Name: in situ chemotaxis assay to examine microbial behavior in aquatic ecosystems
Uploaded: 2020-05-05T14:45:00
Description: Watch this Scientific Journal Video about In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems at JoVE.com

This research was funded in part by the Gordon and Betty Moore Foundation Marine Microbiology Initiative, through grant GBMF3801 to J.R.S. and R.S., and an Investigator Award (GBMF3783) to R.S., as well as an Australian Research Council Fellowship (DE160100636) to J.B.R., an award from the Simons Foundation to B.S.L. (594111), and a grant from the Simons Foundation (542395) to R.S. as part of the Principles of Microbial Ecosystems (PriME) Collaborative.

We recommend executing section 1 prior to field experiments to optimize results.
1. Laboratory optimization
NOTE: The volumes described in the optimization procedure are sufficient for a single ISCA (composed of 20 wells).
<ol>
	<li>Preparation of the chemical of interest 
	NOTE: The optimal concentration for each chemoattractant often needs to be determined under laboratory conditions prior to field deployments. The chemical concentration field will decrease...

<ol>
	<li>Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+microbes+see+a+sea+of+gradients.">Marine microbes see a sea of gradients.</a> Science. 338, 628-633 (2012).</li><li>Raina, J. B., Fernandez, V., Lambert, B., Stocker, R., Seymour, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microbial+motility+and+chemotaxis+in+symbiosis.">The role of microbial motility and chemotaxis in symbiosis.</a> Nature Reviews Microbiology. 17, 284-294 (2019).</li><li>Chet, I., Asketh, P., Mitchell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repulsion+of+bacteria+from+marine+surfaces.">Repulsion of bacteria from marine surfaces.</a> Applied Microbiology. 30, 1043-1045 (1975).</li><li>Smriga, S., Fernandez, V. I., Mitchell, J. G., Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+toward+phytoplankton+drives+organic+matter+partitioning+among+marine+bacteria.">Chemotaxis toward phytoplankton drives organic matter partitioning among marine bacteria.</a> PNAS. 113, 1576-1581 (2016).</li><li>Matilla, M., Krell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+bacterial+chemotaxis+on+host+infection+and+pathogenicity.">The effect of bacterial chemotaxis on host infection and pathogenicity.</a> FEMS Microbiology Reviews. 42, (2018).</li><li>Adler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+in+bacteria.">Chemotaxis in bacteria.</a> Science. 153, 708-716 (1966).</li><li>Adler, J., Dahl, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+measuring+the+motility+of+bacteria+and+for+comparing+random+and+non-random+motility.">A method for measuring the motility of bacteria and for comparing random and non-random motility.</a> Journal of General Microbiology. 46, 161-173 (1967).</li><li>Ahmed, T., Shimizu, T. S., Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+bacterial+chemotaxis.">Microfluidics for bacterial chemotaxis.</a> Integrative Biology. 2, 604-629 (2010).</li><li>Hol, F. J. H., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zooming+in+to+see+the+bigger+picture:+microfluidic+and+nanofabrication+tools+to+study+bacteria.">Zooming in to see the bigger picture: microfluidic and nanofabrication tools to study bacteria.</a> Science. 346, 1251821 (2014).</li><li>Rusconi, R., Garren, M., Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+expanding+the+frontiers+of+microbial+ecology.">Microfluidics expanding the frontiers of microbial ecology.</a> Annual Review of Biophysics. 43, 65-91 (2014).</li><li>Lambert, B. 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=A+microfluidics-based+in+situ+chemotaxis+assay+to+study+the+behaviour+of+aquatic+microbial+communities.">A microfluidics-based in situ chemotaxis assay to study the behaviour of aquatic microbial communities.</a> Nature Microbiology. 2, 1344-1349 (2017).</li><li>Marie, D., Partensky, F., Jacquet, S., Vaulot, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+and+cell+cycle+analysis+of+natural+populations+of+marine+picoplankton+by+flow+cytometry+using+the+nucleic+acid+stain+SYBR+Green+I.">Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I.</a> Applied Environmental Microbiology. 63, 186-193 (1997).</li><li>Rinke, C., 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=Obtaining+genomes+from+uncultivated+environmental+microorganisms+using+FACS-based+single-cell+genomics.">Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics.</a> Nature Protocols. 9, 1038-1048 (2014).</li><li>Gaworzewska, E. T., Carlile, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+chemotaxis+of+Rhizobium+leguminosarum+and+other+bacteria+towards+root+exudates+from+legumes+and+other+plants.">Positive chemotaxis of Rhizobium leguminosarum and other bacteria towards root exudates from legumes and other plants.</a> Microbiology. , (1982).</li><li>Walker, T. S., Bais, H. P., Grotewold, E., Vivanco, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+exudation+and+rhizosphere+biology.">Root exudation and rhizosphere biology.</a> Plant Physiology. 132, 44-51 (2003).</li></ol>

