This work was supported by the Max Planck Society.

1. Preparation of Electro-competent V. parahaemolyticus Cells and Electroporation of Plasmid DNA into Swimmer Cells
NOTE: The following section of the protocol allows for the preparation of electro-competent cells and the subsequent electroporation of plasmid DNA into swimmer cells. This step is important if fluorescent proteins are being ectopically expressed from a plasmid.
<ol>
	<li>Streak out V. parahaemolyticus cells from a -80 &#176;C glycerol stock onto a fresh LB ag...

<ol>
	<li>McCarter, L., Hilmen, M., Silverman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+dynamometer+controls+swarmer+cell+differentiation+of+V.+parahaemolyticus.">Flagellar dynamometer controls swarmer cell differentiation of V. parahaemolyticus.</a> Cell. 54 (3), 345-351 (1988).</li><li>Kawagishi, I., Imagawa, M., Imae, Y., McCarter, L., Homma, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sodium-driven+polar+flagellar+motor+of+marine+Vibrio+as+the+mechanosensor+that+regulates+lateral+flagellar+expression.">The sodium-driven polar flagellar motor of marine Vibrio as the mechanosensor that regulates lateral flagellar expression.</a> Mol. Microbiol. 20 (4), 693-699 (1996).</li><li>Ulitzur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+swarming+in+Vibrio+parahaemolyticus.">Induction of swarming in Vibrio parahaemolyticus.</a> Arch. Microbiol. 101 (4), 357-363 (1974).</li><li>Ulitzur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+temperature,+salts,+pH,+and+other+factors+on+the+development+of+peritrichous+flagella+in+Vibrio+alginolyticus.">Effect of temperature, salts, pH, and other factors on the development of peritrichous flagella in Vibrio alginolyticus.</a> Arch. Microbiol. 104 (3), 285-288 (1975).</li><li>Ulitzur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+swarming+of+Vibrio+alginolyticus.">The mechanism of swarming of Vibrio alginolyticus.</a> Arch. Microbiol. 104 (1), 67-71 (1975).</li><li>Gode-Potratz, C. J., Chodur, D. M., McCarter, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+iron+regulate+swarming+and+type+III+secretion+in+Vibrio+parahaemolyticus.">Calcium and iron regulate swarming and type III secretion in Vibrio parahaemolyticus.</a> J. Bacteriol. 192 (22), 6025-6038 (2010).</li><li>McCarter, L., Silverman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+regulation+of+swarmer+cell+differentiation+of+Vibrio+parahaemolyticus.">Iron regulation of swarmer cell differentiation of Vibrio parahaemolyticus.</a> J. Bacteriol. 171 (2), 731-736 (1989).</li><li>Gode-Potratz, C. J., Kustusch, R. J., Breheny, P. J., Weiss, D. S., McCarter, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+sensing+in+Vibrio+parahaemolyticus+triggers+a+programme+of+gene+expression+that+promotes+colonization+and+virulence.">Surface sensing in Vibrio parahaemolyticus triggers a programme of gene expression that promotes colonization and virulence.</a> Mol. Microbiol. 79 (1), 240-263 (2011).</li><li>Heering, J., Ringgaard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Localization+of+Chemotactic+Signaling+Arrays+during+the+Lifecycle+of+Vibrio+parahaemolyticus.">Differential Localization of Chemotactic Signaling Arrays during the Lifecycle of Vibrio parahaemolyticus.</a> Front Microbiol. 7 (November), 1-12 (2016).