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 in magnitude with distance from the source (ISCA well), which means that the concentration experienced by microorganisms in the environment will be lower than that present inside the device. The optimal concentration to use in the ISCA well depends on the diffusion rate of the chemoattractant. If the concentration in the well is too low (close to the detection limit of the microbes), no positive chemotaxis will be observed. Conversely, if the concentration in the well is too high, the concentration will also be high in the immediate environment and microbial accumulation will occur above the ISCA wells rather than inside. Therefore, dilution series should be carried out in laboratory conditions for each compound in order to determine the optimal concentration for field deployments. Ideally, a concentration range should also be tested in the field to confirm laboratory results.
	<ol>
		<li>Prepare 2.5 L of a suitable medium containing the appropriate salt concentration (e.g., phosphate-buffered saline [PBS] or artificial seawater). Filter the medium with a 0.2 &#181;m filter using a peristaltic or vacuum pump and autoclave.</li>
		<li>Prepare a 10 mM solution of chemoattractant in 1 mL of the sterile medium. Filter the chemoattractant solution with a 0.2 &#181;m syringe filter to remove particles and potential contaminants. 
		NOTE: Ideally, no organic compounds other than the chemoattractant should be present in the final solution.</li>
	</ol>
	</li>
	<li>Concentration range by dilution series
	<ol>
		<li>Dilute the filtered chemoattractant stock solution in series, ranging (for example) from 10 mM to 100 &#181;M. 
		NOTE: At least a 0.7 mL final volume is needed per concentration tested.</li>
	</ol>
	</li>
	<li>Loading the ISCA
	<ol>
		<li>Fill a 1 mL syringe with the filtered chemoattractant and connect it to a 27 G syringe. Holding the ISCA with the port facing upward, slowly inject the substance until a small droplet appears on top of the port. 
		NOTE: 1) Each dilution or substance must be injected with a separate syringe and needle to prevent cross-contamination. 2) This small droplet is important because it ensures that no air bubble is trapped within the port, which could impair the ability of microbes to migrate through the port. 3) It is recommended to fill an entire row per substance or concentration (five wells) to provide an adequate minimal volume for further analyses. 4) One row per ISCA should act as negative control and should be filled with the filtered medium in which the microbes will be suspended. This treatment accounts for the effect of random motility by microbes into the ISCA wells and should be used to normalize the treatments containing a chemoattractant.</li>
	</ol>
	</li>
	<li>Deployment in the laboratory
	<ol>
		<li>Overnight, incubate a 5 mL culture enriched with 1% marine broth (for marine bacteria) or 1% lysogeny broth (LB, for fresh water bacteria). 
		NOTE: Motile bacterial isolates or natural bacterial communities can be used for laboratory deployments.</li>
		<li>Incubate the culture for 12 h at room temperature (RT) and 180 rpm. After 12 h, ensure that the microbial communities are motile by direct observation under a microscope.</li>
		<li>Spin down the culture at 1,500 x g for 10 min and resuspend 1/100 in 150 mL of appropriate medium (e.g., filtered seawater, filtered freshwater).</li>
		<li>Place two small pieces of double-sided adhesive tape on the flat surface of a 200 mL capacity tray (the lids of 1 mL tip boxes have the ideal dimensions for this purpose and can easily be autoclaved). Place one ISCA on top, ensuring that it attaches securely to the tape. Slowly fill the deployment tray with the bacterial solution using a 50 mL serological pipette. 
		NOTE: Fill the tray until the ISCA is submerged under approximately 1&#8211;2 cm of liquid. If using multiple trays, use the same volume across all.</li>
		<li>Leave the ISCA to incubate for 1 h to allow bacterial chemotaxis. After 1 h, remove the medium very gently with a 50 mL serological pipette to minimize turbulent flow.</li>
		<li>Retrieve the ISCA from the deployment tray without touching the upper surface. Use a pipette and disposable wipers to remove remaining liquid on the ISCA surface. 
		NOTE: It is important to avoid touching the ports during this process, as the resulting changes in pressure can remove or add bacteria from the outside environment into the well and thereby bias the bacterial density and composition inside the well.</li>
	</ol>
	</li>
	<li>Retrieval of the samples
	<ol>
		<li>Holding the ISCA with the port facing downward, retrieve the volume of the wells using a sterile 27 G syringe needle attached to a 1 mL syringe. 
		NOTE: Each row (if containing the same substance) can be pooled to provide a working sample of approximately 550 &#181;L. This sample can subsequently be aliquoted into different tubes depending on the required downstream applications.</li>
		<li>Determine the number of bacteria attracted to each chemoattractant concentration by analyzing the samples with flow cytometry12. Choose the concentration of chemoattractant that maximizes chemotaxis for subsequent field deployments.</li>
	</ol>
	</li>
</ol>
2. Preparation for field deployment
NOTE: Preparation of material and construction of the flow-damping enclosure (section 2) must be conducted prior to deployment.
<ol>
	<li>Preparation of materials
	<ol>
		<li>Prepare all materials listed in Table 1. 
		NOTE: Material quantities are provided for one ISCA.</li>
	</ol>
	</li>
	<li>Construction and preparation of the flow-damping enclosure 
	NOTE: The flow-damping enclosure minimizes unwanted turbulences that otherwise prevents the establishment of chemical gradients emanating from the ISCA.
	<ol>
		<li>Cut the pieces for the deployment enclosure with a laser cutter from a 3 mm acrylic sheet. 
		NOTE: The file for the pieces can be found using the following link: &#60;https://figshare.com/articles/Flow_damping_enclosure_for_ISCA_deployments/10630220&#62;.</li>
		<li>Assemble the laser-cut pieces as demonstrated in Figure 2 using acrylic glue. 
		NOTE: Assemble the pieces with care. Holes or misalignment can create leaks upon deployment, which directly impacts data quality.</li>
		<li>Leave the assembled enclosure to dry overnight.</li>
		<li>Wash the enclosure with deionized water.</li>
		<li>Identify potential leakage by pouring deionized water into the enclosure. Fix any potential leaking joints by adding more acrylic glue, then repeat steps 2.2.3&#8211;2.2.5.</li>
		<li>Cut the screw threads into the acrylic piece that will be used to secure the ISCA. This can be achieved using a tap with a diameter and pitch matching the mounting screws.
		<ol>
			<li>First, affix the tap into a tap wrench, then secure the acrylic piece to be tapped in a benchtop vice. For the best results, make sure the acrylic piece is as level as possible. Make sure that the tap is perpendicular to the acrylic piece and start turning the tap wrench (clockwise), applying light pressure to the tap.</li>
			<li>After several full revolutions in the acrylic piece, reverse the rotation of the tap (counterclockwise) for one-quarter of a rotation to clear acrylic from the tap. Repeat the process until the entire depth of the acrylic piece is tapped.</li>
			<li>Finally, remove the tap (turning counterclockwise) and test the threads using a screw.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Procedure in the field
<ol>
	<li>Water filtration
	<ol>
		<li>Collect water from the field site when ready to start the experiment. Filter 5 mL of water per ISCA through a 0.2 &#181;m syringe filter (with a 50 mL syringe) into a 50 mL conical centrifuge tube. 
		NOTE: Approximately 3 mL of filtered water are required to fill all the wells of an ISCA; however, it is recommended to 1) filter 5 mL per device to account for losses during the quadruple filtration process, and 2) preserve aliquots of the filtrates as negative controls for both flow cytometry and molecular procedures.</li>
		<li>Filter the filtrate twice through a 0.2 &#181;m hydrophilic GP filter cartridge (using the same one, both times) with a new 50 mL syringe into a new 50 mL conical centrifuge tube. Filter the filtrate through a 0.02 &#181;m syringe filter (with a new 50 mL syringe) into a new 50 mL conical tube. 
		NOTE: This quadruple filtration should remove nearly all microorganisms and particles from the water. Keep the final filtrate away from any source of heat until use. This water will be used to resuspend all chemicals used in the ISCA, and it should be maintained at the same temperature as the water at the deployment site. Convective flows triggered by differences in temperature between the ISCA wells and outside environment may otherwise occur.</li>
		<li>Use aliquots of the filtrate to resuspend all chemoattractants of interest (typically dry) to the desired concentrations in 15 mL conical centrifuge tubes.</li>
		<li>Filter the resuspended chemoattractants through a 0.2 &#181;m syringe filter with a 10 mL syringe into sterile 15 mL conical centrifuge tubes to remove unwanted particles or water-insoluble compounds (if using extracts). 
		NOTE: Filter gently to prevent particles from passing through the filter. It is important to resuspend the chemoattractants in the ultrafiltered water from the field site and not solubilize them into other solutions. Using water from the field site is necessary to (1) obtain the same salt concentration inside the wells as that in the bulk environmental water to prevent density-driven flow, and (2) guarantee that background nutrient levels are equal inside and outside of the well.</li>
	</ol>
	</li>
	<li>ISCA filling
	<ol>
		<li>Perform section 1.3 to fill the ISCA. 
		NOTE: It is recommended to fill one row (five wells) per substance (i.e., three different substances per ISCA and one ultrafiltered seawater control).</li>
	</ol>
	</li>
	<li>Deployment in the field
	<ol>
		<li>Screw the ISCA (Figure 3A) to piece 9 of the enclosure (Figure 2K and Figure 3B). 
		NOTE: The flow-damping enclosure outlined above can contain two ISCAs side-by-side or one ISCA placed at its center.</li>
		<li>Close the enclosure (Figure 3C) and seal it with adhesive tape (Figure 3D). 
		NOTE: Wrinkles must be avoided to ensure a perfect seal. Seal all sides first, then (in a second step) seal the side holes, which will be used to drain water from the enclosure at the end of sampling. Do not seal the top and bottom holes. Do not place the ISCA upside down, as density-driven flow can occur in wells containing chemoattractants, which will bias the number of cells in the wells.</li>
		<li>Because the enclosure must remain steady during deployment, it is recommended to attach it to manmade structures (e.g., pontoon, ladder) using bungee cords. 
		NOTE: The enclosure can be attached to a deployment arm (here, a modified clamp with a perpendicular platform) using bungee cords before immersion in the water. Alternatively, the enclosure can be filled and secured with a small weight on shallow substrates. If deployments are intended in the pelagic ocean, the enclosure can be attached to a net with a buoy on one side and dive weight on the other.</li>
		<li>Submerge the enclosure completely to start filling. While filling, hold the enclosure firmly to prevent excessive water movement inside. Once the level of the water reaches the top of the enclosure, make sure that no air is trapped inside. 
		NOTE: In case some air bubbles are trapped, tilt the enclosure gently with the vent hole facing upward, which will enable the bubbles to escape.</li>
		<li>Once completely full, seal the bottom and top holes with two plugs, which can be made out of silicon or rubber or by sealing the extremities of 20 &#181;L pipette tips (Figure 4). 
		NOTE: This step prevents flow inside the enclosure during sampling.</li>
		<li>Leave the ISCA in place for sampling for 1&#8211;3 h. 
		NOTE: The ideal deployment time is primarily dictated by the temperature of the water and doubling time of the bacterial community. When the water temperature is above 20 &#176;C, it is not recommended to deploy the ISCA for more than 1 h, because cell division can occur in the wells containing chemoattractants after 1.5&#8211;2.0 h. However, optimal deployment time can be tested prior to the ISCA experiment by amending natural communities with the loaded chemicals and quantifying the number of cells through time.</li>
		<li>Remove the enclosure from the water. Place it over a container enabling the water to be drained from the enclosure.</li>
		<li>Remove the upper part of the adhesive tape from the front holes very gently. 
		NOTE: The flow of the water leaving the enclosure must be at a dripping speed. Proceed one hole at a time, from the top of the enclosure to the bottom. It should take approximately 10&#8211;15 min to drain the enclosure completely.</li>
		<li>Once the waterline passes below the top of the ISCA, remove the bottom plug, and drain the rest of the water.</li>
		<li>While the ISCAs are still attached to the enclosure, carefully remove the water trapped on top of the ISCA with a 1 mL pipette.</li>
		<li>Remove the ISCA without touching the upper surface and use a disposable wipe to remove any remaining liquid on the surface. 
		NOTE: It is important not to touch the ports during this process, as the resulting changes in pressure can remove or add bulk bacteria into the well and bias the bacterial counts.</li>
		<li>Retrieve the samples from the ISCA by repeating step 1.5.1.</li>
	</ol>
	</li>
</ol>
4. Downstream applications
NOTE: Volumes are given based on a 550 &#181;L sample (one row of an ISCA).
<ol>
	<li>Fix 100 &#181;L of well contents with glutaraldehyde (2% final concentration) for flow cytometry to quantify chemotaxis to each attractant. 
	NOTE: Store on ice (or at 4 &#176;C) and analyze the samples on the same day. Alternatively, samples can be flash frozen in liquid nitrogen following fixation if analysis is not feasible on the same day. Flow cytometry is the recommended method to quantify the number of cells in the ISCA wells, as it is straightforward, fast, and accurate12.</li>
	<li>Snap freeze 300 &#181;L of well content in liquid nitrogen for subsequent DNA extraction and analysis11. 
	NOTE: Store the samples at -80 &#176;C until analysis.</li>
	<li>Add 90 &#181;L of well contents to 10 &#181;L of TE-glycerol buffer and snap-freeze the samples for single-cell genomics13.</li>
	<li>Spread 10&#8211;20 &#181;L on agar plates containing the desired medium for bacterial isolation.</li>
</ol>

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The authors declare no conflict of interest.

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 &amp;micro;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 &amp;times; 4 &amp;times; 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 &amp;micro;L</td><td>Fisher Scientific</td><td>21-236-4</td><td>Or different company</td></tr><tr><td>Pipette tips - 200 &amp;micro;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 &amp;micro;m</td><td>Whatman</td><td>WHA68091002</td><td>Anatop filter</td></tr><tr><td>Syringe filter - 0.2 &amp;micro;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 &amp;times; 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 &amp;micro;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 ...

in-situ-chemotaxis-assay-to-examine-microbial-behavior-aquatic

Research

JoVE Journal

Environment

7.0K Views.  ETH Zürich. 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.

Video: In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

Biology

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

My name is Roman Stocker. I'm a second year assistant professor in the Ralph Parsons Laboratory, which is part of the Department of Civil and Environmental Engineering here at MIT. I'm originally from Italy and I studied fluid mechanics.My background is therefore more in the physical sciences, but in the past two years, three years actually, I've moved more into environmental and biological topics. Two years ago, I've started developing microfluidics to look at problems in microbial ecology and using these micro devices, we are able to develop typical ecological questions for things at the scales of microns. Our work is primarily concerned with al organisms, those that can swim around in the ocean and track their sources of food.In particular, bacteria have developed very clever ways of swimming, and I have a small demonstration here. The bacterial head is pretty much an ellipsoid. You imagine this being about two microns in size.At the back of the bacterium, there is one or more flagella. These flagella are helical structures, which the bacterium can rot rotate. That rotation pro produces trust that allows the bacterium to actually swim forward.There is different modes in which bacteria actually do that, and there are different strategies of swimming and it is part of what our research is concerned with. The reason bacteria swim in the ocean is to be able to better exploit sources of nutrients, and that is part of a paradigm shift which has occurred over the last 15 or 20 years. Before that, people used to think that the ocean was a homogeneous soup of nutrients at scales of meters and up to tens of meters, and in that picture of the ocean, these small scale organisms like bacteria were taught to play no substantial role and they were really just passively vectored by the oceanic occurrence.But 10 or 15 years ago, people realized that that is really not what is going on in the ocean and that in fact these small scale organisms are among the most important players in determining the ocean health and therefore even things like our global climate. This is so because of two reasons. The first reason is that they occur everywhere in the ocean and their cumulative biomass by far exceeds that of all other marine organisms.The second reason, and maybe the most fundamental one, is that these small scale organisms by utilizing nutrient sources and in particular carbon, can recycle the nutrients for other components of the food web and therefore substantially intensifying other aspects and other parts of the trophic chain in the ocean. In all of this, our work focuses primarily on interaction between swimming organisms and nutrient patches. Bacteria not only are able to swim, but they also able to sense detect nutrient gradients.That is, they're able to tell when life is getting better and swim up gradients of nutrients. They do that in a process known as a chemotaxis.Properly. Chemo taxes is the ability to sense a chemical gradient and by swimming home in on it.This is really what we study in the lab and microfluidics is a tool that allows us to probe the chemotactic and swimming abilities of bacteria and therefore extrapolate and scale up their role to determine how important they are in the determining the health of the ocean. In the Past year and a half, we've set up micro devices that allow us to expose bacteria to different kind of conditions that you typically find in the ocean. As I said earlier, typical nutrient patches, but also for example, turbulence.The what we have found so far is that marine bacteria seem to have adapted very efficiently to exploit these nutrient patches and to be able to counteract by swimming a large variety of conditions of turbulence conditions in the ocean that would make them very well suited to actually survive in an ocean, which is typically a desert of nutrients from their point of view and in which nutrients occur only in scattered and very rare patches in a three-dimensional water column, and at the same time be able to swim, to fight turbulence and therefore still be able to track their nutrient foods even when the water around them is moving violently. The Earth system initiative is a very interesting initiative and one that brings together a variety of people from different disciplines, all interested in understanding better how our planet actually works. In short, the earth system initiative's goal is to try to do the same thing for the planet as we are already doing and have been doing for a long time.For the human body that