At the scale of aquatic microorganisms, the environment is far from homogenous and is often characterized by physical/chemical gradients that structure microbial communities1,15. The capacity of motile microorganisms to use behavior (i.e., chemotaxis) facilitates foraging within these heterogeneous microenvironments1. Studying chemotaxis directly in the environment has the potential to identify important interspecific interactions and chem...

Diverse microorganisms use motility and chemotaxis to exploit patchy nutrient environments, find hosts, or avoid deleterious conditions1,2,3. These microbial behaviours can in turn influence rates of chemical transformation4 and promote symbiotic partnerships across terrestrial, freshwater, and marine ecosystems2,5.
Chemotaxis has been extensively studied under laboratory conditions for the past 60 years6. The first quantitative method to study chemotaxis, the capillary assay, involves a capillary tube filled with a putative chemoattractant immersed in a suspension of bacteria6. Diffusion of the chemical out of the tube creates a chemical gradient, and chemotactic bacteria respond to this gradient by migrating into the tube7. Since the development of the capillary assay, still widely used today, many other techniques have been developed to study chemotaxis under increasingly controlled physical/chemical conditions, with the most recent involving the use of microfluidics8,9,10.
Microfluidics, together with high-speed video microscopy, enables tracking of the behavior of single cells in response to carefully controlled gradients. Although these techniques have vastly improved our understanding of chemotaxis, they have been restricted to laboratory use and do not translate easily to field deployment in environmental systems. As a consequence, the capacity of natural communities of bacteria to use chemotaxis within natural ecosystems has not been examined; thus, current understanding of the potential ecological importance of chemotaxis is biased toward artificial laboratory conditions and a limited number of laboratory-cultured bacterial isolates. The recently developed ISCA overcomes these limitations11.
The ISCA builds on the general principle of the capillary assay; however, it makes use of modern microfabrication techniques to deliver a highly replicated, easily deployable experimental platform for the quantification of chemotaxis toward compounds of interest in the natural environment. It also allows identification and characterization of chemotactic microorganisms by direct isolation or molecular techniques. While the first working device was self-fabricated and constructed of glass and PDMS11, the latest injection-molded version is composed of polycarbonate, using a highly standardized fabrication procedure (for interest in the latest version of the device, the corresponding authors can be contacted).
The ISCA is credit card-sized and consists of 20 wells distributed in a 5 x 4 well array, each linked to the external aquatic environment by a small port (800 &#181;m in diameter; Figure 1). Putative chemoattractants loaded into the wells diffuse into the environment via the port, and chemotactic microbes respond by swimming through the port into the well. As many factors can influence the outcome of an ISCA experiment in the natural environment, this step-by-step protocol will help new users overcome potential hurdles and facilitate effective deployments.

