The development of this technique was supported by the funds from the Ministry of Science and Technology&#39;s National Key R&#38;D plan (2018YFA0902604), National Natural Science Foundation of China&#39;s Research Fund for International Young Scientists (22050410270) and Shenzhen Institutes of Advanced Technology External Funds (DWKF20190001). We would like to offer our sincere gratitude to Miss Chen Xinyi for her assistance in proofreading the document and laboratory management.

1. Preparation of gradient swarm plates
<ol>
	<li>Preparation of swarm medium 
	NOTE: See the discussion section for a brief comparison of different medium viscosities; 0.7% (w/v) agar concentration of swarm medium was used in this protocol.
	<ol>
		<li>Prepare Lysogeny broth (LB) powder with agar in two conical flasks; each flask contains 2 g of tryptone, 2 g of sodium chloride, 1 g of yeast extract, and 1.4 g of agar. Add double-distilled water (ddH2O) and stir the suspension using a magnetic stir bar. Adjust the final volume to 200 mL by adding additional ddH2O.</li>
		<li>Autoclave the solution at 121 &#176;C for 20 min. Use an air-permeable cap or bottle sealing film with an air vent. 
		NOTE: Agar will dissolve when heated in the autoclave.</li>
		<li>When the temperature drops to 65 &#176;C, mix the solution to ensure homogeneity, and transfer the medium to a 65 &#176;C incubator or water bath for short-term usage.</li>
	</ol>
	</li>
	<li>Preparation of bottom-layer swarm medium 
	NOTE: The bottom-layer medium is the mixture of swarm medium and inducer stock solutions. The formulation of inducer gradient swarm plates is shown in Table 1.
	<ol>
		<li>Prepare a 100 mM resveratrol stock solution by dissolving 114.12 mg of lyophilized resveratrol powder into 5 mL of dimethyl sulfoxide (DMSO), and store the solution at -20 &#176;C.</li>
		<li>Prepare a 20% (w/v) arabinose stock solution by dissolving 6 g of arabinose powder in 30 mL of ddH2O; wait for 10-15 min to allow the arabinose to dissolve; and store the solution at room temperature.</li>
		<li>Take out the medium from the 65 &#176;C incubator and place it at room temperature; allow the swarm medium to cool until the Erlenmeyer flask is cool enough to hold (~50 &#176;C). Do not place the swarm medium at room temperature for long periods, as this will cause the solidification of agar.</li>
		<li>Add the required volume of the inducer stock solution to the swarm medium at 50 &#176;C (Table 1). Use a pipette to dispense the inducer solution instead of pouring it. Gently swirl to mix the inducer with the swarm medium. 
		NOTE: This step is for inducers that cannot be autoclaved. Be careful not to introduce bubbles into the medium.</li>
	</ol>
	</li>
	<li>Preparation of double-layer gradient swarm plates 
	NOTE: The upper-layer medium is LB medium containing 0.7% (w/v) agar.
	<ol>
		<li>Label 13 x 13 cm square Petri dishes with inducer name and strains, and prop the dishes up over the edge of the lids (Figure 1B).</li>
		<li>Add 40 mL warm bottom-layer medium (50 &#176;C) using a 50 mL pipette or a 50 mL centrifuge tube. 
		NOTE: Alternatively, for 13 x 13 cm square Petri dishes, 40 mL bottom-layer and upper-layer medium is suitable; for 10 x 10 cm square Petri dishes, 25 mL bottom layer and upper-layer medium is suitable.</li>
		<li>Allow the bottom-layer medium to cure uncovered for 1 h inside a laminar flow hood. Do not disturb square Petri dishes while the medium solidifies. 
		NOTE: While curing the bottom layer, swarm medium not containing inducers should be maintained in a 65 &#176;C incubator or water bath.</li>
		<li>Once the bottom layer is completely solidified, remove the lids and lay the square Petri dishes inside a laminar flow hood.</li>
		<li>Add 40 mL of warm upper-layer medium (50 &#176;C) using a 50 mL pipette or 50 mL centrifuge tube. 
		NOTE: The upper-layer medium does not contain inducers.</li>
