J.-L.B., A.S., and N.M.H-K. wrote and revised the manuscript. All authors designed the experiments. J.-L.B. performed the experiments and analysis. This work was supported by NIH award K22AI112816 and R21AI139968 grant to A.S. and by the University of California. N.M.H-K. was supported by Lundbeck Fellowships R220-2016-860 and R251-2017-1070. The funders had no role in the decision to submit the work for publication. We have no competing interests to declare.

1. Preparing Swarming Agar Plates for P. aeruginosa Swarming Time-lapse Imaging
<ol>
	<li>Prepare 1 L of 5x M8 minimum media in a glass bottle by adding 64 g of Na2HPO4&#8226;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.</li>
	<li>Prepare 100 mL of 1 M MgSO4 (magnesium sulfate) in a glass bottle by adding 24.6 g of MgSO4&#8226;7H2O in 50 mL ddH2O. Adjust the final volume to 100 mL with additional ddH2O. Autoclave to sterilize. Store at room temperature.</li>
	<li>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.</li>
	<li>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 &#181;m filter. Store at room temperature.</li>
	<li>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.
	<ol>
		<li>Immediately after autoclaving, place the agar solution in a 55 &#176;C water bath for 15 min.</li>
		<li>Remove the agar solution from the water bath and add 40 mL of 5x M8 minimum media, 200 &#181;L 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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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&#8211;50% 1 h prior to the next step.</li>
	<li>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&#8211;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 &#176;C for up to 24 h.</li>
	<li>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.</li>
</ol>
2. Growth of P. aeruginosa and Plating Conditions
<ol>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>Streak P. aeruginosa on an LB-agar Petri dish from a frozen stock stored at -80 &#176;C using sterile loops or wooden sticks. Incubate the Petri dish upside-down overnight at 37 &#176;C. Store LB-agar plate at 4 &#176;C for up to 1 week.</li>
	<li>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&#8211;18 h) at 37 &#176;C in a roller drum set at 100 rpm.</li>
	<li>Pipet 5 &#181;L 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&#8211;45&#176;) 2.5 cm above the spotting area, pipetting down to the first stop, and touching the agar with only the liquid drop (Figure 1B).
	<ol>
		<li>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).</li>
		<li>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).</li>
	</ol>
	</li>
	<li>For phage infection, mix 30 &#181;L of overnight culture of P. aeruginosa from step 2.4 with 6 &#181;L of 1 x 1012 pfu/mL phage DMS3vir20. Proceed immediately to the next step.
	<ol>
		<li>Pipet 6 &#181;L 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&#176;) 2.5 cm above the spotting area, pipetting down to the first stop, and touching the agar with only the liquid drop (Figure 1C).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>For antibiotic treatments, mix 30 &#181;L overnight culture P. aeruginosa from step 2.4 with 6 &#181;L of 3 mg/mL gentamycin, 10 &#181;L of 100 mg/mL kanamycin, or 7.5 &#181;L of 100 mg/mL fosfomycin. Proceed immediately to the next step.
	<ol>
		<li>Pipet 6 &#181;L 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&#176;) 2.5 cm above the spotting area, pipetting down to the first stop, and touching the agar with only the liquid drop (Figure 1D).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Replace the clear Petri dish lids with black lids made in step 1.9 (Figure 2A).</li>
	<li>Place the swarming agar plates on a scanner in an incubator set at 37 &#176;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.</li>
</ol>
3. Image Acquisition with Scanner
<ol>
	<li>Decrease the ambient lighting of the Petri dishes by attaching black matte fabric to a rack 40&#8211;60 cm above the flatbed document scanner. Secure it using zip ties (Figure 2B).</li>
	<li>The scanner will be controlled using a scanning software and an automatic scripting software.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Set the saving path of images by clicking on the folder icon to the right of Scan to open File Save Settings (Figure 3A).
	<ol>
		<li>Select the folder destination for saving images by selecting Other under Location and click on Browse. Choose a folder to save the images.</li>
		<li>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).</li>
		<li>Leave the first option unchecked (&#34;Overwrite any files with the same name&#34;) and check the 3 next options (&#34;Show this dialog box before next scan&#34;, &#34;Open image folder after scanning&#34;, and &#34;Show Add Page dialog after scanning&#34;). Click OK to close the window</li>
		<li>Check the image quality by clicking on Preview. The preview window appears, and the Scan icon becomes functional (Figure 3C).</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Compiling Time-lapse Images and Measuring Swarm Repulsion
<ol>
	<li>Perform movie editing and image analysis using ImageJ.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Set the video compression level to 100 by clicking on Edit | Options | Input/Output&#8230; and adjust JPEG quality to 100.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#34;+&#34; to zoom in closer and &#34;-&#34; to zoom out.</li>
</ol>

