Name: preparation, imaging, and quantification of bacterial surface motility assays
Uploaded: 2015-04-07T13:00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays at JoVE.com

Partial support for this work was provided by the National Institute of Health (R01GM100470 and 1R01GM095959-01A1; to MA and JDS) and a Core Facility grant from the Indiana Clinical and Translational Sciences Institute (funded in part by NIH grant #UL1 TR000006; to JDS).

1. Swarm Assay Media Preparation and Inoculation4,5,7,8,11
<ol>
	<li>Media Preparation 
	NOTE: The medium composition described below is applicable to P. aeruginosa tendril formation studies. Please see media specifications on Table 1 for P. aeruginosa, B. subtilis, and M. xanthus surface motility assays.
	<ol>
		<li>Mix 200 ml of FAB-minus (NH4)2SO4 swarm medium (Materials Table), 0.9 g of Noble agar, and 0.2 g of Casamino acids (Table 1) by stirring with a magnetic stir bar. Use small volumes (100-300 ml) to improve consistency between experiments.</li>
		<li>Autoclave the 200 ml agar/media mixture using an exposure time of 22 min, exposure temperature of 121.1 &#176;C, and a fast vent option. The autoclave settings will allow proper sterilization and agar melting, but prevent agar caramelization. 
		NOTE: Noble agar is prone to caramelization; bacterial motility is altered on caramelized agar.</li>
		<li>Immediately after the sterilization cycle has finalized, close the cap of the media bottle to prevent water loss by evaporation. However, note that tight capping can cause a &#8220;vacuum sealing&#8221;-like effect on the bottle.</li>
		<li>Cool the media to 50 &#176;C while stirring at room temperature (RT) and add 2 ml of sterile 1.2 M glucose. Alternatively, place the media in a 60 &#176;C incubator or water bath until ready to use (up to 15 hr later), and proceed as indicated. To prevent the formation of bubbles in the media, mix thoroughly using the magnetic stir bar; bubbles on the surface of the agar will prevent even swarming. 
		NOTE: For other assays, add at this step heat-sensitive components that cannot be autoclaved such as additional nutrients or dyes, as needed (e.g., addition of 8 &#181;l Invitrogen Syto 63 dye per 100 ml melted agar to image M. xanthus as shown in Representative Results, below). Addition of some dyes may affect baseline swarming behavior, which should be checked against a non-dye control.</li>
		<li>In a laboratory hood, aliquot 7.5 ml of sterile media per 60 mm diameter polystyrene Petri dish and maintain the plates in a single layer (not stacked). For larger swarming surface, aliquot 25 ml of media per 100 mm diameter Petri dish. It is important to fill the dishes on an even horizontal surface. Use a bull&#8217;s eye level to check if the surface is leveled. 
		NOTE: For P. aeruginosa assays, using a specific media volume per plate will improve consistency and reproducibility. For B. subtilis and M. xanthus assays, hand pouring yields results comparable to specific volume aliquots.</li>
	</ol>
	</li>
	<li>Plate curing
	<ol>
		<li>For small plates (60 mm), allow the melted agar medium to cure (both set to semi-solid and dry excess liquid) in the hood uncovered (i.e., without lids) for 30 min. Larger plates (100 mm) require a longer curing time (see Discussion). 
		NOTE: Alternatively, some assays may require plates to cure on the bench top overnight (20-24 hr) covered (i.e., lids on) in a single layer (Table 1). Swarming is sensitive to both excess and inadequate moisture. The humidity, airflow, and temperature of any given lab may necessitate variation to plate curing to promote optimal swarming of you bacterium.</li>
		<li>Inoculate plates immediately after the drying period is over. Do not store the plates for further use.
		<ol>
			<li>Perform the &#8220;ink spread test&#8221; by spotting a test plate with 10 &#181;l mixture of 0.50% (vol/vol) Higgins Waterproof Black India Ink and bacterial inoculum11. If the ink/inoculum mixture spreads readily (i.e. does not retain droplet form) on the surface of the media, the media will need additional time to dry. 
			NOTE: For species that are particularly sensitive to humidity (e.g., P. aeruginosa), perform a quick &#8220;ink spread test&#8221;11 to determine if the plates are dry enough.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Swarm Assay Inoculation
	<ol>
