Name: a microfluidic platform to study bioclogging in porous media
Uploaded: 2022-10-13T15:00:00.000+00:00
Duration: 5 min 10 s
Description: Watch this Scientific Journal Video about A Microfluidic Platform to Study Bioclogging in Porous Media at JoVE.com

The authors acknowledge support from SNSF PRIMA grant 179834 (to E.S.), discretionary funding from ETH (to R.S.), ETH Zurich Research Grant (to R.S. and J.J.M.), and discretionary funding from Eawag (to J.J.M.). The authors would like to thank Roberto Pioli for illustrating the experimental setup in Figure 1B and Ela Burmeister for the silicon wafer preparation.

1. Silicon wafer preparation
<ol>
	<li>Design the geometries of the microfluidic channel in computer-aided design (CAD; see Table of Materials) software and print it onto a transparent film to create the photomask (Figure 1A).</li>
	<li>Fabricate the master mold by soft lithography (under clean-room conditions) following the steps below.
	<ol>
		<li>Bake the silicon wafer at 200 &#176;C for 2 h.</li>
		<li>Place the wafer at the center of a spin-coater and pour SU8 3050 photoresist (see Table of Materials) onto the wafer. Spin coat at 1,700 rpm for 40 s with a 10 s/100 rpm ramp time. 
		NOTE: The spin-coating parameters were set to obtain a target thickness of 100 &#181;m for the SU8 3050.</li>
		<li>After the spin-coating process, soft bake the silicon wafer at 65 &#176;C for 600 s and 95 &#176;C for 2,700 s. Let the wafer cool at room temperature overnight. 
		NOTE: The overnight cooling enhances the adhesion of the SU8 to the wafer.</li>
		<li>Place the photomask (step 1.1) onto the wafer and expose it to UV light, with an exposure energy of 250 mJ/cm2 and at a wavelength of 350 nm.</li>
		<li>Post-exposure, bake the exposed substrate at 65 &#176;C for 60 s and 95 &#176;C for 300 s.</li>
		<li>Develop the silicon wafer to obtain the master mold by immersing it in a beaker filled with mrDev600 developer (see Table of Materials). Gently shake the beaker for 1,800 s to wash out the unpolymerized resist. Then, splash wash by spraying isopropanol on the silicon wafer and air dry.</li>
		<li>Hard bake the silicon wafer at 200 &#176;C for 1,800 s.</li>
	</ol>
	</li>
	<li>Silanize the master mold through vapor deposition of 20 &#181;L of Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane (see Table of Materials) placed on a glass slide next to the mold for 40 min in a vacuum desiccator, creating a gauge pressure of 100 mbar.</li>
</ol>
2. Fabrication of the microfluidic device
NOTE: The fabrication procedure described here is for a microfluidic device with one microfluidic channel. However, the same method can be applied to fabricate a microfluidic device with multiple microfluidic channels in parallel.
<ol>
	<li>Mix the elastomer with its crosslinker at a ratio of 10:1 (see Table of Materials) to prepare a PDMS mixture. Stir the mixture until it gets uniformly mixed and turns opaque due to the enclosed air bubbles.</li>
	<li>Degas the mixture in a vacuum desiccator, creating a gauge pressure of 100 mbar until the entrapped air bubbles are removed and it looks transparent. The time required for degassing is typically 30 min.</li>
	<li>Place the master mold (step 1) in a cell culture dish (see Table of Materials). Pour 20 g of the PDMS mixture on the master mold to produce channels with a final thickness of 5 mm.</li>
	<li>Bake the master mold at 70 &#176;C for 2 h.</li>
	<li>Cut the cured PDMS around the microfluidic channel (at a distance of approximately 3 mm) using a blade, and then peel the PDMS microfluidic channel off the master mold.</li>
	<li>To create the microfluidic channels&#39; inlet and outlet, punch holes with a biopsy punch (diameter of 1.5 mm) at its extremities (top of the triangles, see Figure 1A). Punch one additional hole at the center of the inlet triangle to install the pressure sensor later.</li>
	<li>Wash a glass slide and the microfluidic channel with a commercially available 1% detergent solution (see Table of Materials) for 5 min, then rinse them with deionized water. Thereafter, wash the PDMS microfluidic channel and the glass slide with isopropanol. Then, rinse them again with deionized water. Dry the PDMS microfluidic channel and the glass slide with compressed air at 1 bar for 1 min. 
	NOTE: The porous structure of the PDMS must be completely dry for the bonding to be effective.</li>
	<li>Place the glass slide and the microfluidic channel in a plasma cleaner (see Table of Materials) and ensure the surfaces to be bonded are facing up. Turn on the plasma cleaner and treat the microfluidic channel and glass slide with air plasma at an airflow of 1 SL/h (standard liter per hour) for 1 min. Bond the microfluidic channel to the glass slide immediately after taking them out of the cleaner by putting them in contact with each other. 
	NOTE: Ensure not to touch the treated surfaces, as this might affect the bonding. When fabricating a microfluidic device with multiple microfluidic channels, expose the microfluidic channels simultaneously and bond them in a single step.</li>
	<li>Place the bonded microfluidic device on an 80 &#176;C hot plate for at least 15 min.</li>
	<li>Store the microfluidic device in a clean cell culture dish until the experiment starts.</li>
</ol>
3. Preparation of the bacterial suspension
<ol>