</li><li>Cameron, T. A., Anderson-Furgeson, J., Zupan, J. R., Zik, J. J., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan+synthesis+machinery+in+Agrobacterium+tumefaciens+during+unipolar+growth+and+cell+division.">Peptidoglycan synthesis machinery in Agrobacterium tumefaciens during unipolar growth and cell division.</a> mBio. 5 (3), (2014).</li><li>Development Core Team, R. F. F. S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R:+A+Language+and+Environment+for+Statistical+Computing.">R: A Language and Environment for Statistical Computing.</a> R Foundation for Statistical Computing. 1, 2673 (2008).</li><li>Ringgaard, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ParP+prevents+dissociation+of+CheA+from+chemotactic+signaling+arrays+and+tethers+them+to+a+polar+anchor.">ParP prevents dissociation of CheA from chemotactic signaling arrays and tethers them to a polar anchor.</a> Proc Natl Acad Sci U S A. 111 (2), E255-E264 (2014).</li><li>Ringgaard, S., Schirner, K., Davis, B. M., Waldor, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+ParA-like+ATPases+promotes+cell+pole+maturation+by+facilitating+polar+localization+of+chemotaxis+proteins.">A family of ParA-like ATPases promotes cell pole maturation by facilitating polar localization of chemotaxis proteins.</a> Genes Dev. 25, 1544-1555 (2011).</li><li>Vischer, N. O. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+age+dependent+concentration+of+Escherichia+coli+divisome+proteins+analyzed+with+ImageJ+and+ObjectJ.">Cell age dependent concentration of Escherichia coli divisome proteins analyzed with ImageJ and ObjectJ.</a> Front Microbiol. 6 (JUN), (2015).</li><li>Schneider, C. a., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9 (7), 671-675 (2012).</li><li>Sliusarenko, O., Heinritz, J., Emonet, T., Jacobs-Wagner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput,+subpixel+precision+analysis+of+bacterial+morphogenesis+and+intracellular+spatio-temporal+dynamics.">High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics.</a> Mol. Microbiol. 80 (3), 612-627 (2011).</li><li>Ducret, A., Quardokus, E. M., Brun, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicrobeJ,+a+tool+for+high+throughput+bacterial+cell+detection+and+quantitative+analysis.">MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis.</a> Nat. Microbiol. 1, 16077 (2016).</li><li>Carpenter, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biol. 7 (10), R100 (2006).</li><li>Morales-Soto, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+Imaging,+and+Quantification+of+Bacterial+Surface+Motility+Assays.">Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays.</a> J. Vis. Exp. (98), e52338 (2015).</li><li>Paintdakhi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oufti:+An+integrated+software+package+for+high-accuracy,+high-throughput+quantitative+microscopy+analysis.">Oufti: An integrated software package for high-accuracy, high-throughput quantitative microscopy analysis.</a> Mol. Microbiol. 99 (4), 767-777 (2016).</li><li>de Jong, I. G., Beilharz, K., Kuipers, O. P., Veening, J. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Cell+Imaging+of+Bacillus+subtilis+and+Streptococcus+pneumoniae+using+Automated+Time-lapse+Microscopy.">Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy.</a> J. Vis. Exp. (53), e1-e6 (2011).</li></ol>