		<li>Cure the double-layer plates on the benchtop covered and undisturbed for 1 h. Store the prepared plates at 4 &#176;C for up to 24 h. 
		&#8203;NOTE: Longer curing times would reduce the moisture content and restrict swarming motility.</li>
	</ol>
	</li>
</ol>
2. Growth of E. coli K12 and P. aeruginosa PAO1
<ol>
	<li>Prepare 500 mL of LB medium by adding 5 g of tryptone, 5 g of NaCl, and 2.5 g of yeast extract into ddH2O, and top up the solution to 500 mL. Autoclave the solution on liquid cycle for 20 min at 121 &#176;C, and store it at 4 &#176;C.</li>
	<li>Prepare 100 mL of 1.5% (w/v) LB-agar medium by adding 1 g of tryptone, 1 g of NaCl, 0.5 g of yeast extract, and 1.5 g of agar into ddH2O and top up the solution to 100 mL. Autoclave the solution on liquid cycle for 20 min at 121 &#176;C. Transfer the medium to a 50 &#176;C water bath to prevent the agar from solidifying.</li>
	<li>When the LB-agar medium flask is comfortable to hold, add 20 mL of LB-agar medium into a Petri dish (10 cm in diameter) using a 25 mL pipette. Leave the plate at room temperature overnight, and store the LB-agar plate at 4 &#176;C.</li>
	<li>Take stock cultures stored at -80 &#176;C, streak E. coli K12, E. coli K12-YdeH, P. aeruginosa PAO1, and P. aeruginosa PAO1-YdeH strains on LB-agar Petri dishes using disposable inoculation loops. Incubate the Petri dishes inverted overnight at 37 &#176;C.</li>
	<li>Pick single colonies for different strains from the Petri dishes, inoculate each colony into 5 mL of LB medium, and incubate the culture at 37 &#176;C in a laboratory orbital shaker set at 220 rpm.</li>
	<li>When the culture density reaches OD600nm ~1.0, remove the culture from the shaker and place it at room temperature. Adjust the culture density to OD600nm = 1.0, as described in step 3.2.1.</li>
</ol>
3. Inoculation and incubation of gradient swarm plates
<ol>
	<li>Preparation of inoculation wells 
	NOTE: 3D printing cover models capable of generating wells separated by a standard distance can be used instead of the method described below (Supplemental Figure S1).
	<ol>
		<li>Mark the well positions on A4 paper shown in Figure 1C. Set three test concentrations in one square Petri dish with two or three replicates.</li>
		<li>Place the marked A4 paper under a solidified gradient plate. Push the broader side of a 100 &#181;L pipette tip into the semisolid medium surface at the marked position. Press the pipette tip until it reaches the bottom of the upper-layer medium.</li>
		<li>When the tip touches the bottom, apply no vertical force to the tip; gently rotate the tip to isolate the content of the cylindrical well.</li>
		<li>Horizontally move the pipette tips along a very small distance to allow airflow into the narrow place set aside. Press the tip with the index finger to block the gas flow inside the tip while holding the pipette using the thumb and middle finger.</li>
		<li>Pull the tip out vertically, keeping the well content in the tip while pulling it out. 
		NOTE: If the well content slips, apply slightly more pressure with the index finger to completely seal the tip.</li>
		<li>Repeat steps 3.1.2 to 3.1.5 in every marked position. Cover the swarm plate before inoculation.</li>
	</ol>
	</li>
	<li>Gradient plate inoculation and incubation
	<ol>
		<li>Adjust the overnight growth culture density to OD600nm = 1.0.</li>
		<li>Pipette 80 &#181;L of the overnight growth culture into every well. Do not spill the bacterial culture outside the wells.</li>
		<li>Wrap the plates with sealing film. For long-term observation (3-5 days), wrap the plates with sterile laboratory rubber tape. 
		NOTE: Rubber tape is less likely to break.</li>
		<li>Place a beaker filled with ddH2O in the incubator to maintain humidity inside the incubator. Incubate the gradient swarm plates at 37 &#176;C. 
		NOTE: Do not incubate the swarm plates upside-down; this will cause the bacterial culture to leak from the wells.</li>