	<li>Grow a population of Bacillus subtilis NCIB 3610 20 h prior to the start of the experiment by directly inoculating 3 mL of nutrient broth no. 3 culture medium (see Table of Materials) from a frozen glycerol stock in a 15 mL culture tube. Incubate in a shaking incubator at 30 &#176;C and 200 rpm overnight (for 16 h).</li>
	<li>Make a subculture from the overnight culture 4 h prior to the start of the experiment by adding 3 &#181;L of the overnight culture in 3 mL of fresh culture medium (1:1,000 dilution) in a 15 mL culture tube. Incubate the subculture in a shaking incubator at 30 &#176;C at 200 rpm for 3.5-4 h to obtain an optical density at 600 nm (OD600) of 0.1.</li>
</ol>
4. Biofilm growth experiment
<ol>
	<li>Turn on the box incubator of the microscope 3 h before the experiment to ensure a stable temperature of 25 &#176;C. Mount the syringe pump and the pressure sensors (see Table of Materials).</li>
	<li>Connect the inlet and outlet tubing to the microfluidic device. Directly insert a needle (with an outer diameter of 0.6 mm) into the inlet tubing to secure the connection between the tubing and the syringe.</li>
	<li>Place the microfluidic device, 30 mL of deionized water, and 30 mL of culture medium in a vacuum desiccator and degas them for at least 1 h. Then, slowly pull the culture medium and the deionized water into two separate 30 mL syringes. 
	NOTE: This step is crucial to prevent bubble formation in the channel while flushing with the culture medium.</li>
	<li>Mount the microfluidic device on the microscope and place the outlet tubing in a waste container.
	<ol>
		<li>Connect the syringe filled with deionized water to the microfluidic channel through the microfluidic tubing and slowly inject the water until it exits from the pressure sensor outlet. Fill the pressure sensor with water and flush all the bubbles from the tubing connecting the microfluidic channel and the pressure sensor. Close the outlet of the pressure sensor with the screws dedicated to the pressure sensor. 
		NOTE: The described filling procedure ensures that the pressure changes at the microfluidic channels&#39; inlet will be precisely recorded. When running an experiment with multiple microfluidic channels, connect each channel to a separate syringe to ensure equal flow conditions in all channels.</li>
	</ol>
	</li>
	<li>Fill the rest of the microfluidic channel with the deionized water.</li>
	<li>Place a 1.2 &#181;m filter (see Table of Materials) on the culture media syringe. Then, remove the water syringe and carefully connect the culture media syringe to the inlet microfluidic tubing. Mount the syringe on the syringe pump and flush the channel with the culture medium at a flow rate of 2 mL/h for 1 h. 
	NOTE: The filter prevents bacterial cells from entering the syringe during loading. Flushing the microfluidic channel with the culture media will remove the remaining bubbles in the porous structure.</li>
	<li>Set the syringe pump at the desired flow rate (here 1 mL/h) during the experiment and set the pressure reading of the pressure sensors to zero. 
	NOTE: By setting the initial pressure reading to zero, only the pressure difference caused by biofilm development during the experiment will be measured.</li>
	<li>Pipette 1 mL of the bacterial culture at an OD600 of 0.1 in a 1.5 mL centrifuge vial. Load the bacterial culture into the microfluidic channel by placing the outlet tube into the centrifuge vial. After waiting for 5 min to remove any potential air bubbles from the tubes&#39; outlet, withdraw 150 &#181;L of bacterial solution at a flow rate of 1 mL/h, until the microfluidic channel is filled with the bacterial culture.</li>
	<li>Carefully remove the culture media syringe filter and place the outlet into the waste container. Leave the bacterial cells at zero-flow conditions in the microfluidic channel for 3 h to allow their surface attachment in the porous medium. 
	NOTE: Leaving the bacterial cells at zero-flow conditions for 3 h was optimized for the attachment of the bacterial strain used while assuring a well-oxygenated bacterial culture. Other bacterial strains might require more or less time.</li>
	<li>To start the experiment, start the flow by setting the syringe pump to the desired flow rate (here 1 mL/h) and start the pressure reading at 1 Hz.</li>
	<li>Acquire images of the growing biofilm&#160;at the desired time interval, optical configuration, and magnification. 
	&#8203;NOTE: In the present study, images at 4x magnification in the bright-field mode in 18 positions spanning the entire domain of the porous medium were acquired every 6 min for 24 h.</li>
</ol>
5. Image analysis
<ol>
	<li>Reconstruct the entire porous medium from the image sequences recorded by stitching the images from the 18 positions using image analysis software (see Table of Materials) and a stitching algorithm24.</li>
	<li>Save the stitched image sequences as a sequence of singular images. 
	NOTE: If the files are too big, at this point, the images can be rescaled and binned to a reasonable size for further processing.</li>
	<li>Create a mask of the pillars of the porous medium to remove them from the analysis.</li>
	<li>Remove the background of the images by dividing them by their background with the image taken at t = 0 h (Figure 2A) and binarize the images at a threshold suitable to segment the biofilm (Figure 2B).</li>
	<li>Compute the saturation of the biofilm by calculating the area covered by the biofilm (number of pixels attributed to the biofilm) compared to the void area of the porous domain (Figure 3).</li>
</ol>

