementary Figure S2).
      NOTE: Automatic scanning runs until the user manually stops the script. To stop the script, select Idle scanning (imported), right click Idle scanning (imported), and left click on Enabled. The check mark will be removed.

4. Compiling Time-lapse Images and Measuring Swarm Repulsion

1. Perform movie editing and image analysis using ImageJ.
2. Import all the scanned images to ImageJ by clicking on File | Import | Image Sequence and select the images. In the Sequence Options window, check Convert to RGB to keep images in color. Number of images indicates the number of images selected.
3. Keep Starting image at 1 to start from the first picture in the folder and Scale images at 100% to conserve original size of the images. Leave Use virtual stack unchecked. Click OK and wait for images to load (Figure 4A).
4. Set the video compression level to 100 by clicking on Edit | Options | Input/Output… and adjust JPEG quality to 100.
5. Save the file as an .avi by clicking on File | Save As | AVI. Adjust Compression to JPEG and Frame Rate to 5 fps (Figure 4B). Save the .avi time-lapse in the desired folder.
6. To quantify swarm repulsion distances, open an image near the end of the swarming period in ImageJ. Click on File | Open and select the image. Adjust the scale by clicking on Analyze | Set Scale and setting Distance in pixels to 118, Known distance to 1, Pixel aspect ratio to 1.0, and Unit of length to cm (Figure 4C). Leave Global unchecked. Click OK to close the window.
7. Click on the Straight icon and measure from the center of the colony at the satellite position to the edge of the swarming population. Select Analyze | Measure to make a new window appear with the measurements (Length) (Figure 4D).
   NOTE: Use "+" to zoom in closer and "-" to zoom out.

Results

The steps to grow P. aeruginosa, stress the cells, and image the swarming agar plates are represented in Figure 1. We inoculated a single colony of wild-type P. aeruginosa UCBPP-PA14 strain from an LB-agar plate in 2 mL of LB broth overnight at 37 °C and spotted 5 µL in the center of the swarming agar plate. Time-lapse imaging of this plate reveals initial growth in the form of a colony at the center and then spreading of tendrils radially from the colony (Video 1). For...

Discussion

This protocol focuses on minimizing the variability in swarming agar plates and providing a simple and low-cost method to acquire time-lapse images of P. aeruginosa swarming and responding to stress. This procedure can be extended to image other bacterial systems by adapting the media composition and growth conditions. For P. aeruginosa, although M9 or FAB minimal medium can be used to induce swarming^16,21, the protocol presented here uses M8 me...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Bacto agar, dehydrated	BD Difco	214010	For LB-agar plate and swarming agar plate
Casamino acids	BD Difco	223050	For swarming media
D-Glucose	Fisher Chemical	D16500	Dextrose. For swarming media
Fosfomycin disodium salt	Tokyo Chemical Industry	F0889	Stock concentration: 200 mg/mL. Dissolved in ddH2O
Gentamycin sulfate	Sigma-Aldrich	G1914	Stock concentration: 3 mg/mL. Dissolved in ddH2O
Kanamycin sulfate	Sigma-Aldrich	60615	Stock concentration: 100 mg/mL. Dissolved in ddH2O
LB-Miller	BD Difco	244620	For LB broth and LB-agar plates
Magnesium sulfate heptahydrate	Sigma-Aldrich	230391	For swarming media
Potassium phosphate monobasic	Sigma-Aldrich	P0662	For 5x M8 media
Sodium chloride	Sigma-Aldrich	S9888	For 5x M8 media
Sodium phosphate dibasic heptahydrate	Fisher Chemical	S373	For 5x M8 media
Strains
Pseudomonas aeruginosa	Siryaporn lab	AFS27E.118	PA14 strain
DMS3vir	O'Toole lab	DMS3vir20	Bacteriophage
Supplies
Aluminium oxide sandpaper	3M	150 Fine	For black lids
Black fabric	Joann	PRD7089	Black fabric
Black spray paint	Krylon	5592 Matte Black	For black lids
Erlenmeyer flask	Kimax	26500	250 mL
Glass storage bottles	Pyrex	13951L	250 mL, 500 mL, 1,000 mL
8 inches zip ties	Gardner Bender	E173770	For attaching black matte fabric
Petri dishes (100 mm x 15 mm)	Fisher	FB0875712	100 mm x 15 mm polystyrene plates
Wooden sticks	Fisher	23-400-102	For streaking and inoculating bacteria
Equipment
Autoclave	Market Forge Industries	STM-E	For sterilizing reagents
25 mL pipette	USA Scientific, Inc.	1072-5410	To pipet 20 mL for swarming agar plates
Dehumidifier	Frigidaire	FAD704DWD 70-pint	For maintaing room relative humidity at about 45%
ImageJ	NIH	v1.52a	Software for image analysis
Incubator	VWR	89032-092	For growth of bacteria at 37 °C
Isotemp waterbath	Fisher	15-462-21Q	For cooling media to 55 °C
Laminar flow hood	The Baker Company	SG603A	For drying plates
P-20 pipet	Gilson	F123601	Spotting on swarming agar plates
Pipette Controller	BrandTech	accu-jet	To pipet 20 mL for swarming agar plates
Roller Drum	New Brunswick	TC-7	For growth of bacteria at 100 rpm
Scanner	Epson	Epson Perfection V370 Photo	Scanner for imaging plates
Scanner automation software	RoboTask Lite	v7.0.1.932	For 30 min internals imaging
Scanner image acquisition software	Epson	v9.9.2.5US	Software for imaging plates