		<li>Inoculate 6 ml of broth culture media (see Table 1 for details) with an isolated colony from a fresh (&#60; 5 days old if left at room temperature) Lysogeny Broth (LB) plate culture. Incubate broth cultures overnight (&#8804;18 hr) at 30 &#176;C or 37 &#176;C with horizontal shaking (240 rpm).</li>
		<li>Inoculate swarm plates by spotting with 1-5 &#181;l of overnight broth culture, or by &#8220;poking&#8221; the agar with a sterile tooth pick or wire inoculation needle. 
		NOTE: We prefer the latter method because it decreases the likelihood of spattering the inoculum and prevents adding additional moisture to the swarm surface area.</li>
	</ol>
	</li>
	<li>Swarm Assay Incubation
	<ol>
		<li>For general assay, incubate swarm assay plates at 30 &#176;C or 37 &#176;C (or even 42 &#176;C for B. subtilis; Table 1)&#8212;this is bacterium specific. Invert the plates during incubation so that excess moisture condenses on the lid, not the agar. 
		NOTE: Temperature can affect phenotype as well as kinetics. For P. aeruginosa swarms, incubation at 37 &#176;C leads to faster growth and swarm expansion than incubation at 30 &#176;C; however, the morphology of these swarms often differs with this change in temperature.</li>
		<li>For time-lapse imaging, incubate swarm plates at the appropriate temperature prior to transfer into the imaging station (see Table 1 for details). 
		NOTE: This pre-imaging incubation allows swarms to start their development and become established before being moved to a new environment, which may or may not be optimal for swarming motility.</li>
	</ol>
	</li>
</ol>
2. Macroscopic Imaging of Surface Motility Assays7,8
<ol>
	<li>For time-lapse imaging, after the pre-imaging incubation period place swarm assay plates on a clear imaging plate inside a commercial in vivo imaging station. Image up to six, 60 mm diameter or four, 100 mm diameter plates at a time. Since the camera captures images from beneath the imaging plane, invert the plates so that the optical path is not obstructed8. Alternatively, incubate at 30 &#176;C or 37 &#176;C (Table 1) for the duration of the experiment, and remove the plates to be imaged from the incubator at set time intervals.</li>
	<li>Place the lids of the Petri dishes upright on top of the plate counterpart that holds the inoculated media. Fill the lids of the Petri dishes with water to prevent excessive drying during imaging, and enclose the entire set up using another clear tray to maintain humidity throughout the experiment.</li>
	<li>Using Molecular Imaging (MI) software12, run assay(s) at room temperature using the imaging settings described in Table 2. For time-lapse imaging, set up a protocol with the necessary steps and specifications.</li>
</ol>
3. Data Processing and Interpretation7,8
<ol>
	<li>Image Processing
	<ol>
		<li>Use MI software to batch export images as 16-bit TIFF files: File &#62; Export or Export Multiple &#62; Select file(s) to export and export location.</li>
		<li>Use ImageJ to open a single image or import a time-lapse series:
		<ol>
			<li>Open a single image: File &#62; Open</li>
			<li>Import time-lapse image sequence: File &#62; Import Sequence, and select &#8220;Sort names numerically&#8221;.
			<ol>
				<li>For larger time-lapse files, select &#8220;Use virtual stack&#8221; in the &#8220;Import Sequence&#8221; window to stack the exported images into appropriate categories (i.e., GFP, RFP, etc.).</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>If required, change images from 16-bit files to 8-bit files: Image &#62; Type &#62; 8-bit 
		NOTE: Some ImageJ tools require 8-bit images.</li>
		<li>Determine if the intensity signal for an image or time-lapse sequence needs to be inverted. Place the cursor on a bright spot in the image (e.g., fluorescently labeled growth) and note the signal intensity &#8220;Value&#8221; from the ImageJ toolbar. Then, place the cursor in a dark spot outside the plate area and note the signal intensity. If the signal intensity for the dark spot is larger than the intensity for the bright spot, the image signal intensity needs to be inverted (follow substeps 1-2 below).
		<ol>
			<li>Invert the intensity signals: Edit &#62; Invert</li>
			<li>Invert the lookup table: Image &#62; Lookup Tables &#62; Invert LUT</li>
		</ol>
		</li>
		<li>Subtract the background: Process &#62; Subtract Background, and use a &#8220;Rolling ball radius&#8221; with a pixel radius that is one half of one image dimension (e.g., 1,000 pixels for a 2,000 x 2,000 pixel image).</li>