		<li>Image the swarm plate immediately after inoculation, recording this as the 0 h time point.</li>
	</ol>
	</li>
</ol>
4. Imaging bacterial surface swarming
<ol>
	<li>Take the swarm plates out, one at a time, from the incubator every 12 h, holding the plate horizontally, and place them in the gel imaging system (see the Table of Materials). 
	NOTE: Do not leave fingerprints on the surface of plates; hold the side of the swarm plate with clean gloves.</li>
	<li>Select gel imaging mode; expose the swarm plate to white light; and adjust the focal length to give the clearest view of swarms. 
	NOTE: Use the same focal length for all plates in a given batch.</li>
	<li>Enhance the brightness of the swarms for clear observation by adjusting the exposure time to 300 ms. Adjust the threshold to minimize interference from the background light. 
	NOTE: Threshold is adjusted on the operating interface of the gel imaging system. Increase values on the left to minimize interference from background light; decrease values on the right to enhance the brightness of the swarms. In this protocol, the region is usually between 6,000 and&#160;50,000.</li>
	<li>Save the image file for further analysis. Record the imaging time, inducer type, gradient orientation, and strains in a .txt file.</li>
</ol>
5. Quantify the swarm area using ImageJ software
<ol>
	<li>Import the image file acquired using the gel imaging system.</li>
	<li>Set the scale bar using ImageJ software and apply it to all the images.
	<ol>
		<li>Create a line segment marking the length of the board by clicking on the line tool.</li>
		<li>Click Analyze | Set Scale to open the Set Scale window.</li>
		<li>Type the actual length in Known distance and Unit of length. 
		NOTE: Because 13 x 13 cm square Petri dishes were used in this work, the actual length is &#39;130&#39;, and the unit of length is &#39;mm.&#39;</li>
		<li>Check the Global box.</li>
		<li>Insert a scale bar by clicking Analyze | Tools | Scale Bar, type Width in mm, Height in pixels, Font size, and select Color, Background, and Location from the dropdown menu. Alternatively, choose Bold Text, Hide Text, Serif Font, and Overlay by ticking the checkboxes. 
		NOTE: The choice of those parameters is determined by users and the properties of the images. In this protocol, Width in mm was set to 25, Height in pixels to 20, Font size to 80, and the scale bar was placed in the lower right corner by selecting Location | Lower Right. Other parameters can be chosen by the user.</li>
	</ol>
	</li>
	<li>Click Process | Shadows to enhance the sharpness of the image, especially the boundaries (Supplemental Figure S2A). Click Process | Batch to process images. 
	NOTE: The purpose of this step is to provide more precise boundary demarcation.
	<ol>
		<li>Process an image as a reference by clicking on Process | Shadows | South.</li>
		<li>Click Process | Batch | Macro to open the Batch Process window. Look for the following commands displayed in the window: 
		run(&#34;South&#34;); 
		run(&#34;Save&#34;); 
		&#8203;close();</li>
		<li>Type the folder address of the original images and the output file address by clicking Process in the Batch Process window. 
		NOTE: It is recommended to export images with shadows to another folder and retain a copy of the original images.</li>
	</ol>
	</li>
	<li>Use Wand (tracing) tool to select swarms individually and adjust the tolerance (double-click Wand (tracing) tool) until the generated line fits the swarm boundary correctly (Supplemental Figure S2B). 
	NOTE: First, click Wand (tracing) tool and select a swarm on one image. If the boundaries have not been depicted correctly, double-click the Wand (tracing) tool to open the Wand Tool windows, where the Tolerance can be adjusted.</li>
	<li>Click on Analyze | Measure to export the area value.</li>
	<li>Repeat steps 5.1.1-5.1.5 until all swarms are measured, save the results to a .csv file for further analysis.</li>
</ol>