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The authors declare no conflict of interest.

Microfluidic porous media analogs coupled with pressure sensors provide a suitable tool to study biofilm development in porous media. The versatility in the design of the microfluidic porous medium, specifically the arrangement of the pillars, including diameter, irregular shapes, and pore size, allows the investigation of many geometries. These geometries range from single pores to highly complex, irregularly arranged obstacles mimicking different natural (e.g., soils) and industrial (e.g., membranes and filters) porous...

Biofilms - bacterial colonies embedded in a self-secreted matrix of extra-polymeric substances (EPS) - are ubiquitous in natural porous media, such as soils and aquifers1, and technical and medical applications, like bioremediation2, water filtration3 and medical devices4. The biofilm matrix is comprised of polysaccharides, protein fibers, and extracellular DNA5,6, and strongly depends on the microorganisms, the availability of nutrients, as well as the environmental conditions7. Yet, the functions of the matrix are universal; it forms the scaffold of the biofilm structure, protects the microbial community from mechanical and chemical stresses, and is largely responsible for the biofilms&#39; rheological properties5.
In porous media, the growth of biofilms can clog pores, causing the so-called bioclogging. Biofilm development is controlled by the fluid flow and pore size, defined as the distance separating two pillars, of the porous medium8,9,10. Both the pore size and the fluid flow control the nutrient transport and local shear forces. In turn, the growing biofilm clogs pores, affecting the velocity distribution of the fluid11,12,13, the mass transport, and the hydraulic conductivity of the porous medium14,15. The changes in hydraulic conductivity are reflected through increased pressure in confined systems16,17,18,19. Current microfluidic studies in biofilm development and bioclogging focus on studying the impact of flow velocities in homogeneous geometries16,20 (i.e., with a singular pore size)&#160;or heterogeneous porous media12,21,22. However, to disentangle the effects of flow rates and pore size on biofilm development and the resulting pressure changes in the bioclogged porous medium, a highly controllable and versatile experimental platform allowing the study of different porous media geometries and environmental conditions in parallel is required.
The present study introduces a microfluidic platform that combines pressure measurements with simultaneous imaging of the evolving biofilm within the porous medium. Because of its gas-permeability, bio-compatibility, and flexibility in the channel geometry design, a microfluidic device made of polydimethylsiloxane (PDMS) is a suitable tool for studying biofilm development in porous media. Microfluidics allow&#160;the control of physical and chemical conditions (e.g., fluid flow and nutrient concentration) with high precision to mimic the environment of microbial habitats23. Further, microfluidic devices can easily be imaged with micrometric resolution using an optical microscope and coupled with online measurements (e.g., the local pressure).
In this work, the experiments focus on studying the impact of pore size in a homogeneous porous medium analog under controlled imposed flow conditions. The flow of a culture medium is imposed using a syringe pump, and the pressure difference through the microfluidic channel is measured simultaneously with pressure sensors. Biofilm development is initiated by seeding a planktonic culture of Bacillus subtilis in the microfluidic channel. Regular imaging of the evolving biofilm and image analysis allows one to obtain pore scale resolved information on the surface coverage under various experimental conditions. The correlated information of pressure change and the extent of bioclogging provides crucial input for permeability estimations of bioclogged porous media.

<table><tbody><tr><td>Acrodisc 25 mm Syringe Filter, 1.2 &amp;micro;m Versapor Membrane</td><td>Pall Corporation</td><td>PN4190</td><td>1.2 &amp;micro;m filters</td></tr><tr><td>BD 10 mL Syringe (Luer-Lock)</td><td>BD</td><td>300912</td><td>used to fill the channel with deionised water</td></tr><tr><td>Box Incubator</td><td>Life Imaging Services</td><td></td><td>used to have a stable temperature during the biofilm growth experiment</td></tr><tr><td>Cell density meter CO8000</td><td>WPA biowave</td><td></td><td>OD meter</td></tr><tr><td>Centrifuge vial</td><td>Eppendorf</td><td>30120086</td><td>1.5 mL</td></tr><tr><td>CETONI Base 120</td><td>CETONI GmbH</td><td></td><td>syringe pump</td></tr><tr><td>CorelCAD</td><td>CorelDRAW</td><td></td><td>software used to design the microfluidic channel geometries</td></tr><tr><td>Culture tubes (14 mL, sterile)</td><td>greiner bio-one</td><td></td><td>Culture tubes</td></tr><tr><td>Drying oven, VENTI-Line</td><td>VWR</td><td></td><td>Oven to cure the PDMS</td></tr><tr><td>Handy</td><td>Migros</td><td></td><td>Detergent solution</td></tr><tr><td>Hot plate with temperature control</td><td>VRW</td><td></td><td>to cure the PDMS-glass bonding after plasma treatment</td></tr><tr><td>ImageJ</td><td>FIJI&amp;nbsp;</td><td></td><td>Image analysis software</td></tr><tr><td>Innova 42 Inc Shaker (New Brunswick)</td><td>Eppendorf</td><td></td><td>Incubator</td></tr><tr><td>Isopropanol (&gt; 99.8%)</td><td>Sigma Aldrich</td><td>67-63-0</td><td></td></tr><tr><td>Masterflex transfer tubing</td><td>Masterflex</td><td>HV-06419-05</td><td>0.020&#039;&#039; ID, 0.06&#039;&#039; OD</td></tr><tr><td>Micro Slides, Plain, 75 x 60 mm</td><td>Corning</td><td>2947-75X50</td><td>Glass slides</td></tr><tr><td>Microfluidic pressure sensor (1 bar)</td><td>Elveflow</td><td></td><td>Pressure sensors</td></tr><tr><td>Miltex Biopsy puncher, diameter 1.5 mm</td><td>Integra</td><td></td><td>Puncher to make the inlet and outlet holes of the microfluidic channel</td></tr><tr><td>mrDev600 developer</td><td>Microresist</td><td></td><td></td></tr><tr><td>Nikon Eclipse Ti2</td><td>Nikon Instruments</td><td></td><td>Microscope</td></tr><tr><td>Nutrient broth n&amp;deg;3</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Omnifix Syringe with Luer-Lock</td><td>B.Braun</td><td></td><td>syringes of different volume</td></tr><tr><td>Plasma chamber Zepto</td><td>Diener Electronic</td><td>ZEPTO-1&amp;nbsp;</td><td>used to plasma bond the PDMS and the glass slide</td></tr><tr><td>Precision wipes (Kimtech Science)</td><td>Kimberly Clark</td><td>KCP-7552</td><td>to dry the glass slide</td></tr><tr><td>Scale</td><td>VWR-CH</td><td>611-2605</td><td>used to weigh the elastomer to crosslinking agent ratio</td></tr><tr><td>Silicon wafer (10 cm)</td><td>Silicon Materials Inc.&amp;nbsp;</td><td></td><td>N//Phos &amp;lt;100&gt; 1-10 &amp;Omega; cm</td></tr><tr><td>Spincoater, Spin module SM150</td><td>Sawatec</td><td></td><td></td></tr><tr><td>SU8 3050 Photoresist</td><td>Kayakuam</td><td></td><td></td></tr><tr><td>S&amp;uuml;ss MA6 Mask aligner</td><td>SUSS MicroTec Group</td><td></td><td>used to align the chrome-glass mask</td></tr><tr><td>Sylgard 184</td><td>Dow Corning</td><td></td><td>silicone elastomer kit; curing agent</td></tr><tr><td>Techni Etch Cr01</td><td>Technic</td><td></td><td>Technic</td></tr><tr><td>Tissue culture dish 150</td><td>TPP</td><td>93150</td><td></td></tr><tr><td>Trichloro (1H, 1H, 2H, 2H perfluorooctyl) silane</td><td>Sigma Aldrich</td><td>Sigma Aldrich</td><td>used to silanize the silicane wafer</td></tr><tr><td>Veeco Dektak 6 M</td><td>Veeco</td><td></td><td>Profilometer</td></tr></tbody></table>