		<li>Artificially color an image or time-lapse sequence: Image &#62; Lookup Tables, and select the appropriate color from the list options.</li>
		<li>For movies with two or more channels, merge and balance the colors prior to saving as a movie (Image Processing, Step 8). To merge images together, open all image stacks in ImageJ, then select Image &#62; Color &#62; Merge Channels, and assign each stack to a color channel.</li>
		<li>Save time-lapse sequence as AVI or QuickTime Movie: File &#62; Save as, and choose desired format and specifications.</li>
	</ol>
	</li>
	<li>Data Analysis
	<ol>
		<li>Acquiring Bacterial Surface Growth Area to Quantify Expansion Rate
		<ol>
			<li>Open image(s) in ImageJ</li>
			<li>To calculate the diameter of the plate in pixels, draw a line across the center of an assay plate with the &#8220;Straight&#8221; tool from the and measure its length: Analyze &#62; Measure</li>
			<li>The default measurement unit in ImageJ is the pixel. Obtain a conversion factor by dividing the diameter of the assay plate (e.g., 60 for a 60 mm plate) by the pixel length obtained in the previous step.</li>
			<li>Change the unit of measurement from pixel to mm: Image &#62; Properties
			<ol>
				<li>Change the &#8220;Unit of Length&#8221; to &#8220;mm&#8221;, and the &#8220;Pixel Width&#8221;, &#8220;Pixel Height&#8221;, and &#8220;Voxel Depth&#8221; to the conversion factor calculated in the previous step. Select the &#8220;Global&#8221; box to maintain this conversion factor across multiple images. 
				NOTE: If ImageJ is closed and reopened, or the field of view of an image is changed (i.e., one image is zoomed in more than another), the conversion factor must be recalculated. Alternatively, perform all analyses in pixels and then converted to mm.</li>
			</ol>
			</li>
			<li>For every frame, trace and measure the swarm area using the &#8220;Freehand Selections&#8221; tool in the toolbar to trace the outline of the swarm and measure the area using: Analyze &#62; Measure. This will generate a measurements log that can be saved for further analysis in Microsoft Excel or similar programs: File &#62; Save As</li>
		</ol>
		</li>
		<li>Acquiring Bacterial Surface Growth Intensity to Quantify Surface Growth Rate
		<ol>
			<li>Once the background is subtracted (Image Processing, Step 5), use the last frame of the sequence to determine the maximum area of swarm (Data Analysis, Step 1).</li>
			<li>Use the &#8220;Oval&#8221; selection tool from the toolbar to draw a box around the bacterial surface growth.</li>
			<li>Set the mean intensity measurement of pixels in the box using: Analyze &#62; Set Measurements, and select &#34;Mean gray value&#34;.</li>
			<li>To obtain intensity signal measurements for each frame in the time-lapse sequence, while on the first frame of the sequence go to: Analyze &#62; Measure. This will generate a measurements log that can be saved for further analysis in Microsoft Excel or similar programs: File &#62; Save As</li>
		</ol>
		</li>
		<li>Alternative to the previous section (Data Analysis, Step 2). Use the ImageJ Macros plugin to setup and run a Macros surface growth intensity measurement script.
		<ol>
			<li>Setup an automated measurement script to analyze multiple frames simultaneously: Plugin &#62; New &#62; Macro, and paste the provided script (below) into the box and save as an ImageJ Macros text file: File &#62; Save, and save to the ImageJ application folder under &#8220;macros&#8221;. 
			numberOfFrames = N 
			for(i=0; i&#60; numberOfFrames; i++){ 
			run(&#39;Measure&#39;); 
			run(&#39;Next Slice [&#62;]&#39;); 
			} 
			NOTE: Here the variable &#8220;N&#8221; is for an undefined number of frames.</li>
			<li>Edit the &#8220;numberOfFrames&#8221; in the Macros plugin for each experiment to reflect the number of frames in the image sequence prior to running the script. Use: Plugin &#62; Macros &#62; Edit, and type in the correct number of frames in the sequence and save (File &#62; Save).</li>
			<li>Follow substeps 1-3 in Data Analysis-Step 2, and while on the first frame of the sequence run the Macros plugin: Plugin &#62; Macros &#62; Run. This will generate a measurements log that can be saved for further analysis in Microsoft Excel or similar programs: File &#62; Save As</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
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Publication fees for this article were partially sponsored by Bruker Corporation.