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The authors have no conflicts of interest to disclose.

Investigation of bacterial swarming motility on semisolid gradient plates can be a challenging task18,19,20, as it involves multiple factors such as substrate viscosity, humidity, and medium components. Among these factors, agar concentration plays a decisive role in determining microbial reversion to either swimming or swarming motility. As the agar concentration increases from 0.3% (w/v) to 1% (w/v), bacterial swimming motilit...

Bacterial swarming motility refers to the collective migration of bacterial cells across the surface of a substance. In addition to semisolid agar plates specially prepared in the laboratory1, this phenotype is also observed on some soft substrates such as animal tissues2, hydrated surfaces3, and plant roots4. While a semisolid surface is considered one of the fundamental conditions for bacterial swarming, some species also require an energy-rich medium to support their swarming motility5. Flagella rotation powers both swimming and swarming motility-swimming describes the unicellular motility within a liquid environment, whereas swarming is the synchronous movement of a microbial population across semisolid surfaces.
Substrate viscosity influences bacterial motility; studies of pathogenic microbes, such as Helicobacter pylori,&#160;have shown that the pathogen&#39;s motility changes depending on the mucin layer viscosity, which is influenced by environmental acidification in the human host6. To replicate these environments, earlier studies using agar concentration above 0.3% (w/v) restrict bacterial swimming motility to effect a gradual shift into surface swarming. The use of agar concentration above 1% (w/v) prevents the swarming motility of many species7. The colony patterns formed on the surface are diverse, including featureless mat8, bull&#39;s eye9, dendrites10, and vortex11.
Although the relevance of such patterns remains unclear, those patterns seem to be dependent on environmental and chemical cues12. Environmental cues cover different aspects, including temperature, salinity, light, and pH, whereas chemical cues include the presence of microbial quorum sensing molecules, biochemical byproducts, and nutrients. Autoinducer quorum sensing signaling molecules such as AHL (N-hexanoyl-L homoserine lactone) can impact surface swarming by regulating the production of surfactant13,14. Resveratrol, a phytoalexin compound, could restrict bacterial swarming motility15.
In the present work, we investigate the effect of gradient concentrations of resveratrol on wild-type Escherichia coli K12 strain and investigate arabinose-inducible swarming motility of engineered E. coli K12-YdeH and Pseudomonas aeruginosa PAO1-YdeH species. The production of the YdeH enzyme is induced by arabinose via the araBAD promoter, resulting in cellular c-di-GMP perturbation and affecting bacterial swarming motility16,17. This inducible swarming behavior is studied using arabinose gradient swarm plates with E. coli K12-YdeH and P. aeruginosa PAO1-YdeH strains.
The gradient swarm plates are prepared by successively solidifying double-layer medium (Figure 1B). The bottom layer comprises the medium added with the inducer, poured on one side of a propped-up Petri dish. Upon the solidification of the bottom layer, the Petri dish is returned to a flat surface, where the upper layer containing the medium without the inducer is added from the other side of the plate. After the swarm plates are completely solidified, isometrically arranged holding-wells are produced by boring holes on the swarm plates following a fixed layout (Figure 1C) or by imprinting the wells using a 3D printed model of the plate cover containing pegs during the medium curing process (Supplemental Figure S1). A gel imaging system is used to capture the swarming morphologies at different time points (Figure 2). Analysis of surface swarming using ImageJ software (Supplemental Figure S2) provides quantitative results of the surface swarming process (Figure 3).
Thus, we propose a simple method to test surface swarming motility within a concentration range of inducers. This method can be used to test multiple concentration responses of other inducers by mixing the inducer into the bottom-layer medium and can be applied to other motile species (e.g., Bacillus subtilis, Salmonella enterica, Proteus mirabilis, Yersinia enterocolitica). This approach could provide reliable qualitative and quantitative results for screening a single chemical inducer, and separate plates may be employed to evaluate more chemical inducers.

<table><tbody><tr><td>Agar</td><td>Sigma-Aldrich</td><td>V900500</td><td>500 g</td></tr><tr><td>Ampicillin</td><td>Solarbio</td><td>A8180</td><td>25 g, &amp;ge; 85% (GC)</td></tr><tr><td>Centrifuge tube</td><td>Corning</td><td>430790</td><td>15 mL</td></tr><tr><td>Cryogenic vial</td><td>Corning</td><td>430488</td><td>2 mL</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Aladdin</td><td>D103272</td><td>AR, &gt; 99% (GC)</td></tr><tr><td>L(+)-Arabinose</td><td>Aladdin</td><td>A106195</td><td>98% (GC), 500 g</td></tr><tr><td>Petri dishes</td><td>Bkman</td><td>B-SLPYM90-15</td><td>Plastic Petri dishes,circular,90 mm x 15 mm</td></tr><tr><td>Resveratrol</td><td>Aladdin</td><td>R107315</td><td>99% (GC), 25 g</td></tr><tr><td>Sodium chloride</td><td>Macklin</td><td>S805275</td><td>AR, 99.5% (GC), 500 g</td></tr><tr><td>Square Petri dishes</td><td>Bkman</td><td>B-SLPYM130F</td><td>Plastic Petri dishes, square, 13 mm x 13 mm</td></tr><tr><td>Tryptone</td><td>Thermo Scientific Oxoid</td><td>LP0042</td><td>500 g</td></tr><tr><td>Yeast extract</td><td>Thermo Scientific Oxoid</td><td>LP0021</td><td>500 g</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biochemical incubator</td><td>Blue pard</td><td>LRH-70</td><td></td></tr><tr><td>Tanon 5200multi imaging system</td><td>Tanon</td><td>5200CE</td><td></td></tr><tr><td>Thermostatic water bath</td><td>Jinghong</td><td>DK-S28</td><td></td></tr></tbody></table>

