Environmental Microfluidics Group, MIT - Massachusetts Institute of Technology
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Seymour, J. R., Marcos, Stocker, R. Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers. J. Vis. Exp. (4), e203, doi:10.3791/203 (2007).
1. Create a Mask
Using a CAD software, design the channel for high-resolution printing on a transparency. This will be the "mask".
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°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 http://www.microchem.com/
After coating the wafer, bake it first at 65°C and then at 95°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.
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°C and then 95°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
Bake for at least 12 hours in an oven at 65°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.
Exp #1: Investigating the chemotactic response of marine microbes to micro-scale nutrient layers
1) Setting up the experiment
2) Running the experiment
Exp #2: Investigating the effects of shear on marine bacteria swimming in a vortexZ
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 (< 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 experienced by swimming microbes in the ocean. Recent advances in microfluidics (Whitesides et al. 2001) have enabled detailed analyses of microbial ecology within complex microhabitats (Mao et al. 2003, Park et al. 2003, Keymer et al. 2006, Marcos and Stocker 2006). The microfluidic devices described here allowed us to examine both the chemotactic response of marine microbes to a diffusing nutrient patch (Blackburn et al. 1997, 1998) and the swimming behaviour of microbes within turbulent shear, at a single cell level.
The soft lithographic fabrication process involved in making the microfluidic channels allows for intricate details to be created within the channel architecture, permitting the precise control of flows and gradients within the channels. Flexibility afforded by the fabrication technique allows for channels of various dimensions to be created for comparative studies. The image analysis system applied here permits the visualization of individual cells and nutrient gradients, providing a platform for the detailed quantitative analysis of microbial swimming and chemotactic behaviour at both a single-cell and population level.
We have applied this microfluidic channel as a sensitive chemotaxis assay for a variety of swimming marine microbes and have found that many species are capable of rapidly responding to a diffusing patch of nutrients, forming dense aggregations of cells within the high nutrient concentrations inside the patch. During the chemotactic accumulation of cells within the nutrient patch, some species have also exhibited marked behavioural shifts, including changes in swimming speed and turning frequency. Our observations provide experimental support for the hypothesis that marine microbes can utilise short-lived nutrient patches in the ocean as important growth habitats.
Using different channel geometry, we are able to generate stable microvortices on scales relevant to microbial dynamics in the aquatic environment. This setup allows us to observe the behaviour of bacteria swimming in response to different shear rates. In contrast to the random swimming behaviour under quiescent flow condition, under the influence of a strong shear, bacteria both follow streamlines of the flow field and are aligned with them. This setup offers valuable insight on the fundamental interaction between microorganisms and their fluid dynamical environment.
In each of these experiments, microfluidics has proven to be an effective tool for studying microbial behaviour within dynamic microhabitats. With an increasing recognition of the importance of microbial dynamics within natural habitats, and novel applications of microfluidic technology, we suggest that the further coupling of microfluidics with microbial ecology will yield important new insights.
We would like to thank Microsystems Technology Laboratories at MIT for allowing us to film part of this video in the clean room facility.
|PDMS, Sylgard 184||Silicone Elastomer Kit||Dow Corning||http://www.ellsworth.com/sylgard.html|
|Nikon Eclipse TE2000-E inverted microscope||Microscope||Nikon Instruments|
|PEEK tubing (0.762 mm ID, 1.59 mm OD)||Tool||Upchurch Scientific||www.upchurch.com|
|Syringes (Luer-Lok Tip)||Tool||BD Biosciences|
|Fitting Part P-704-01||Tool||Upchurch Scientific||To connect tubing to Luer-Lok Tip Syringes|
|Syringe Pump (PHD 2000 Programmable)||Equipment||Harvard Apparatus|
|CCD Camera (PCO 1600)||Equipment||Cook|
1. Azam, F., Malfatti, F. Microbial structuring of marine ecosystems. Nature Reviews Microbiology 5, 782-791 (2007).
2. Blackburn, N., Azam, F., Hagstrom, A. Spatially explicit simulations of a microbial food web. Limnology and Oceanography 42, 613-622 (1997)
3. Blackburn, N., Fenchel, T., Mitchell, J.G. Microscale nutrient patches in plankton habitats shown by chemotactic bacteria. Science 282, 2254-2256 (1998)
4. Keymer, J.E., Galajda, P., Muldoon, C., Park, S., Austin, R.H. Bacterial metapopulations in nanofabricated landscapes. Proceedings of the National Academy of Science 103, 17290-17295 (2006)
5. Mao, H., Cremer, P.S., Manson, M.D. A sensitive, versatile microfluidic assay for bacterial chemotaxis. Proceedings of the National Academy of Science 100, 5449-5454 (2003)
6. Marcos, Stocker, R. Microorganisms in vortices: a microfluidic setup. Limnology and Oceanography: Methods 4, 392-398 (2006)
7. Park, S., Wolanin, P.M., Yuzbahyan, E.A., Lin, H., Darnton, N.C., Stock, J.B., Silberzan, P., Austin, R. Influence of topology on bacterial social interaction. Proceedings of the National Academy of Sciences 100, 13910-13915 (2003)
8. Whitesides, G.M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D.E. Soft lithography in biology and biochemistry. Annual Review of Biomedical Engineering 3, 335-373 (2001)