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.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bacto agar, dehydrated</td><td>BD Difco</td><td>214010</td><td>For LB-agar plate and swarming agar plate</td></tr><tr><td>Casamino acids</td><td>BD Difco</td><td>223050</td><td>For swarming media</td></tr><tr><td>D-Glucose</td><td>Fisher Chemical</td><td>D16500</td><td>Dextrose. For swarming media</td></tr><tr><td>Fosfomycin disodium salt</td><td>Tokyo Chemical Industry</td><td>F0889</td><td>Stock concentration: 200 mg/mL. Dissolved in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Gentamycin sulfate</td><td>Sigma-Aldrich</td><td>G1914</td><td>Stock concentration: 3 mg/mL. Dissolved in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Kanamycin sulfate</td><td>Sigma-Aldrich</td><td>60615</td><td>Stock concentration: 100 mg/mL. Dissolved in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>LB-Miller</td><td>BD Difco</td><td>244620</td><td>For LB broth and LB-agar plates</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>230391</td><td>For swarming media</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P0662</td><td>For 5x M8 media</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S9888</td><td>For 5x M8 media</td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Fisher Chemical</td><td>S373</td><td>For 5x M8 media</td></tr><tr><td>&lt;strong&gt;Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Pseudomonas aeruginosa&lt;/em&gt;</td><td>Siryaporn lab</td><td>AFS27E.1&lt;sup&gt;18&lt;/sup&gt;</td><td>PA14 strain</td></tr><tr><td>DMS3vir</td><td>O&#039;Toole lab</td><td>DMS3vir&lt;sup&gt;20&lt;/sup&gt;</td><td>Bacteriophage</td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Aluminium oxide sandpaper</td><td>3M</td><td>150 Fine</td><td>For black lids</td></tr><tr><td>Black fabric</td><td>Joann</td><td>PRD7089</td><td>Black fabric</td></tr><tr><td>Black spray paint</td><td>Krylon</td><td>5592 Matte Black</td><td>For black lids</td></tr><tr><td>Erlenmeyer flask</td><td>Kimax</td><td>26500</td><td>250 mL</td></tr><tr><td>Glass storage bottles</td><td>Pyrex</td><td>13951L</td><td>250 mL, 500 mL, 1,000 mL</td></tr><tr><td>8 inches zip ties</td><td>Gardner Bender</td><td>E173770</td><td>For attaching black matte fabric</td></tr><tr><td>Petri dishes (100 mm x 15 mm)</td><td>Fisher</td><td>FB0875712</td><td>100 mm x 15 mm polystyrene plates</td></tr><tr><td>Wooden sticks</td><td>Fisher</td><td>23-400-102</td><td>For streaking and inoculating bacteria</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Autoclave</td><td>Market Forge Industries</td><td>STM-E</td><td>For sterilizing reagents</td></tr><tr><td>25 mL pipette</td><td>USA Scientific, Inc.</td><td>1072-5410</td><td>To pipet 20 mL for swarming agar plates</td></tr><tr><td>Dehumidifier</td><td>Frigidaire</td><td>FAD704DWD 70-pint</td><td>For maintaing room relative humidity at about 45%</td></tr><tr><td>ImageJ</td><td>NIH</td><td>v1.52a</td><td>Software for image analysis</td></tr><tr><td>Incubator</td><td>VWR</td><td>89032-092</td><td>For growth of bacteria at 37 &amp;deg;C</td></tr><tr><td>Isotemp waterbath</td><td>Fisher</td><td>15-462-21Q</td><td>For cooling media to 55 &amp;deg;C</td></tr><tr><td>Laminar flow hood</td><td>The Baker Company</td><td>SG603A</td><td>For drying plates</td></tr><tr><td>P-20 pipet</td><td>Gilson</td><td>F123601</td><td>Spotting on swarming agar plates</td></tr><tr><td>Pipette Controller</td><td>BrandTech</td><td>accu-jet</td><td>To pipet 20 mL for swarming agar plates</td></tr><tr><td>Roller Drum</td><td>New Brunswick</td><td>TC-7</td><td>For growth of bacteria at 100 rpm</td></tr><tr><td>Scanner</td><td>Epson</td><td>Epson Perfection V370 Photo</td><td>Scanner for imaging plates</td></tr><tr><td>Scanner automation software</td><td>RoboTask Lite</td><td>v7.0.1.932</td><td>For 30 min internals imaging</td></tr><tr><td>Scanner image acquisition software</td><td>Epson</td><td>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 °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). F...