Achieving reproducible swarming in a laboratory can be challenging, as swarm assays are highly sensitive to environmental factors, such as humidity and available nutrients. The most critical aspect of a surface motility plate assay is moisture on the agar surface. Prior to inoculation, swarm media must be dry enough to prevent bacterial cells from swimming across the surface liquid, but not so dry as to inhibit swarming motility5. Incubation should take place in a sufficiently humid environment: too little moisture can result in the assay drying out during incubation, while too much moisture can lead to artificial or artifactual surface spreading. Unless a humidity-controlled incubator is at hand, incubator and laboratory humidity can vary dramatically. Consequently, an additional water reservoir, a humidifier, or a dehumidifier within the incubator might be required to prevent over drying or the accumulation of excess moisture while keeping the relative humidity near 80%. Maintaining this ideal humidity may prove challenging if seasonal humidity changes are significant. If this is the case, the swarm assay protocol will require some adjustments to account for seasonal changes in humidity. We have found that modifying the swarm media drying time is the simplest way to adjust for seasonal humidity changes. Constant humidity monitoring, both inside and outside of the incubator, is recommended. Further, it is recommended that researchers calibrate and validate their instruments, incubators, scales, etc. as minor errors in temperature, volume or amounts of media components can impact reproducibility of these assays.
It should also be noted that the type and size of the plate used in the assay can affect plate moisture, and thus swarming. Airtight plates do not vent off excess moisture, thus encouraging swimming motility. In contrast, open-faced plates allow too much moisture to escape. A Petri dish provides an ideal environment because it vents off enough excess moisture to prevent liquid build up, but retains enough moisture to prevent the media from drying out. This method details a surface motility assay protocol that allows for high quality imaging. To keep the agar clear for imaging 60 mm diameter dishes are filled with 7.5 ml of agar media. If detailed imaging is not required, volumes up to 20 ml can also provide reproducible results.
While swarming motility can be achieved on a wide array of agar concentrations, the optimal range of agar required for swarming depends on the species. Overall, higher agar concentrations inhibit swarming motility, and consequently the time needed to produce an image-ready swarm increases. P. aeruginosa generally swarms on agar concentrations between 0.4-0.7%1, however we find that optimal swarming occurs in a much narrower range (0.4-0.5%). Others, such as B. subtilis and S. enterica swarm at 0.6% agar, and Vibrio parahaemolyticus at 1.5% agar10. The required agar concentration is also determined by the type and brand of agar. Higher purity agars, like Noble agar, strongly enhance swarming in P. aeruginosa and are preferred over granulated agar13,14. However, these purified versions of agar are also more prone to caramelization during the autoclave sterilization cycle; depending on the instrument, a shortened/modified sterilization sequence (to possibly alter the exhaust cycle to prevent prolonged heat exposure) may be required to prepare swarm media using Noble agar.
Media composition also plays a role in the observed swarm phenotype3. P. aeruginosa swarming motility studies are usually performed using minimal nutrient media. We prefer FAB medium4,8 (Materials Table), but other media, such as M9, LB, or slight variations to these common media, have been used successfully9,15,16. Tendril formation is best achieved on FAB minimal medium supplemented with glucose as the carbon source and casamino acids (CAA), but without an additional nitrogen source (i.e., (NH4)2SO4)6,13. If tendril formation or morphology is not the main focus of the study, then FAB minimal medium (Materials Table; Table 1) devoid of CAA is recommended so that the effects of specific carbon sources and/or additional nutrients can be studied in detail. Other species, such as B. subtilis (presented here), are versatile swarmers, capable of swarming on LB and granulated agar. These species swarm readily, requiring only ~10 hr to develop a full swarm. This fast swarming rate makes following the progression of the swarm potentially difficult but our protocol makes such tracking very feasible. The ability to perform swarm time-lapse imaging provides a substantial ease in swarm data acquisition, particularly from such avid swarmers.
We introduce a robust, comprehensive, two-phase protocol and guidelines aimed at enhancing the execution and reproducibility of bacterial surface motility research and have primarily emphasized aspects important to examine flagellar-mediated swarming. This swarm assay protocol details important aspects of media composition and handling of surface motility plates to provide for greater consistency and reproducibility within and among research groups. This will improve the basis of comparison among different research studies. In addition, the presented approach and protocol provides means to make research on swarming and surface motility less susceptible to environmental variations by making researchers aware that such factors affect their work and providing possible solutions (e.g., how small changes in agar affect swarming4,5). Furthermore, the protocol provided to quantify macroscopic aspects of swarming, provides an opportunity to measure many attributes of bacterial surface growth that were previously unquantifiable.
We have not examined all surface motile bacteria in the development of this protocol. As such, it is expected that protocol modifications will be required for species not presented here. The efficiency of this protocol is restricted by the inherent limits of the equipment and materials employed. For instance, temperature related studies are not possible as yet with the Bruker imaging station, since temperature control is not a feature of the equipment. In addition, the use of dyes (such as Nile Red to stain rhamnolipids) can have kinetic and concentration limitations8. This technique strongly relies on the processing and analysis of digital images; improved automation of data analysis (e.g., using additional Macros script function in ImageJ) would reduce the time needed for analysis and expand the usefulness of the data. Finally, due to the robustness of the imaging protocol, future applications should aim at expanding this technique to examine less uniform growth surfaces that are more relevant to surfaces colonized by environmental and pathogenic bacteria.

