This study was supported by grant # 0649883 from the National Science Foundation, by the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE), and by the Searle Systems Biology and Bioengineering Undergraduate Research Experience (Searle SyBBURE).

I. Microfluidic Device Fabrication
 <ol>
 <li>The first step in creating a microfluidic device is to draw a two dimensional layout of the device in a computer assisted drawing (CAD) program. We have used AutoCAD, but other drawing programs are also available, such as CleWin, or CorelDraw.</li>
 <li>The next step is to fabricate a photolithographic mask. Depending on the device dimensions, required resolution and budget, these masks could be fabricated in chrome (highest resolution, high cost), created on photographic film, or even printed directly on an overhead transparency using a high resolution printer. Here, we have used Cr masks manufactured by Advance Reproductions Corp., North Andover, MA.</li>
 <li>The next step is to transfer the pattern from the mask onto a photosensitive epoxy to create a raised-relief mold.
 <ol>
 <li>First, a layer of negative SU8 photoresist is spun onto a silicone wafer to the desired thickness, and baked to volatilize excess solvent. Photoresist formulation and spin speeds control the thickness of the deposited layer. </li>
 <li>Then, the pattern is exposed to a pre-determined dose of UV light through the mask. A post exposure bake cross-links the light-exposed regions of the photoresist coating, rendering them insoluble.</li>
 <li>Next is the development step, where unexposed resist is removed with a chemical developer, leaving a positive raised-relief structure called a master.</li>
 <li>A third baking step called a hard bake step is optional to further fix the structures.</li>
 </ol></li>
 <li>PDMS molding
 <ol>
 <li>We use the silicone elastomer poly(dimethyl siloxane) for replica molding of microfluidic devices 1. First, the base material is mixed in a 10:1 weight ratio with the curing agent, poured over the mold in a shallow container such as a petri dish, and degassed under vacuum until all the visible bubbles are gone. </li>
 <li>The master with the uncured PDMS is then placed in a carefully leveled oven for at least 4h at 65&deg;C for cross-linking of the polymer to occur.</li>
 <li>After PDMS is solidified, the molded section is cut out of the petri dish. Holes are punched through the top of the device to access the microfluidic spaces in the finished device.</li>
 </ol>
 </li>
 <li>Attachment to glass
 <ol>
 <li>Clean PDMS is irreversibly bonded to clean glass by exposure to oxygen plasma. First, the molded and punched PDMS and a clean glass slide are placed bonding surface up into a plasma cleaner. We used a PDC-32G Harrick plasma cleaner.</li>
 <li>After the chamber is closed, the operator turns on a vacuum pump and then the radio frequency or rf source. Plasma will self ignite in the chamber, evidenced by a slightly purple glow in the chamber.</li>
 <li>After exposure to plasma for 30 seconds, the rf source and then the vacuum pump are turned off.</li>
 <li>Quickly, the glass slide and PDMS device are removed from the chamber and brought into direct contact (molded PDMS surface side down). This will form an irreversible bond, and will also make the surface properties hydrophilic.</li>
 <li>If desired, different surface chemistries can be achieved with PDMS through immobilization of proteins on the surface by adsorption or covalent bonding. </li>
 <li>The device can then be filled with fluid such as deionized water, growth media, or artificial groundwater by capillary forces or use of gentle pressure with a syringe. </li>
 </ol>
</li>
 </ol>
II. Flow Quantification of Microfluidic Device by Gravimetric Analysis
 <ol>
 <li>To calibrate pressure-driven flow in the device, micro-structured flow cells were first preloaded with DI water.</li>
 <li>A liquid containing reservoir, such as a plastic syringe or the flow module (shown in Figure 1) is connected to the upstream inlet well, and the height of fluid in the reservoir is maintained above the height at the downstream well. </li>
 <li>Samples are collected at the waste well at regular intervals and weighed on an analytical balance.</li>
 <li>The slope of the curve plotting total volume vs. total time gives the average volumetric flow rate (Figure 2). We have found reproducible, linear average velocities for a wide range of pressure heads and for different pressure-maintaining systems.</li>
 </ol>
III. Flow Visualization, Velocity Mapping, and Hydrophilic / Hydrophobic Interaction
 <ol>
 <li>For model calibration, 3 &mu;m fluorescent latex beads in both Carboxy YG and Plain YG surfaces were acquired from Polysciences, Inc. and were diluted in DI water to the concentration of 0.01 % solids. </li>
 <li>Beads flowing through the habitats were visualized on a Zeiss Axiovert 25 fluorescence microscope equipped with a Mightex BCE-B013-U monochrome camera. </li>
 <li>Individual frames were captured in rapid succession and assembled into movies with the ImageJ software package (Figure 3).</li>
 </ol>
IV. Microfluidic Device (EcoChip) for Modeling Bacterial Growth and Transport
<ol>
 <li>Vibrio sp. GFP Kan, a non-pathogenic green fluorescent protein-expressing organism was grown for 14 hours in Luria-Bertani medium to a concentration of approximately 109 cells/ml.</li>
 <li>Bacteria in the growth medium were introduced into EcoChip devices and allowed to colonize the device and establish flocs and films overnight for 19 hours.</li>
 <li>Then the growth media was removed from the wells and all of the habitats were flushed with artificial seawater (please see Wang et al. 2 for ASW recipe) so that different densities of bacteria were remaining in different habitats. </li>
 <li>One habitat was kept sealed with no flow, while slow flow conditions were maintained in the 3 other habitats. </li>
 <li>Fluorescent and white light pictures of the bacterial growth were taken every 15-20 hours for a period of several days (Figure 4).</li>
</ol>
V. Representative Results
The flow module provides simple means for regulating and distributing flows through multiple habitats. Gravimetric analysis proved to be an easy and straightforward way to determine the flow rates through the habitat structures, which depend on the hydraulic resistance of the habitats and the pressure differences between input and the output wells. For the bead flow experiments we have observed much larger bead accumulation at the device surfaces for the uncarboxylated beads (Figure 2). Additionally, larger diameter beads, 6 and 10 mm, were much more likely to become entrained in the smaller pore openings and begin to accumulate in the device. Faster flow rates reduced particle retention and entrainment. 
During the bacteria growth experiments, the influence of flow conditions is clearly evident. Continuous shearing forces cause bacteria to aggregate together and form flocs, and not to be found as individual cells. Transport of large bacterial flocs is an important environmental process which is extremely difficult to study in a macroscopic system. 
<img src="/files/ftp_upload/1741/1741fig1.jpg" alt="Figure 1" /> Figure 1. Schematic showing features and operation of flow module.
<img src="/files/ftp_upload/1741/1741fig2.jpg" alt="Figure 2" /> Figure 2. Flow rate through the structured habitat as determined by gravimetric analysis for different column heights. Column height ratio: 60 / 40 = 1.5, determined flow rate ratio: 1.04 / 0.71 = 1.46. Resulted flow rates for H = 40 mm is V = 0.71 &mu;L/min and for H = 60 mm is V = 1.04 &mu; L/min. Estimated average linear velocities are 3.1 mm/s and 4.6 mm/s respectively. 
<img src="/files/ftp_upload/1741/1741fig3.jpg" alt="Figure 3" /> Figure 3. Transport of 3um latex beads with and without carboxylation flowing through the structured habitats.
<img src="/files/ftp_upload/1741/1741fig4.jpg" alt="Figure 4" /> Figure 4. Change in bacterial growth and biofilm formation as a function of seed density in presence of a slow flow.

