Name: combining fluidic devices with microscopy and flow cytometry to study microbial transport in porous media across spatial scales
Uploaded: 2020-11-25T14:00:00
Description: Watch this Scientific Journal Video about Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales at JoVE.com

We acknowledge the help of Antoine Wiedmer with the setup of the robotic dispenser and the dispenser.py script.

1. Bacterial culture conditions
<ol>
	<li>Working under a laminar flow hood, use 100 &#956;L of a glycerol stock of GFP-tagged Pseudomonas putida KT2440 (1 &#215; 107 mL-1, stored at -80 &#176;C) to inoculate 5 mL of Luria-Bertani (LB) medium. Incubate at 30 &#176;C while shaking at 250 rpm overnight.</li>
	<li>The next day, resuspend 100 &#956;L of the overnight culture in 5 mL LB medium and incubate under the same conditions for 5h (exponential phase). Sample a 1 mL aliquot into a 2 mL ...

<ol>
	<li>Stevik, K., Aa, K., Ausland, G., Fredrik Hanssen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retention+and+removal+of+pathogenic+bacteria+in+wastewater+percolating+through+porous+media:+a+review.">Retention and removal of pathogenic bacteria in wastewater percolating through porous media: a review.</a> Water Research. 38 (6), 1355-1367 (2004).</li><li>Ribet, D., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+bacterial+pathogens+colonize+their+hosts+and+invade+deeper+tissues.">How bacterial pathogens colonize their hosts and invade deeper tissues.</a> Microbes and Infection. 17 (3), 173-183 (2015).</li><li>Ginn, T. R., Wood, B. D., Nelson, K. E., Scheibe, T. D., Murphy, E. M., Clement, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processes+in+microbial+transport+in+the+natural+subsurface.">Processes in microbial transport in the natural subsurface.</a> Advances in Water Resources. 25 (8), 1017-1042 (2002).</li><li>Tufenkji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+microbial+transport+in+porous+media:+Traditional+approaches+and+recent+developments.">Modeling microbial transport in porous media: Traditional approaches and recent developments.</a> Advances in Water Resources. 30 (6-7), 1455-1469 (2007).</li><li>Foppen, J. W., Van, M. H., Schijven, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+and+modelling+straining+of+Escherichia+coli+in+saturated+porous+media.">Measuring and modelling straining of Escherichia coli in saturated porous media.</a> Journal of contaminant hydrology. 93 (1-4), 236-254 (2007).</li><li>Battin, T. J., Besemer, K., Bengtsson, M. M., Romani, A. M., Packmann, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecology+and+biogeochemistry+of+stream+biofilms.">The ecology and biogeochemistry of stream biofilms.</a> Nature Reviews Microbiology. 14 (4), 251-263 (2016).</li><li>Scheidweiler, D., Peter, H., Pramateftaki, P., Anna, P., de Battin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+biophysical+underpinnings+to+the+success+of+multispecies+biofilms+in+porous+environments.">Unraveling the biophysical underpinnings to the success of multispecies biofilms in porous environments.</a> The ISME Journal. 1, (2019).</li><li>Carrel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms+in+3D+porous+media:+Delineating+the+influence+of+the+pore+network+geometry,+flow+and+mass+transfer+on+biofilm+development.">Biofilms in 3D porous media: Delineating the influence of the pore network geometry, flow and mass transfer on biofilm development.</a> Water Research. 134, 280-291 (2018).</li><li>Bhattacharjee, T., Datta, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+hopping+and+trapping+in+porous+media.">Bacterial hopping and trapping in porous media.</a> Nature Communications. 10 (1), 2075 (2019).</li><li>Scheidweiler, D., Miele, F., Peter, H., Battin, T. J., de Anna, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trait-specific+dispersal+of+bacteria+in+heterogeneous+porous+environments:+from+pore+to+porous+medium+scale.">Trait-specific dispersal of bacteria in heterogeneous porous environments: from pore to porous medium scale.</a> Journal of The Royal Society Interface. 17 (164), 20200046 (2020).</li><li>Morales, V. L., Parlange, J. Y., Steenhuis, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+preferential+flow+paths+perpetuated+by+microbial+activity+in+the+soil+matrix?+A+review.">Are preferential flow paths perpetuated by microbial activity in the soil matrix? A review.</a> Journal of Hydrology. 393 (1), 29-36 (2010).</li><li>Creppy, A., Cl&#233;ment, E., Douarche, C., D'Angelo, M. V., Auradou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+motility+on+the+transport+of+bacteria+populations+through+a+porous+medium.">Effect of motility on the transport of bacteria populations through a porous medium.</a> Physical Review Fluids. 4 (1), 013102 (2019).</li><li>Camesano, T. A., Logan, B. E. <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+Fluid+Velocity+and+Cell+Concentration+on+the+Transport+of+Motile+and+Nonmotile+Bacteria+in+Porous+Media.">Influence of Fluid Velocity and Cell Concentration on the Transport of Motile and Nonmotile Bacteria in Porous Media.</a> Environmental Science &amp; Technology. 32 (11), 1699-1708 (1998).</li><li>Lutterodt, G., Basnet, M., Foppen, J. W. A., Uhlenbrook, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+surface+characteristics+on+the+transport+of+multiple+Escherichia+coli+isolates+in+large+scale+columns+of+quartz+sand.">The effect of surface characteristics on the transport of multiple Escherichia coli isolates in large scale columns of quartz sand.</a> Water Research. 43 (3), 595-604 (2009).</li><li>Bozorg, A., Gates, I. D., Sen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+biofilm+on+bacterial+transport+and+deposition+in+porous+media.">Impact of biofilm on bacterial transport and deposition in porous media.</a> Journal of Contaminant Hydrology. 183 (Supplement C), 109-120 (2015).</li><li>Long, T., Ford, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Transverse+Migration+of+Bacteria+by+Chemotaxis+in+a+Porous+T-Sensor.">Enhanced Transverse Migration of Bacteria by Chemotaxis in a Porous T-Sensor.</a> Environmental Science &amp; Technology. 43 (5), 1546-1552 (2009).</li><li>Rusconi, R., Garren, M., Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+Expanding+the+Frontiers+of+Microbial+Ecology.">Microfluidics Expanding the Frontiers of Microbial Ecology.</a> Annual Review of Biophysics. 43 (1), 65-91 (2014).</li><li>Xia, Y., Whitesides, G. M. <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.">Soft Lithography.</a> Annual Review of Materials Science. 28 (1), 153-184 (1998).</li><li>Crocker, J. C., Grier, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+Digital+Video+Microscopy+for+Colloidal+Studies.">Methods of Digital Video Microscopy for Colloidal Studies.</a> Journal of Colloid and Interface Science. 179 (1), 298-310 (1996).</li><li>del Giorgio, P. A., Bird, D. F., Prairie, Y. T., Planas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+determination+of+bacterial+abundance+in+lake+plankton+with+the+green+nucleic+acid+stain+SYTO+13.">Flow cytometric determination of bacterial abundance in lake plankton with the green nucleic acid stain SYTO 13.</a> Limnology and Oceanography. 41 (4), 783-789 (1996).</li></ol>