This paper reports a method for microscopy imaging of V. parahaemolyticus swarmer cells, followed by downstream analyses intended to resolve and quantify the subcellular localization and other features of the studied fluorescently labeled proteins. Although several tools exist for microscopy image processing and analysis14,15,16,17,18, the analysis of...

Bacteria constantly experience changes to their external environment and have developed several techniques to change and adapt their behavior accordingly. One such mechanism involves differentiation into distinct cell types that better complement the altered environment. Differentiation often involves major changes in the regulation of the cell cycle, cell morphology, and the spatiotemporal organization of the cells. One organism that can undergo differentiation is Vibrio parahaemolyticus. V. parahaemolyticus belongs to the Vibrionaceae, whichis a family of proteobacteria that usually inhabit fresh or salt water. Vibrionaceae are widely distributed in the environment and include several species that cause intestinal tract infections in humans, also including Vibrio cholerae.V. parahaemolyticus is a dimorphic organism and is able to differentiate into two distinct cell types as a response to accommodate changes to its external milieu. In aqueous environments, it exists as a short rod-shaped swimmer cell with a single polar flagellum positioned at the old cell pole. Upon surface contact, differentiation into a swarmer cell is triggered. Swarmer cell differentiation involves two major changes: swarmer cell morphogenesis through inhibition of cell division, and the induction of a second flagella system. This results in the formation of a peritrichous and highly elongated rod-shaped swarmer cell, which can either continue the swarmer life-style, where division events results in progeny swarmer cells, or alternatively differentiate back into swimmer cells.
Several factors have been reported to induce or influence swarmer differentiation. The primary stimulus appears to be driven by mechanosensing where V. parahaemolyticus uses the polar flagellum as a tactile sensor that detects inhibition of the rotation upon surface contact, but several other factors are involved as well1. In the lab, rotation of the polar flagellum can be artificially inhibited by addition of phenamil, which blocks the sodium-channel driven flagellar rotation, thereby inducing swarmer differentiation2. Furthermore, in an earlier study, Vibrio alginolyticus was induced to swarm on solid media when cells were propagated on growth medium in a Petri dish sealed with clear plastic tape. Alkali-saturated filter paper prevented swarming under these conditions, hence suggesting that one or more volatile acids might be involved in induction of swarming. Thus, sealing the Petri dish with plastic tape likely allowed for the accumulation of volatile acids, formed as by-products of cellular metabolism, within the head space of the plate. The same effect was achieved when H2O2 was added to the growth medium in un-sealed Petri dishes in order to artificially produce volatile acids by hydrolysing media components3,4,5. Nevertheless, the identity of such volatile acids remains unknown. Moreover, it has been shown that excess availability of calcium6 and iron-limitation7 both enhance swarmer differentiation and proliferation over solid surfaces. Cells can be starved for iron by adding the compound 2,2&#180;-Bipyridyl to the growth medium, which has been shown to influence swarmer differentiation8. The factors known to regulate differentiation are now implemented in the design of a protocol that reproducibly induces V. parahaemolyticus differentiation into swarmer cells and their proliferation on solid agar surfaces.
V. parahaemolyticus differentiation involves major changes in the regulation of cell division, cellular morphology, and the positioning of macromolecular machines such as flagella and chemotaxis apparatuses &#8211; processes that all require the specific localization of proteins in accordance with the cell cycle. Thus, the ability to study the intracellular localization of such proteins is essential to the understanding of the aforementioned cellular processes. In order to perform such studies fluorescence microscopy on single cells is required. This can be particularly challenging when imaging cells that exist within dense bacterial populations, as is the case for swarmer cells. There have been attempts to induce swarmer differentiation in liquid media, which could potentially generate single cells for microscopy studies. And although on a transcriptional level these cells have partially induced the swarmer-differentiation program, they do not undergo the same distinct morphological changes as fully differentiated swarmer cells grown on solid medium8. This paper offers a robust and reproducible protocol of a method to induce swarmer cell differentiation on an agar surface. The protocol produces a population of easily accessible swarmer cells readily available for single cell microscopy and subsequent analysis. Furthermore, the protocol enables localization studies of fluorescently labeled proteins in both the swimmer and swarmer cell-types (sections 1, 2, 3, 4). Additionally, the protocol describes the subsequent workflow on how to process and analyze the generated data from fluorescence microscopy experiments, which allows for demographic analysis of bacterial societies (section 5).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB-medium</td>
			<td>Roth</td>
			<td>X968.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Agar, granulated</td>
			<td>BD</td>
			<td>214510</td>
			<td>For preparation of LB agar with LB-medium</td>
		</tr>
		<tr>
			<td>Difco Heart Infusion Agar</td>
			<td>BD</td>
			<td>244400</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Agarose NEEO, ultra quality</td>
			<td>Roth</td>
			<td>2267.3</td>
			<td>For preparation of microscopy agarose pads</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Additives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-arabinose</td>
			<td>Roth</td>
			<td>5118.3</td>
			<td>Used to induce pBAD derivatives</td>
		</tr>
		<tr>
			<td>2,2`-Bipyridyl</td>
			<td>Sigma Aldrich</td>
			<td>D216305-25G</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Roth</td>
			<td>5239.1</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Roth</td>
			<td>K029.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Roth</td>
			<td>3886.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>-</td>
			<td>-</td>
			<td>Standard recipe for Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>D(+) Sucrose</td>
			<td>Roth</td>
			<td>4621.1</td>
			<td>For preparation of electrocompetent cells</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Roth</td>
			<td>3783.5</td>
			<td>For preparation of electrocompetent cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Hardware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 150x20mm with cams</td>
			<td>Sarstedt</td>
			<td>82.1184.500</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>kelvitron t (B 6420)</td>
			<td>Heraeus</td>
			<td>-</td>
			<td>Used for drying HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Microbial System</td>
			<td>BioRad</td>
			<td>1652662</td>
			<td>Used for elctroporation of plasmid DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph Offline (version 7.7.5.0)</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>Used for microscopy image analysis</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>-</td>
			<td>Used for microscopy data analsis</td>
		</tr>
	</tbody>
</table>