<ol>
	<li>Whitesides, G., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. <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><li>Wang, W., Shor, L. M., LeBoeuf, E. J., Wikswo, J. P., Kosson, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobility+of+protozoa+through+narrow+channels.">Mobility of protozoa through narrow channels.</a> Applied and Environmental Microbiology. 71, 4628-4637 (2005).</li></ol>

The EcoChip system is adaptable to the needs of an individual experiment. New masters can be created relatively easily, and once a master is fabricated, additional exactly replicated devices can be cast as needed. The flow module is simple to use, requires no special equipment or complex connections, and can be modeled as a simple falling head pressure-driven flow system. Additional extensions to this work are ongoing, and include creating humic acid coated channels, and systematically varying the aqueous chemistry of th...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>SU8-2025</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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window on a microworld: simple microfluidic systems for studying microbial transport in porous media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Watch this Scientific Journal Video about Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media at JoVE.com

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

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Research

JoVE Journal

Biology

10.8K Görüntüleme.  Vanderbilt University. Mikroakışkan cihazlar gerçek zamanlı olarak ve uygun fiziksel ölçeklerde karmaşık doğal süreçler görselleştirmek için kullanılabilir. Biz yeraltı bakteri büyüme ve taşımacılık eğitimi için doğal gözenekli ortamın temel özellikleri taklit basit bir mikroakışkan cihaz geliştirdik.

Video: Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

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 ...

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-&beta;-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.