Many bacteria move on surfaces using various means of self-propulsion. Some motility phenotypes can be researched in the laboratory using plate assays that are affected by the liquid environment associated with the semi-solid plate assay composition. A subset of useful surface motility plate assays further involve a gas phase&#8212;typically room air. Accordingly, the outcome of any particular surface motility assay, demands careful control of the interface of three phases: the local environmental solid surface, liquid environment, and gas environment properties.
The most commonly studied motility mode in such a three-phase assay is known as swarming. Swarming motility is the coordinated group movement of bacterial cells that are propelled by their flagella through thin liquid films on surfaces1. It is typically studied in laboratories using semi-solid plate assays containing 0.4%-0.8% (wt/vol) agar1. An array of human pathogens exploit this motility behavior to explore and colonize the human host. For instance, Proteus mirabilis uses swarming motility to move up the urethra, reaching and colonizing the bladder and kidneys2. Swarming motility is generally considered a precursor step to biofilm formation, the primary cause of pathogenesis in many human pathogens3.
The swarming phenotype is highly varied among bacterial species; experimental success and reproducibility strongly rely on factors such as nutrient composition, agar type and composition, sterilization protocol (e.g., autoclaving), semi-solid media curing, and ambient moisture (e.g., seasonal changes), among others3-5. The variability of surface motility responses emphasizes the challenges encountered in these studies and the significant influence media and environment can exert. For some swarming species, such as Pseudomonas, swarming motility can occur on a variety of media compositions, although the observed phenotype and accompanying swarm expansion rate will vary greatly3. Combined, these factors can make surface motility studies extremely challenging. Seasonal variability within a lab can influence these three-phase assays: assays may function better in the humid-air of summer and worse in the dry air of winter. Here we present general guidelines to circumvent some of the most notable challenges when performing surface motility plate studies.
For some surface motility studies, the development of specific phenotypes is of great interest. Most, but not all, published studies to examine swarming of P. aeruginosa show the formation of tendrils or fractals radiating from an inoculation center3-9. Differences between P. aeruginosa strains have been documented5,8, but much of the presence or absence of tendrils can be attributed to the specific medium and protocol used for these swarm motility plate assays. Here we include details on how to promote tendril-forming swarms for P. aeruginosa. Because P. aeruginosa is just one of many swarming bacteria, we also include details for our method to examine swarming of Bacillus subtilis and gliding of Myxococcus xanthus. Like P. aeruginosa, current research on B. subtilis and M. xanthus spans an array of topics as researchers are working to discern aspects of sporulation, motility, stress response, and transitional behavior1,10. There is a need to quantify the patterns and dynamics of the specific behavior(s) for these cells in swarming groups.
Surface motility data acquisition, analysis, and interpretation can be cumbersome and qualitative. We have developed a protocol for the detailed macroscopic analysis of bacterial swarms that provides in addition to swarm zone morphology and size (e.g., diameter), quantitative dynamic information regarding swarm expansion rate and bacterial or bioproduct density distribution7. Furthermore, this method can take advantage of available fluorescent proteins, luminescence, and dyes to obtain a comprehensive view of bacterial interactions8, as well as to track the synthesis of bioproducts (e.g., P. aeruginosa rhamnolipid7,8) within a swarm.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Materials table</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Amount</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagentsa:</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FAB Minimal Media:</td>
			<td></td>
			<td></td>
			<td></td>
			<td>Prepare every ~4 weeks. Top to 1 L with nanopure H2O.</td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sigma</td>
			<td>A4418</td>
			<td>2 g</td>
			<td>Not used in P. aeruginosa tendril formation studies.</td>
		</tr>
		<tr>
			<td>Na2HPO4 x 7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S9390</td>
			<td>9 g</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4&#160;</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td>3 g</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BDH</td>