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 differentiation and generation of swarming colonies
Figure 1 provides a schema of the important steps involved in producing swarming colonies of V. parahaemolyticus (Figure 1A-C). A swimmer culture was spotted on swarm-agar and incubated at 24 oC, inducing swarmer differentiation and proliferation over the solid agar surface. A representative stereo-microscopy image...

Watch this Scientific Journal Video about Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells at JoVE.com

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.

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 ...

The overall goal of this method is to induce cellular differentiation of the bacterium Vibrio parahaemolyticus and the formation of a population of differentiated swarmer cells easily available for single cell analysis by fluorescence microscopy. This method will help scientists understand how bacteria regulate the intracellular organization during cellular differentiation and morphogenesis. Since imaging cells within bacterial communities can be challenging, the main advantage of this technique is that it produces a population of differentiated swarmer cells that exist in a single cell layer readily available for single cell microscopy analysis.The implications of this technique extend toward a better understanding of how bacteria regulate changes in the cell cycle, cell morphology, and intracellular organization during cellular differentiation. Demonstration of this method is critical as reliable induction of cellular differentiation and the formation of cells available for single cell analysis are difficult to achieve. To prepare the agar solution for making swarming plates, suspend four grams of heart infusion, or H.I.agar powder, and 100 milliliters of double distilled water.Carefully boil the H.I.agar solution in a microwave oven, shaking the bottle from time to time until the powder is dissolved. Autoclave the agar solution at 121 degrees Celsius for 20 minutes, cool down the agar solution to 60 t0 65 degrees Celsius with constant stirring. The next step involves handling 2, 2'Bipyridyl, which is highly toxic, so gloves and safety goggles must be worn.Add 2, 2'Bipyridyl to a final concentration of 50 micromolar. Add calcium chloride to a final concentration of four millamolar. Inoculate a small amount of V.parahaemolyticus cells from the edge of a single colony into 5 millilters of LB broth.Incubate the culture at 37 degrees Celsius in a shaker incubator until it reaches an optical density at 600 nanometers of 0.8. This usually takes around two to three hours. While the cell culture is growing, pipette 30 to 35 milliliters of the final H.I.agar solution into a round 150 millimeter petri dish, and let the agar solidify.Right before spotting the cell culture, dry the agar plate upright with an open lid at 37 degrees Celsius for at least 10 minutes, or until any liquid residues have disappeared, but no longer. Spot one microliter of cell culture at the center of the H.I.agar swarming plate and let the spot dry. Seal the plate with clear plastic tape, taking care to avoid the formation of air bubbles and folds to the tape.Incubate the plate at 24 degrees Celsius overnight. This will result in the formation of a V.Parahaemolyticus swarm colony with swarm flares extending from the center of the colony, as shown in this time lapse video clip. The first step in this procedure is to prepare agarose slides for microscopy.Add one gram of agarose to 100 milliliters of a 20%PBS and a 10%LB broth solution. Carefully boil the solution in the microwave to dissolve the agarose, and then let it cool down to 65 to 70 degrees Celsius while mixing with a magnetic stir bar. Place a clean microscope slide on to a perfectly level section of a working bench.Fix the slide to the bench by covering both ends of the slide with two layers of general purpose laboratory labeling tape. The distance between the two pieces of tape should be around three centimeters. Pipette about 250 microliters of the previously prepared agarose solution onto