<table>
	<tbody>
		<tr>
			<td>Acrylic glue</td>
			<td>Evonik</td>
			<td>1133</td>
			<td>Acrifix 1S 0116</td>
		</tr>
		<tr>
			<td>Acrylic sheet</td>
			<td>McMaster-Carr</td>
			<td>8505K725</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Adhesive tape</td>
			<td>Scotch</td>
			<td>3M 810</td>
			<td>Scotch Magic tape</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Systec</td>
			<td>D-200</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Fisher Scientific</td>
			<td>75002451</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Bungee cord</td>
			<td>Paracord Planet</td>
			<td>667569184000</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Centrifuge tube - 2 mL</td>
			<td>Sigma Aldrich</td>
			<td>BR780546-500EA</td>
			<td>Eppendorf tube</td>
		</tr>
		<tr>
			<td>Conical centrifuge tube - 15 mL</td>
			<td>Fisher Scientific</td>
			<td>11507411</td>
			<td>Falcon tube</td>
		</tr>
		<tr>
			<td>Conical centrifuge tube - 50 mL</td>
			<td>Fisher Scientific</td>
			<td>10788561</td>
			<td>Falcon tube</td>
		</tr>
		<tr>
			<td>Deployment arm</td>
			<td>Irwin</td>
			<td>1964719</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Deployment enclosure plug</td>
			<td>Fisher Scientific</td>
			<td>21-236-4</td>
			<td>See alternatives in manuscript</td>
		</tr>
		<tr>
			<td>Disposable wipers</td>
			<td>Kimtech - Fisher Scientific</td>
			<td>06-666</td>
			<td>Kimwipes</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Beckman</td>
			<td>C09756</td>
			<td>CYTOFlex</td>
		</tr>
		<tr>
			<td>Glutaraldehyde 25%</td>
			<td>Sigma Aldrich</td>
			<td>G5882</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Green fluorescent dye</td>
			<td>Sigma Aldrich</td>
			<td>S9430</td>
			<td>SYBR Green I - 1:10,000 final dilution</td>
		</tr>
		<tr>
			<td>Hydrophilic GP filter cartridge - 0.2 &#181;m</td>
			<td>Merck</td>
			<td>C3235</td>
			<td>Sterivex filter</td>
		</tr>
		<tr>
			<td>In Situ Chemotaxis Assay (ISCA)</td>
			<td>-</td>
			<td>-</td>
			<td>Contact corresponding authors</td>
		</tr>
		<tr>
			<td>Laser cutter</td>
			<td>Epilog Laser</td>
			<td>Fusion pro 32</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Luria Bertani Broth</td>
			<td>Sigma Aldrich</td>
			<td>L3022</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Marine Broth 2216</td>
			<td>VWR</td>
			<td>90004-006</td>
			<td>Difco</td>
		</tr>
		<tr>
			<td>Nylon slotted flat head screws</td>
			<td>McMaster-Carr</td>
			<td>92929A243</td>
			<td>M 2 &#215; 4 &#215; 8 mm</td>
		</tr>
		<tr>
			<td>Pipette set</td>
			<td>Fisher Scientific</td>
			<td>05-403-151</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Pipette tips - 1 mL</td>
			<td>Fisher Scientific</td>
			<td>21-236-2A</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Pipette tips - 20 &#181;L</td>
			<td>Fisher Scientific</td>
			<td>21-236-4</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Pipette tips - 200 &#181;L</td>
			<td>Fisher Scientific</td>
			<td>21-236-1</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Sea salt</td>
			<td>Sigma Aldrich</td>
			<td>S9883</td>
			<td>For artificial seawater</td>
		</tr>
		<tr>
			<td>Serological pipette - 50 mL</td>
			<td>Sigma Aldrich</td>
			<td>SIAL1490-100EA</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Syringe filter - 0.02 &#181;m</td>
			<td>Whatman</td>
			<td>WHA68091002</td>
			<td>Anatop filter</td>
		</tr>
		<tr>
			<td>Syringe filter - 0.2 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>10695211</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Syringe needle 27G</td>
			<td>Henke Sass Wolf</td>
			<td>4710004020</td>
			<td>0.4 &#215; 12 mm</td>
		</tr>
		<tr>
			<td>Syringes - 1 mL</td>
			<td>Codau</td>
			<td>329650</td>
			<td>Insulin Luer U-100</td>
		</tr>
		<tr>
			<td>Syringes - 10 mL</td>
			<td>BD</td>
			<td>303134</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Syringes - 50 mL</td>
			<td>BD</td>
			<td>15899152</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Tube rack - 15 mL</td>
			<td>Thomas Scientific</td>
			<td>1159V80</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Tube rack - 50 mL</td>
			<td>Thomas Scientific</td>
			<td>1159V80</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Uncoated High-Speed Steel General Purpose Tap</td>
			<td>McMaster-Carr</td>
			<td>8305A77</td>
			<td>Or different company</td>
		</tr>
		<tr>
			<td>Vacuum filter - 0.2 &#181;m</td>
			<td>Merck</td>
			<td>SCGPS05RE</td>
			<td>Steritop filter</td>
		</tr>
	</tbody>
</table>

in situ chemotaxis assay to examine microbial behavior in aquatic ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

This section presents laboratory results using the ISCA to test the chemotactic response of marine microbes to a concentration range of glutamine, an amino acid known to attract soil bacteria14. The concentration of glutamine that elicited the strongest chemotactic response in the laboratory tests was used to perform a chemotaxis assay in the marine environment.
To perform the laboratory tests, seawater communities sampled from coastal water in Sydney, Australia, were e...

Watch this Scientific Journal Video about In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems at JoVE.com

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...