			<td>BDH8014</td>
			<td>3 g</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 x 6H2O solution (198 g/L)</td>
			<td>Fisher Scientific</td>
			<td>M33</td>
			<td>1 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 x 2H2O solution (14 g/L)</td>
			<td>Fisher Scientific</td>
			<td>C79</td>
			<td>1 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Trace metal solution (see below)</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>1 ml</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trace Metal Solution:</td>
			<td></td>
			<td></td>
			<td></td>
			<td>Top to 1 L with nanopure H2O. Maintain in a glass bottle, stirring and covered with foil.</td>
		</tr>
		<tr>
			<td>CaSO4 x 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>255548</td>
			<td>200 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>MnSO4 x H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M7634</td>
			<td>20 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4 x 5H2O</td>
			<td>Fisher Scientific</td>
			<td>C493</td>
			<td>20 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4 x 7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>Z4750</td>
			<td>20 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>CoSO4 x 7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C6768</td>
			<td>10 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>NaMoO4 x 2H2O</td>
			<td>Sigma</td>
			<td>S6646</td>
			<td>10 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>H3BO3</td>
			<td>Fisher Scientific</td>
			<td>A74</td>
			<td>5 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>FeSO4 x 7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>F7002</td>
			<td>200 mg</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CTT Media:</td>
			<td></td>
			<td></td>
			<td></td>
			<td>Prepare as needed. Top to 100 ml with nanopure H2O.</td>
		</tr>
		<tr>
			<td>Tris-HCl, 1 M solution (adjust to pH 8.0)</td>
			<td>Amresco</td>
			<td>0234</td>
			<td>1 ml</td>
			<td>Prepare a 1 M stock solution in nano pure H2O. Adjust pH to 8.0 and filter sterilize (0.2 &#956;m pore).</td>
		</tr>
		<tr>
			<td>K2HPO4, 1 M solution (adjust to pH 7.6)</td>
			<td>Sigma-Aldrich</td>
			<td>P3786</td>
			<td>0.1 ml</td>
			<td>Prepare a 1 M stock solution in nano pure H2O. Adjust pH to 7.6 and filter sterilize (0.2 &#956;m pore).</td>
		</tr>
		<tr>
			<td>MgSO4 solution</td>
			<td>Fisher Scientific</td>
			<td>M65</td>
			<td>0.8 ml</td>
			<td>Prepare a 1 M stock solution in nano pure H2O. Filter sterilize (0.2 &#956;m pore).</td>
		</tr>
		<tr>
			<td>Casitone</td>
			<td>BD Diagnostics</td>
			<td>225930</td>
			<td>1 g</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Additional Reagents:</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth, Lennox</td>
			<td>BD Diagnostics</td>
			<td>240230</td>
			<td>2 % (wt/vol)</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>30 mM for overnight broth cultures; 12 mM for swarm media</td>
			<td>Prepare a 1.2 M filter sterilized stock solution in nano pure H2O. Add to media after autoclaving.</td>
		</tr>
		<tr>
			<td>Casamino acids (CAA)</td>
			<td>Amresco</td>
			<td>J851</td>
			<td>0.10 % (wt/vol)</td>
			<td>Recommended for P. aeruginosa tendril formation studies. Add to media prior to autoclaving.&#160;</td>
		</tr>
		<tr>
			<td>Agar, Noble</td>
			<td>Sigma-Aldrich</td>
			<td>A5431</td>
			<td>0.45 % (wt/vol)</td>
			<td>Preferred Noble agar for P. aeruginosa surface motility studies. Add to media prior to autoclaving.</td>
		</tr>
		<tr>
			<td>Agar, Noble&#160;</td>
			<td>Affymetrix</td>
			<td>10907</td>
			<td>1.50 % (wt/vol)</td>
			<td>Used in M. xanthus surface motility studies. Not recommended for P. aeruginosa motility studies. Add to media prior to autoclaving.&#160;</td>
		</tr>
		<tr>
			<td>Agar, Granulated</td>
			<td>Fisher Scientific</td>
			<td>BP1423</td>
			<td>0.60 % (wt/vol)</td>
			<td></td>
		</tr>
		<tr>
			<td>Higgins Waterproof Black India Ink</td>
			<td>Higgins</td>
			<td>HIG44201</td>
			<td>0.50 % (vol/vol)</td>
			<td>Mix ink with inoculum to test swarm media surface moisture.</td>
		</tr>
		<tr>
			<td>SYTO&#174; 64 Red Fluorescent Nucleic Acid Stain</td>
			<td>Invitrogen</td>
			<td>S-11346</td>
			<td></td>
			<td>Use 4 &#956;l (for P. aeruginosa) or 8 &#181;l (for M. xanthus) of SYTO&#174; 64 per 100 ml of molten agar (added after autoclaving).</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Relevant Materials and Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, sterile, 150 mm x 15 mm (Dia.x H)</td>
			<td>VWR</td>