the non-covered glass surface.Place a second microscope slide in the same orientation as the bottom slide on top and slightly press down so that the residual agarose can flow out to the sides. Let the agarose solidify for one to two minutes. Use a scalpel to cut off any residual agarose that is attached to the sides of the microscope slide.When the agarose has solidified, slowly pull the top glass slide off of the bottom one in a horizontal movement. Slowly remove the tape from the ends of the microscope slide. The agarose slide should be used as soon as possible, as it will slowly begin to dry out.Immediately before the transfer of swarmer cells to the microscope slide, Use a scalpel to cut the plastic tape off the previously prepared swarmer plate. During the excision of the swarming agar, it is important to direct the cut from the outside to the inside of the swarming colony to avoid contaminating the swarming flares with cells that originate from the middle of the colony. Use a fresh scalpel to cut out a three millimeter by 10 millimeter piece of swarming agar from the most outer edge of a swarming colony, making sure to direct the cuts from the outside of the swarming colony edge to the inside of the colony.Transfer the piece of swarming agar to the microscope agarose slide with the cells facing the agarose pad to imprint the swarming cells onto the agarose pad. After waiting for about 30 seconds, remove the agar piece carefully from the agarose pad. Place a glass coverslip on the agarose pad where the cells were imprinted.The cells are now ready for fluorescence microscopy. Stereo-microscopy imaging of a V.parahaemolyticus swarmer colony in increasing magnification shows swarm flares extending from the periphery of the swarm colony. Cells in the flares are grouped in mono or bilayers only, while cells in the middle of the swarm colony are stacked in multiple layers.DIC microscopy imaging revealed the flares only consist of a fully differentiated population of swarmer cells, while cells from the middle of the swarm colony are significantly shorter, and likely represent a mixture of swimmer and swarmer cells. As proof of principle, DIC and fluorescence microscopy imaging was performed on wild-type V.Parahaemolyticus swimmer and swarmer cells, ectopically expressing an essential chemotaxis protein, YFP-CheW. In swimmer cells, YFP-CheW localizes in a unipolar and bipolar manner, but in swarmer cells, YFP-CheW is always bipolarly localized and also forms clusters positioned randomly along the cell length.Changes in the localization pattern of YFP-CheW during swimmer to swarmer cell differentiation, were also shown by displays of foci distribution and demographic analyses of fluorescence intensity profiles. After its development, this technique paved the way for researchers in the field of microbial cell biology to explore how the bacteria Vibrio parahaemolyticus regulates its internal organization during its distinct differentiated states of swimmer and swarmer cells. After watching this video, you should have a good understanding of how to induce cellular differentiation of the bacteria Vibrio parahaemolyticus and how to prepare a population of differentiated swarmer cells for single cell analysis by fluorescence microscopy.Don't forget that Vibrio parahaemolyticus is a human pathogen and proper procedures and regulations on how to handle BSL 2 pathogens in your region should be applied.

induction-cellular-differentiation-single-cell-imaging-vibrio

Research

JoVE Journal

Immunology and Infection

9.2K Views.  Max Planck Institute for Terrestrial Microbiology. The overall goal of this method is to induce cellular differentiation of the bacterium Vibrio parahaemolyticus and the formation of a population of differentiated swarmer cells easily available for single cell analysis by fluorescence microscopy. This method will help scientists understand how bacteria regulate the intracellular organization during cellular differentiation and morphogenesis. Since imaging cells within bacterial communities can be challenging, the main advantage of this techni...