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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

8.1K Views.  University of California, Irvine. Dit protocol kan worden gebruikt om time-lapse films van Pseudomonas aeruginosa zwermen produceren en om de afstoting van zwerm vast te leggen door bacteriofhage of antibiotica. De time-lapse films vangen de gedetailleerde dynamiek van de zwermen reactie op antibiotica en bacteriofaag en de techniek vereist alleen een ontvochtiger, computer, incubator, en document scanner. Voor te bereiden zwermen agar platen voor P.aeruginosa zwermen time-lapse imaging, gebruik maken van een 25 milliliter pi...

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

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Immunology and Infection

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are affected both by the doubling time of the microorganism and by any delay in growth upon transfer from one condition to another, the lag. The ScanLag method enables the characterization of these two independent properties at the level of colonies originating each from a single cell, generating a two-dimensional distribution of the lag time and of the growth time. In ScanLag, measurement of the time it takes for colonies on conventional nutrient agar plates to be detected is automated on an array of commercial scanners controlled by an in house application. Petri dishes are placed on the scanners, and the application acquires images periodically. Automated analysis of colony growth is then done by an application that returns the appearance time and growth rate of each colony. Other parameters, such as the shape, texture and color of the colony, can be extracted for multidimensional mapping of sub-populations of cells. Finally, the method enables the retrieval of rare variants with specific growth phenotypes for further characterization. The technique could be applied in bacteriology for the identification of long lag that can cause persistence to antibiotics, as well as a general low cost technique for phenotypic screens.

ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are ...

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...

Biochemistry

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of signals to create complex, dynamic patterns as a response to environmental cues. These patterns are self-organizing and emergent; they cannot be predicted by observing the behavior of the individual cells. Using a time-lapse microcinematography tracking assay, we identified a distinct emergent pattern in M. xanthus called chemotaxis, defined as the directed movement of a swarm up a nutrient gradient toward its source 1.
In order to efficiently characterize chemotaxis via time-lapse microcinematography, we developed a highly modifiable plate complex (Figure 1) and constructed a cluster of 8 microscopes (Figure 2), each capable of capturing time-lapse videos. The assay is rigorous enough to allow consistent replication of quantifiable data, and the resulting videos allow us to observe and track subtle changes in swarm behavior. Once captured, the videos are transferred to an analysis/storage computer with enough memory to process and store thousands of videos. The flexibility of this setup has proven useful to several members of the M. xanthus community.

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

To study Myxococcus xanthus swarm behavior, we have designed a time-lapse microcinematography protocol that can be modified for different assays. It employs standard growth conditions adapted for microscopy, and yields reproducible results by the use of inexpensive, reusable silicone gaskets. We have used this method to quantify multicellular chemotaxis.

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of ...

Multi-scale Analysis of Bacterial Growth Under Stress Treatments

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to characterize the response of bacterial cells to stress-inducing treatments such as exposure to antibiotics or other antimicrobial compounds, irradiation, non-physiological pH, temperature, or salt concentration. Different stress treatments might disturb different cellular processes, including cell division, DNA replication, protein synthesis, membrane integrity, or cell cycle regulation. These effects are usually associated with specific phenotypes at the cellular scale. Therefore, understanding the extent and causality of stress-induced growth or viability deficiencies requires a careful analysis of several parameters, both at the single-cell and at the population levels. The experimental strategy presented here combines traditional optical density monitoring and plating assays with single-cell analysis techniques such as flow cytometry and real time microscopy imaging in live cells. This multiscale framework allows a time-resolved description of the impact of stress conditions on the fate of a bacterial population.

This protocol allows a time-resolved description of bacterial growth under stress conditions at the single-cell and the cell population levels.

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

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

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