			<td>25384-326</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, sterile, 100 mm x 15 mm (Dia.x H)</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, sterile, 60 mm x 15 mm (Dia.x H)</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>In-Vivo Xtream</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
			<td>Use for the macroscopic imaging of surface motility studies. http://www.bruker.com/products/preclinical-imaging/opticalx-ray-imaging/in-vivo-xtreme/overview.html</td>
		</tr>
		<tr>
			<td>Bruker MI software</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
			<td>http://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/PreclinicalImaging/Brochures/MI-software-brochure.pdf</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td></td>
			<td></td>
			<td>http://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td colspan="2">aSee MSDS of reagents for handeling and disposal information.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Variation in plate preparation can greatly influence swarming motility. The curing or drying time after pouring of melted agar medium affects the thin liquid film present on surface motility assays and the bacterial motility over time. Changes in nutrient composition also affect swarming for several bacteria. Figure 1A shows a short-term effect of drying time upon spreading of India Ink and spreading of an initial inoculum of Bacillus subtilis11. Figure 1B shows the effect of drying time and Figure1C shows the effects of ammonium sulfate [(NH4)2SO4] upon subsequent tendril development by swarming P. aeruginosa5.
Quantifiable data can be obtained from endpoint images of surface motility using multiple imaging strategies. Figure 2 shows representative surface growth results for P. aeruginosa swarming and its associated GFP fluorescence image; B. subtilis swarming and its associated bioluminescence image; and Myxococcus xanthus surface growth and the associated red fluorescence image of SYTO 64-stained cells.
Expansion of data acquisition beyond just inspection and imaging of end-point results allows for the study of dynamic behavior(s) for surface growing bacteria. Figure 37 shows an example of P. aeruginosa swarming (imaged for GFP expressing cells) and its associated rhamnolipid production (imaged using Nile Red lipid stain)&#8212;the quantification of data from these images is also displayed to show the expansion rate of P. aeruginosa swarming. Video 1 shows a time-lapse of B. subtilis swarming imaged using luminescence of a lux-expressing strain. Video 28 shows a time-lapse of P. aeruginosa (green&#8212;expressing GFP) and Salmonella enterica serovar Typhimurium (red&#8212;expressing lux) in a competitive swarm assay.
<img alt="Figure 1" src="/files/ftp_upload/52338/52338fig1.jpg" /> 
Figure 1: Examples of factors in surface motility assay preparation that affect assay outcome. Effect of (A) agar drying time on agar surface moisture and spreading of inoculum for B. subtilis (Ref8), (B) agar drying time on P. aeruginosa swarming (Reprinted from Ref5 with permission), and (C) presence or absence of ammonium sulfate on P. aeruginosa swarming and tendril formation.
<img alt="Figure 2" src="/files/ftp_upload/52338/52338fig2.jpg" /> 
Figure 2: Alternative approaches for imaging surface growth and motility of bacteria using a Bruker imaging station. Side by side image of a camera (left) and Bruker image (right) showing (A) P. aeruginosa expressing GFP&#8212;imaged using Green Fluorescence settings, (B) B. subtilis expressing lux bioluminescence reporter&#8212;imaged using Luminescence settings, and (C) M. xanthus stained with SYTO 64&#8212;imaged using Red Fluorescence II settings. See Table 2 for setting details.
<img alt="Figure 3" src="/files/ftp_upload/52338/52338fig3.jpg" /> 
Figure 3: Qualitative and quantitative analysis of a surface motility assay. (A) Time-lapse analysis of cell density distribution, rhamnolipid production (Nile Red lipid stain imaged using the Red Fluorescence I settings; scale bar&#160;= 15&#160;mm), and (B) quantification of the of expansion rate from cell density distribution images of a P. aeruginosa swarm. (Reprinted from Ref6 with permission.)
<a href="https://www.jove.com/files/ftp_upload/52338/52338vid1.mov" target="_blank">Video 1.</a> Time-lapse imaging of a B. subtilis swarm. B. subtilis expressing lux and recorded using the Luminescence settings. See Table 2 for setting details.
<a href="https://www.jove.com/files/ftp_upload/52338/52338vid2.mov" target="_blank">Video 2.</a> Interspecies competition visualized by time-lapse imaging. Swarms of P. aeruginosa (green; expressing GFP and recorded using the Green Fluorescence settings) and S. enterica serovar Typhimurium (red; expressing lux and recorded using the Luminescence settings). See Table 2 for setting details. (Reprint with permission from Ref7.)
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>P. aeruginosa&#160;</td>