Our protocol enables us to robustly quantify microbial chemotaxis directly in their home habitat. And thereby we can isolate microorganisms and we can better quantify and understand the metabolic potential. The in situ chemotaxis assay, or ISCA in short, is a flexible, user-friendly, and simple device that allows new insight into microbial behaviors and therefore the role and function of microorganisms in any environment that has a liquid phase.Demonstrating the procedure for deployment in both the laboratory and in the field is Estelle Clerc, who is currently a PhD student in my research group. Before preparing the chemoattractant, filter the medium with a 0.2 micrometer filter, and autoclave the filtered solution. Next, prepare a 10 millimolar solution of chemoattractant in 1 milliliter of the sterile medium, and filter the chemoattractant solution with a 0.2 micrometer syringe filter to remove any particles and potential contaminants.Then, dilute the filtered chemoattractant stock solution in series, according to the experimental protocol. To fill an ISCA, attach a 27 gauge needle to a 1 milliliter syringe, and load one syringe for each concentration of the filtered chemoattractant. Holding the device with the port facing upwards, slowly inject each concentration of the substance into each of the five wells of one row of the device until a small droplet appears on top of each port.When all of the wells have been filled, transfer 1.5 milliliter of the marine or freshwater bacteria culture of interest in 150 milliliters of the appropriate bacterial culture medium. Next, place two small pieces of double-sided adhesive tape on the flat surface of a 200 milliliter capacity tray. And secure one ISCA onto each piece of tape.Using a 50 milliliter serological pipette, slowly fill the deployment tray with the bacterial solution. And allow the bacteria to respond to the chemoattractant of interest for one hour. To end the analysis, use a new 50 milliliter pipette to gently remove the medium from the ISCA tray.To retrieve the samples, first position the ISCA so that the port faces downward. Next, use a sterile 1 milliliter syringe equipped with a 27 gauge needle to draw the solution from each well. Pulling the samples from each row of the same concentration into a single tube.Then, analyze the samples by flow cytometry to determine the number of bacteria attracted to each chemoattractant concentration. To prepare an ISCA for field deployment, first, collect 5 milliliters of water per ISCA from the field side and filter the water through a 0.2 micrometer syringe filter into a 50 milliliter conical tube. Pass the syringe filtered water through a hydrophilic GP filter cartridge two times, collecting the filtrate in a new 50 milliliter conical centrifuge tube, after each rinse.Then, filter the water through a 0.02 micrometer syringe filter into a new 50 milliliter tube. Use aliquots of the filtrate to resuspend all of the chemoattractants of interest to the desired concentrations in individual 15 milliliter conical tubes. Then use 10 milliliter syringes to filter the resuspended chemoattractants through individual 0.2 micrometer syringe filters into sterile 15 milliliter conical centrifuge tubes to remove any unwanted particles or water insoluble compounds.For ISCA field deployment, screw each ISCA onto piece 9 of the device enclosure and close the enclosure. Seal the enclosure with adhesive tape and use bungee cords to secure the enclosure to a deployment arm and human made structure. Alternatively, the enclosure can be secured with a small weight on a shallow substrate or attached to a net in the Pelagic ocean.Submerge the enclosure completely to start the filling, holding the enclosure firmly to prevent excessive water movement inside. Once the level of the water has reached the top of the enclosure, make sure that no air is trapped inside. When the enclosure is completely full, seal the bottom and top holes with two plugs.Then, leave the ISCA in place for 1 to 3 hours. At the end of the sampling period, remove the enclosure from the water. Place the enclosure over a container to drain out the water, and carefully remove the upper part of the adhesive tape from the front holes.Once the water line has passed below the top of the ISCA, remove the bottom plug and drain the rest of the water. While the ISCAs are still attached to the enclosure, use a 1 milliliter pipette, or a 1 milliliter syringe, to carefully remove the water trapped on top of each ISCA and remove the ISCAs without touching the upper surface. Then use a disposable wipe to remove any remaining liquid on the surface, and retrieve the samples as demonstrated for the laboratory sample retrieval.In this representative in vitro analysis, 1 millimolar was determined to be the optimal glutamine deployment concentration as it induced a significant chemotactic response that was 18 fold higher than the filtered seawater control. Higher and lower concentrations of glutamine also induced significant, but weaker, chemotactic response. In this field deployment analysis, five ISCA replicates filled with 1 millimolar glutamine were deployed for 1 hour at a coastal site near Sydney, Australia.And the glutamine filled ISCA attracted nearly three times more bacteria than the control wells filled with filtered seawater. The most important thing to remember when deploying the ISCA, both in the lab and in the field, is to avoid creating strong fluid flows which will prevent chemotaxis. ISCA derived samples can be used to identify specific organisms or genes, to characterize the genomic potential of chemotactic organisms, and for targeted isolation of specific strains on selective media.

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