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.

The workflow consisting of the preparation of gradient swarm plates, inoculation, and incubation is shown in Figure 1B. To generate gradient swam plates, the bottom-layer medium is poured into propped-up dishes at ~4.3&#176; from the horizontal plane (Supplemental Figure S3), followed by pouring the upper-layer medium after the bottom layer is completely solidified. The composition of the double-layer medium is shown in Table 1. Then, bacterial culture cultu...

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Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

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 ...

quantifying-bacterial-surface-swarming-motility-on-inducer-gradient

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

3.4K Views.  Southern University of Science and Technology. Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses. 

Video: Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

Genetics

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Biology

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Immunology and Infection

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 ...

Biophysical Characterization of Flagellar Motor Functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

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 ...

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

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 ...

Visualizing Bacterial Motility Based on a Color Reaction

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or dissemination of many pathogenic species. Therefore, it is important to detect bacterial motility. Bacterial growth conditions, such as oxygen, pH, and temperature, can affect bacterial growth and the expression of bacterial flagella. This can lead to reduced motility or even loss of motility, resulting in the inaccurate evaluation of bacterial motility. Based on the color reaction of 2,3,5-triphenyl tetrazolium chloride (TTC) by intracellular dehydrogenases of living bacteria, TTC was added to traditional semisolid agar for bacterial motility detection. The results showed that this TTC semisolid agar method for the detection of bacterial motility is simple, easy to operate, and does not involve large and expensive instruments. The results also showed that the highest motility was observed in semisolid medium prepared with 0.3% agar. Compared with the traditional semisolid medium, the results are easier to evaluate and more accurate.

Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome

The intestines of animals are colonized by commensal microbes, which impact host development, health, and behavior. Precise quantification of colonization is essential for studying the complex interactions between host and microbe both to validate the microbial composition and study its effects. Drosophila melanogaster,&#160;which has a low native microbial diversity and is economical to rear with defined microbial composition, has emerged as a model organism for studying the gut microbiome. Analyzing the microbiome of an individual organism requires identification of which microbial species are present and quantification of their absolute abundance. This article presents a method for the analysis of a large number of individual fly microbiomes. The flies are prepared in 96-well plates, enabling the handling of a large number of samples at once. Microbial abundance is quantified by plating up to 96 whole fly homogenates on a single agar plate in an array of spots and then counting the colony forming units (CFUs) that grow in each spot. This plating system is paired with an automated CFU quantification platform, which incorporates photography of the plates, differentiation of fluorescent colonies, and automated counting of the colonies using an ImageJ plugin. Advantages are that (i) this method is sensitive enough to detect differences between treatments, (ii) the spot plating method is as accurate as traditional plating methods, and (iii) the automated counting process is accurate and faster than manual counting. The workflow presented here enables high-throughput quantification of CFUs in a large number of replicates and can be applied to other microbiology study systems including in vitro and other small animal models.

Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome

This method quantifies microbial abundance using a 96-well plate format to plate colony forming units (CFUs) and is applied to the Drosophila microbiome in whole fly homogenate samples. CFUs are counted with an automated image analysis software provided here.

The intestines of animals are colonized by commensal microbes, which impact host development, health, and behavior. Precise quantification of colonization ...

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Summary

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Chapters in this video

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Biophysical Characterization of Flagellar Motor Functions

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

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Visualizing Bacterial Motility Based on a Color Reaction

Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome