We would like to thank Microsystems Technology Laboratories at MIT for allowing us to film part of this video in the clean room facility.

Preparation
1. Create a Mask&nbsp;
<blockquote>
Using a CAD software, design the channel for high-resolution printing on a transparency. This will be the "mask".
</blockquote>
In the clean room:
2. Clean and bake the wafer
First, squirt the wafer with Acetone, then quickly with Methanol, then with Isopropanol. Finally, dry the wafer using Nitrogen.
Bake the wafer in the oven (130&deg;C) for 5 min.
3. Coating the wafer
Place the wafer at the center of the spin-coating machine. Pour photoresist (SU-8) from the bottle onto the wafer. Let the SU-8 flow and relax for ~ 10 s. Turn on the spin-coater and ramp its speed up from 0 to 500 rpm over 5 s; keep at 500 rpm for 10 s; ramp up to final speed over 10 s and maintain at final speed for 30 s. The final speed depends on the targeted coating thickness and the SU-8 used. The details can be found at <a href="http://www.microchem.com/">http://www.microchem.com/</a>
4. Soft-bake
After coating the wafer, bake it first at 65&deg;C and then at 95&deg;C. The baking time varies with targeted thickness and type of photoresist used. Then, let the wafer sit at room temperature for at least 5 min.
5. Exposure
Place the mask on top of the wafer and expose the wafer to UV light for the time recommended in the SU-8 manual.
6. Post-exposure bake
Bake the wafer at 65&deg;C and then 95&deg;C following the SU-8 manual's instructions.
7. Developing the wafer to obtain the "master" (mold)
Prepare a beaker filled with the developer (PMMA). Immerse the wafer into the beaker while very gently oscillating the beaker until the unexposed part of the photoresist is washed away.
In our lab:
8. Prepare PDMS and pour it onto the wafer
Mix the PDMS with its curing agent at 10:1 ratio into a cup. Stir and mix it homogenously: this will generate lots of bubbles and make the mixture look opaque. Pour the mixture on the "master".
9. De-bubble in vacuum chamber
To remove the bubbles, placed the master and PDMS mixture that is covering it into a vacuum chamber until all bubbles are gone.
10. Baking in oven
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Bake for at least 12 hours in an oven at 65&deg;C to harden the PDMS.
11. Punch holes
Peel off the PDMS from the master and punch holes for inlets and outlets of the channels.
In cleanroom (not shown)
12. Plasma bonding
Channels are bonded to a glass slide after treating both the PDMS layer and the glass slide with oxygen plasma for 1 min.
Experiments:
Exp #1: Investigating the chemotactic response of marine microbes to micro-scale nutrient layers
1) Setting up the experiment
<ol>
<li>Add organisms and substrates to glass syringes</li>
<li>Place microfluidic channel onto microscope stage and attach tubing to appropriate inlets and outlets</li>
<li>Connect tubing to waste reservoir. Make sure tubing is entirely submerged in the fluid in waste reservoir to avoid pressure oscillations</li>
<li>Place syringes onto syringe pump and connect to valves and tubing</li>
<li>Set up microscope: light conditions, magnification, etc.</li>
<li>Focus on appropriate position in the channel</li>
<li>De-bubble channel using larger syringe filled with artificial seawater</li>
<li>Set appropriate flow rate on syringe pump. In this case 2 ml/min, which corresponds to a mean flow velocity of 220 &micro;m s-1 in the channel</li>
</ol>
2) Running the experiment
<ol>
<li>Start syringe pump to establish a nutrient gradient in the channel</li>
<li>Once flow has stabilized and a band of nutrients has developed, stop the flow on the syringe pump and begin recording time from this point</li>
<li>Nutrient band begins to diffuse laterally</li>
<li>At regular time intervals, use image analysis software to record sequences of frames to create 'movies'</li>
<li>Discriminate swimming organisms by taking time-difference images&nbsp;between two subsequent&nbsp;frames, so that&nbsp;only moving objects are now visualized, allowing us to differentiate motile cells from non-moving particles and&nbsp;background noise</li>
<li>Take movies to determine positions of cells in the channel with reference to the position of the nutrient patch</li>
<li>Record movies at regular intervals for 10-20 min to analyze the positions and swimming patterns of organisms</li>
<li>By using the image analysis software to superimpose the positions of organisms in different frames (assigning to each pixel the maximum light intensity recorded in that pixel over the duration of the movie), we can&nbsp;obtain trajectory information for swimming cells&nbsp; </li>
</ol>
Exp #2: Investigating the effects of shear on marine bacteria swimming in a vortexZ
<ol>
<li>Using different channel geometry, we can observe the behavior of bacteria swimming in a vortex at different shear rates</li>
</ol>

<ol>
	<li>Azam, F., Malfatti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+structuring+of+marine+ecosystems.">Microbial structuring of marine ecosystems.</a> Nature Reviews Microbiology. 5, 782-791 (2007).</li><li>Blackburn, N., Azam, F., Hagstrom, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+explicit+simulations+of+a+microbial+food+web.+.">Spatially explicit simulations of a microbial food web. .</a> Limnology and Oceanography. 42, 613-622 (1997).</li><li>Blackburn, N., Fenchel, T., Mitchell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+nutrient+patches+in+plankton+habitats+shown+by+chemotactic+bacteria.">Microscale nutrient patches in plankton habitats shown by chemotactic bacteria.</a> Science. 282, 2254-2256 (1998).</li><li>Keymer, J. E., Galajda, P., Muldoon, C., Park, S., Austin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+metapopulations+in+nanofabricated+landscapes.">Bacterial metapopulations in nanofabricated landscapes.</a> Proceedings of the National Academy of Science. 103, 17290-17295 (2006).</li><li>Mao, H., Cremer, P. S., Manson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive,+versatile+microfluidic+assay+for+bacterial+chemotaxis.">A sensitive, versatile microfluidic assay for bacterial chemotaxis.</a> Proceedings of the National Academy of Science. 100, 5449-5454 (2003).</li><li>Marcos, R., Stocker, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microorganisms+in+vortices:+a+microfluidic+setup.">Microorganisms in vortices: a microfluidic setup.</a> Limnology and Oceanography: Methods. 4, 392-398 (2006).</li><li>Park, S., Wolanin, P. M., Yuzbahyan, E. A., Lin, H., Darnton, N. C., Stock, J. B., Silberzan, P., Austin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+topology+on+bacterial+social+interaction.">Influence of topology on bacterial social interaction.</a> Proceedings of the National Academy of Sciences. 100, 13910-13915 (2003).</li><li>Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+in+biology+and+biochemistry.">Soft lithography in biology and biochemistry.</a> Annual Review of Biomedical Engineering. 3, 335-373 (2001).</li></ol>

An understanding of how marine microbes interact with their local chemical and physical environment is imperative for a more complete and precise perception of the role of planktonic microorganisms in the oceans nutrient and carbon cycles (Azam and Malfatti 2007). However, due to the small scales (&lt; mm) over which many important microbial interactions take place, technical limitations have prevented detailed examinations of microbial behaviour within the heterogeneous bio-physico-chemical landscape predicted to be ex...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PDMS, Sylgard 184</td>
<td>Silicone Elastomer Kit</td>
<td>Dow Corning, Midland, MI, USA</td>
<td>&nbsp;</td>
<td>http://www.ellsworth.com/sylgard.html</td>
</tr>
<tr>
<td>SU8-2100</td>
<td>Photoresist</td>
<td>MicroChem, Newton, MA, USA</td>
<td>&nbsp;</td>
<td>www.microchem.com
</td>
</tr>
<tr>
<td>Nikon Eclipse TE2000-E inverted microscope</td>
<td>Microscope</td>
<td>Nikon, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PEEK tubing (0.762 mm ID, 1.59 mm OD)</td>
<td>Tool</td>
<td>Upchurch Scientific, Oak Harbor, WA, USA</td>
<td>&nbsp;</td>
<td>www.upchurch.com</td>
</tr>
<tr>
<td>Syringes (Luer-Lok Tip)</td>
<td>Tool</td>
<td>BD, Franklin Lakes, NJ, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fitting Part P-704-01</td>
<td>Tool</td>
<td>Upchurch Scientific, Oak Harbor, WA, USA</td>
<td>&nbsp;</td>
<td>To connect tubing to Luer-Lok Tip Syringes</td>
</tr>
<tr>
<td>Syringe Pump (PHD 2000 Programmable)</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CCD Camera (PCO 1600)</td>
<td>Equipment</td>
<td>Cooke, Romulus, MI, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers at JoVE.com

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

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 ...

chemotactic-response-marine-micro-organisms-to-micro-scale-nutrient

Research

JoVE Journal

Biology

13.2K Views.  MIT - Massachusetts Institute of Technology. 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.