is understanding its health, understanding the health of the planet these days is particularly important because of concerns of global warming, for example, and greenhouse gases and several of the efforts within the earth system initiative are geared towards understanding the health of the planet better. Our work fits into that by looking at one component of the carbon cycle in the ocean. We try to understand in particular how small scale organisms, as I mentioned earlier, swimming bacteria in particular can affect these large scale cycles, which has repercussions on the global carbon cycle in the ocean and therefore on the global carbon cycle worldwide, including the Atmosphere.All of our work So far in this field has been allowed and actually triggered by one key technological advance, and that is microfluidics. In the past 10 years, microfluidics has been developed into a really interesting and cool tool in particular in the Boston Cambridge area. The originator of the microfluidics technology in its current form is Professor George Whitesides at Harvard who introduced the process of soft lithography and the use of PDMS for micro devices fabrication.Since then, the application of microfluidics has grown exponentially in a whole range of fields. From chemical engineering to the health sciences, we have started applying it to microbial ecology and to address some of these small scale problems in in the ocean regarding the swimming of microorganisms. In a nutshell, it is difficult, if not impossible to study these processes directly in the ocean because of their small scales and because of the variability that the ocean necessarily presents, we are still not able to visualize bacteria which are about a micron in size, directly in situ in the ocean, and at the same time, people have tried to model these processes, for example, numerically or theoretically, but because this is biology, you necessarily have to rely on assumptions.For example, you will have to make assumptions about the swimming strategy of a bacteria or the chemo taxi strategy of a bacterium, and therefore real experiments are needed to make progress in this field. Microfluidics allows us to take small regions, small parts of the ocean into the lab, and at the same time expose bacteria and microorganisms to environmental conditions that are realistic, both in terms of nutrients and in terms of flows. At the same time, by using video microscopy and cell tracking, we can study the behavior of individual bacteria in a very carefully controlled manner that allows us to make progress in these questions in a way that would not have been possible about 10 years ago, and all of that is due to technological advances, specifically micro Verdicts.There are two main challenges I see lying ahead of us in this field. The first one, as I mentioned earlier, is to be able at some point to take these questions to the ocean. The reason that one ultimately needs to go to the ocean is to capture all the variability that is inherent in these processes.For example, we still have no idea of what really leaks out of a settling particle, what kind of nutrients are present, what kind of chemical compounds do bacteria chemo tax towards in the lab. We can only study the simplest of these conditions, but what really occurs in the ocean is a collection of complicated and very variable processes that we need to ultimately explore direct directly in C two. This is a big challenge from the point of view of sampling from a point of view of imaging and from the point of view of doing chemical and biological analysis on scales, which as I mentioned a few times are only in the order of a few micrometers and in terms of volumes of picoliters.The second challenge that I see more easier to tackle is that visualizing behavior in three dimensions. Everything we do currently in the lab is two dimensional. We use conventional microscopy to visualize trajectories and accumulation of microorganisms, but we have great hope in a technique known as digital holography for giving us in the timeframe of maybe three to five years the ability to track these things in three dimensions and therefore more fatefully and more realistically, realistically be able to capture the ecology In the next Five years.In addition to trying to go three dimensional in the sampling and perhaps trying to make the first, first naive steps possibly towards the sampling things in the ocean, we definitely want to link these studies with ongoing efforts to understand genomics of these organisms. There is a big push in particular as part of the earth system initiative in understanding the genetic composition and the variability in terms of genetics of these bacteria out there in the field. At the same time, genomics lacks the ability of directly linking the findings with the behavior of the organisms, and that is what we hope that microfluidics and these more behavioral, classical ecological studies will be able to bring to the table.We have a great setup and a great team of people looking at the genomics of marine bacteria, and by collaborating with them, we hope to be able to link these two fundamental questions to get a better understanding of what bacteria out there in the ocean and what their role is in determining the health of the ocean and health of the planet.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Behavior

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 ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Bioengineering

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

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