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The authors have nothing to disclose.

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 swarming16,21, the protocol presented here uses M8 me...

Swarming is a collective form of coordinated bacterial motility that increases antibiotic resistance and production of virulence factors in the host1,2,3. This multicellular behavior occurs on semi-solid surfaces that resemble those of mucous layers covering epithelial membranes in the lungs4,5. 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 sensing6,7,8. Many species of bacteria are capable of swarming, including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli9,10,11,12. 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 moisture13,14. In addition to surface properties, growth temperature and ambient humidity affect several aspects of swarming dynamics, including swarming rate and patterns12,13,14,15. 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 area6,16. 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 threats18,19. 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 &#176;C incubator. The scanner is controlled by a software that automatically scans the plates at regular intervals over the swarm growth period, typically 16&#8211;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.

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			<td height="21" style="height:21px;width:228px;">Reagents</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Bacto agar, dehydrated</td>
			<td style="width:157px;">BD Difco</td>
			<td style="width:120px;">214010</td>
			<td>For LB-agar plate and swarming agar plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Casamino acids</td>
			<td style="width:157px;">BD Difco</td>
			<td style="width:120px;">223050</td>
			<td>For swarming media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">D-Glucose</td>
			<td style="width:157px;">Fisher Chemical</td>
			<td style="width:120px;">D16500</td>
			<td>Dextrose. For swarming media</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:228px;">Fosfomycin disodium salt</td>
			<td style="width:157px;">Tokyo Chemical Industry</td>
			<td style="width:120px;">F0889</td>
			<td>Stock concentration: 200 mg / mL. Dissolved in ddH2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:228px;">Gentamycin sulfate</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:120px;">G1914</td>
			<td>Stock concentration: 3 mg / mL. Dissolved in ddH2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:228px;">Kanamycin sulfate</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:120px;">60615</td>
			<td>Stock concentration: 100 mg / mL. Dissolved in ddH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">LB-Miller</td>
			<td style="width:157px;">BD Difco</td>
			<td style="width:120px;">244620</td>
			<td>For LB broth and LB-agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Magnesium sulfate heptahydrate</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:120px;">230391</td>
			<td>For swarming media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Potassium phosphate monobasic</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:120px;">P0662</td>
			<td>For 5X M8 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Sodium chloride</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:120px;">S9888</td>
			<td>For 5X M8 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Sodium phosphate dibasic heptahydrate</td>
			<td style="width:157px;">Fisher Chemical</td>
			<td style="width:120px;">S373</td>
			<td>For 5X M8 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Strains</td>
			<td style="width:157px;"></td>
			<td style="width:120px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Pseudomonas aeruginosa</td>
			<td style="width:157px;">Siryaporn lab</td>
			<td style="width:120px;">AFS27E.118</td>
			<td>PA14 strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">DMS3vir</td>
			<td style="width:157px;">O&#39;Toole lab</td>
			<td style="width:120px;">DMS3vir20</td>
			<td>Bacteriophage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Supplies</td>
			<td style="width:157px;"></td>
			<td style="width:120px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Aluminium oxide sandpaper</td>
			<td style="width:157px;">3M</td>
			<td style="width:120px;">150 Fine</td>
			<td>For black lids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Black fabric</td>
			<td style="width:157px;">Joann</td>
			<td style="width:120px;">PRD7089</td>
			<td>Black fabric</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Black spray paint</td>