a microfluidic platform to study bioclogging in porous media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

For the present study, a microfluidic device with three parallel microfluidic channels with different pore sizes was used (Figure 1) to study biofilm formation in porous media systematically. The biofilm formation process was visualized using bright-field microscopy. The bacterial cells and the biofilm appeared in the images as darker pixels (Figure 2). In addition, a gradual clogging process was observed; during a 24 h experiment, the initially randomly growing...

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A Microfluidic Platform to Study Bioclogging in Porous Media

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

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Environment

1.8K Views.  ETH Zurich. The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

Video: A Microfluidic Platform to Study Bioclogging 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 ...

Biology

Microfluidic Devices for Characterizing Pore-scale Event Processes in Porous Media for Oil Recovery Applications

Microfluidic devices are versatile tools for studying transport processes at a microscopic scale. A demand exists for microfluidic devices that are resistant to low molecular-weight oil components, unlike traditional polydimethylsiloxane (PDMS) devices. Here, we demonstrate a facile method for making a device with this property, and we use the product of this protocol for examining the pore-scale mechanisms by which foam recovers crude oil. A pattern is first designed using computer-aided design (CAD) software and printed on a transparency with a high-resolution printer. This pattern is then transferred to a photoresist via a lithography procedure. PDMS is cast on the pattern, cured in an oven, and removed to obtain a mold. A thiol-ene crosslinking polymer, commonly used as an optical adhesive (OA), is then poured onto the mold and cured under UV light. The PDMS mold is peeled away from the optical adhesive cast. A glass substrate is then prepared, and the two halves of the device are bonded together. Optical adhesive-based devices are more robust than traditional PDMS microfluidic devices. The epoxy structure is resistant to swelling by many organic solvents, which opens new possibilities for experiments involving light organic liquids. Additionally, the surface wettability behavior of these devices is more stable than that of PDMS. The construction of optical adhesive microfluidic devices is simple, yet requires incrementally more effort than the making of PDMS-based devices. Also, though optical adhesive devices are stable in organic liquids, they may exhibit reduced bond-strength after a long time. Optical adhesive microfluidic devices can be made in geometries that act as 2-D micromodels for porous media. These devices are applied in the study of oil displacement to improve our understanding of the pore-scale mechanisms involved in enhanced oil recovery and aquifer remediation.

The goal of this procedure is to easily and rapidly produce a microfluidic device with customizable geometry and resistance to swelling by organic fluids for oil recovery studies. A polydimethylsiloxane mold is first generated, and then used to cast the epoxy-based device. A representative displacement study is reported.

Microfluidic devices are versatile tools for studying transport processes at a microscopic scale. A demand exists for microfluidic devices that are ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Bioengineering

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

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.

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 ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

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 ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...