			<td>P. aeruginosa tendril formation studies</td>
			<td>B. subtilis</td>
			<td>M. xanthus</td>
		</tr>
		<tr>
			<td>Overnight broth culture media</td>
			<td>FAB plus 30 mM Glucose</td>
			<td>FAB plus 30 mM Glucose</td>
			<td>LB</td>
			<td>CTT</td>
		</tr>
		<tr>
			<td>Overnight broth culture incubation temperature</td>
			<td>37 &#176;C</td>
			<td>37 &#176;C</td>
			<td>37 &#176;C</td>
			<td>30 hr at 30 &#176;C</td>
		</tr>
		<tr>
			<td>Swarm media</td>
			<td>FAB</td>
			<td>FAB minus (NH4)2SO4</td>
			<td>2% (wt/vol) LB</td>
			<td>CTT</td>
		</tr>
		<tr>
			<td>Swarm media: additional components</td>
			<td>12 mM Glucosea&#160;</td>
			<td>10% (wt/vol) CAA, 12 mM Glucosea&#160;</td>
			<td>n/a</td>
			<td>SYTO&#174; 64a</td>
		</tr>
		<tr>
			<td>Agar type</td>
			<td>Agar, Noble</td>
			<td>Agar, Noble</td>
			<td>Granulated agar</td>
			<td>Agar, Noble Affymetrix</td>
		</tr>
		<tr>
			<td>Agar concentration (wt/vol)</td>
			<td>0.45%</td>
			<td>0.45%</td>
			<td>0.60%</td>
			<td>1.50%</td>
		</tr>
		<tr>
			<td>Swarm plate size</td>
			<td>60 mm</td>
			<td>60 mm</td>
			<td>100 mm</td>
			<td>150 mm</td>
		</tr>
		<tr>
			<td>Media volume per plate</td>
			<td>7.5 ml</td>
			<td>7.5 ml</td>
			<td>Hand Poured</td>
			<td>Hand Poured</td>
		</tr>
		<tr>
			<td>Swarm media setting/drying method</td>
			<td>Hood; plates uncovered</td>
			<td>Hood; plates uncovered</td>
			<td>Benchtop; plates covered</td>
			<td>Benchtop; plates covered</td>
		</tr>
		<tr>
			<td>Swarm media setting/drying time&#160;</td>
			<td>30 min</td>
			<td>30 min</td>
			<td>Overnight (20 -24 hr)</td>
			<td>Overnight (20 -24 hr)</td>
		</tr>
		<tr>
			<td>Swarm assay incubation temperature</td>
			<td>30 or 37 &#176;C</td>
			<td>30 &#176;C</td>
			<td>37 &#176;C</td>
			<td>30 &#176;C</td>
		</tr>
		<tr>
			<td>Incubation for time lapse imaging&#160;</td>
			<td>30 &#176;C for at least 4 hr</td>
			<td>30 &#176;C for at least 4 hr</td>
			<td>37 &#176;C for 2 hr</td>
			<td>RT for 12 hr</td>
		</tr>
		<tr>
			<td>Time-lapse capture length</td>
			<td>24 hr</td>
			<td>24 hr</td>
			<td>10 hr</td>
			<td>66 hr</td>
		</tr>
		<tr>
			<td>Time-lapse setting</td>
			<td>1 frame/10 min</td>
			<td>1 frame/10 min</td>
			<td>1 frame/6 min</td>
			<td>1 frame/10 min</td>
		</tr>
		<tr>
			<td colspan="3">aAdded after autoclaving.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Specifications for Surface Motility Assay Preparation. Includes surface motility assay preparation specifications for P. aeruginosa, B. subtilis, and M. xanthus.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Signal</td>
			<td>Green Fluorescence</td>
			<td>Red Fluorescence I&#160;</td>
			<td>Red Fluorescence II</td>
			<td>Luminescence</td>
		</tr>
		<tr>
			<td>Protein or dye</td>
			<td>Green Fluorescence Protein (GFP)</td>
			<td>mCherry protein or Nile Red rhamnolipid stain</td>
			<td>SYTO&#174; 64</td>
			<td>Luciferase from lux operon</td>
		</tr>
		<tr>
			<td>Excitation wavelength (nm)</td>
			<td>480 &#177; 10</td>
			<td>540 &#177; 10</td>
			<td>590 &#177; 10</td>
			<td>Off</td>
		</tr>
		<tr>
			<td>Emission wavelength (nm)</td>
			<td>535 &#177; 17.5</td>
			<td>600 &#177; 17.5</td>
			<td>670 &#177; 17.5</td>
			<td>No filter</td>
		</tr>
		<tr>
			<td>Exposure time (sec)</td>
			<td>30</td>
			<td>60</td>
			<td>60</td>
			<td>240</td>
		</tr>
		<tr>
			<td>f-stop</td>
			<td>4.0</td>
			<td>4.0</td>
			<td>2.5</td>
			<td>1.1</td>
		</tr>
		<tr>
			<td>FOV (mm)</td>
			<td>190</td>
			<td>190</td>
			<td>140</td>
			<td>120</td>
		</tr>
		<tr>
			<td>Focal plane (mm)</td>
			<td>27.5</td>
			<td>27.5</td>
			<td>12.2</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Binning (pixels)</td>
			<td>None&#160;</td>
			<td>2 x 2&#160;</td>
			<td>None</td>
			<td>8 x 8</td>
		</tr>
	</tbody>
</table>
Table 2: Imaging Specification. Bruker imaging station specifications for red and green fluorescence, and luminescence imaging of bacterial surface growth.

Watch this Scientific Journal Video about Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays at JoVE.com

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

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 ...

preparation-imaging-quantification-bacterial-surface-motility

Research

JoVE Journal

Biology

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Gastrointestinal Motility Monitor GIMM

Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

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 ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

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.

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 ...

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...