			<td style="width:157px;">Krylon</td>
			<td style="width:120px;">5592 Matte Black</td>
			<td>For black lids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Erlenmeyer flask</td>
			<td style="width:157px;">Kimax</td>
			<td style="width:120px;">26500</td>
			<td>250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Glass storage bottles</td>
			<td style="width:157px;">Pyrex</td>
			<td style="width:120px;">13951L</td>
			<td>250 mL, 500 mL, 1000 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">8 inches zip ties</td>
			<td style="width:157px;">Gardner Bender</td>
			<td style="width:120px;">E173770</td>
			<td>For attaching black matte fabric</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Petri dishes (100 mm x 15 mm)</td>
			<td style="width:157px;">Fisher</td>
			<td style="width:120px;">FB0875712</td>
			<td>100 x 15 mm polystyrene plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Wooden sticks</td>
			<td style="width:157px;">Fisher</td>
			<td style="width:120px;">23-400-102</td>
			<td>For streaking and inoculating bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Equipment</td>
			<td style="width:157px;"></td>
			<td style="width:120px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Autoclave</td>
			<td style="width:157px;">Market Forge Industries</td>
			<td style="width:120px;">STM-E</td>
			<td>For sterilizing reagents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">25 mL pipette</td>
			<td style="width:157px;">USA Scientific, Inc.</td>
			<td style="width:120px;">1072-5410</td>
			<td>To pipet 20 mL for swarming agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Dehumidifier</td>
			<td style="width:157px;">Frigidaire</td>
			<td style="width:120px;">FAD704DWD 70-pint</td>
			<td>For maintaing room relative humidity at about 45%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">ImageJ</td>
			<td style="width:157px;">NIH</td>
			<td style="width:120px;">v1.52a</td>
			<td>Software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Incubator</td>
			<td style="width:157px;">VWR</td>
			<td style="width:120px;">89032-092</td>
			<td>For growth of bacteria at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Isotemp waterbath</td>
			<td style="width:157px;">Fisher</td>
			<td style="width:120px;">15-462-21Q</td>
			<td>For cooling media to 55 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Laminar flow hood</td>
			<td style="width:157px;">The Baker Company</td>
			<td style="width:120px;">SG603A</td>
			<td>For drying plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">P-20 pipet</td>
			<td style="width:157px;">Gilson</td>
			<td style="width:120px;">F123601</td>
			<td>Spotting on swarming agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Pipette Controller</td>
			<td style="width:157px;">BrandTech</td>
			<td style="width:120px;">accu-jet</td>
			<td>To pipet 20 mL for swarming agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Roller Drum</td>
			<td style="width:157px;">New Brunswick</td>
			<td style="width:120px;">TC-7</td>
			<td>For growth of bacteria at 100 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Scanner</td>
			<td style="width:157px;">Epson</td>
			<td style="width:120px;">Epson Perfection V370 Photo</td>
			<td>Scanner for imaging plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Scanner automation software</td>
			<td style="width:157px;">RoboTask Lite</td>
			<td style="width:120px;">v7.0.1.932</td>
			<td>For 30-min internals imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Scanner image acquisition software</td>
			<td style="width:157px;">Epson</td>
			<td style="width:120px;">v9.9.2.5US</td>
			<td>Software for imaging plates</td>
		</tr>
	</tbody>
</table>

time-lapse imaging of bacterial swarms and the collective stress response

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.

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 &#176;C and spotted 5 &#181;L 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</str...

Watch this Scientific Journal Video about Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response at JoVE.com

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

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.

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations ...

time-lapse-imaging-bacterial-swarms-collective-stress

Research

JoVE Journal

Biology

7.9K Views.  University of California, Irvine. 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.

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

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...