Here we suggest two means to study the transport of microbes through porous systems at the single-cell and population level. While the study of transport phenomena using BTC modeling has provided valuable insights into the spread of pathogens or contaminants at the ecosystem scales, difficulties to scale from laboratory experiments to field conditions still exist. The tools described here allow researchers to experimentally resolve the spatial and temporal scales in order to better understand the ecological strategies of...

Studies aiming to understand the transport of microbes through porous media have mainly been driven by concerns of contamination1, the transmission of disease2 and bioremediation3. In this regard, bacteria have mostly been treated as particles in transport models4 and processes such as filtration, straining, gravitational settling or remobilization from biofilms have been identified as drivers of retention or transport of microbes5. However, studying the transport of bacteria through porous landscapes can also inform us on the ecological strategies underpinning their success in these complex environments. Yet, this requires novel experiments and mathematical models operating at the single cell, population or microbial community level.
Natural porous environments, such as those found in the hyporheic zone of streams and rivers, are densely colonized by diverse communities of biofilm-forming microbes6. Biofilms form structures that modify the flow and thus the transport and dispersal of bacteria in the liquid phase7,8. The transport of bacteria at pore scale depends to the constrained space availability in the porous matrix and motility-related dispersal may be an effective way to increase the individual fitness through reduced competition for resources in less densely populated areas. On the other hand, motile bacteria can also reach more isolated regions of the porous matrix and the extended exploration of such areas may provide ecological opportunities to motile populations10. At larger spatial scales, biofilm growth diverts the flow paths also leading to (partial) clogging of pores and, thus, to the establishment of even more channelized and heterogeneous flow conditions11. This has consequences for nutrient supply and dispersal capacity, frequency and distance. Preferential flow, for instance, can generate so-called &#34;fast-tracks&#34; and motile bacteria can attain even higher velocities than the local flow along these tracks12. This is an effective way to increase the exploration of novel habitats.
A variety of tools avail themselves for the study of transport of motile and non-motile bacteria (and particles) in porous media. Numerical models have great predictive capacities important for applications, however are often limited by inherent assumptions4. Laboratory-scale experiments13, 14 combined with breakthrough curve (BTC) modeling have provided important insights in the importance of bacterial cell surface properties for sticking efficiency15. Typically, BTCs (i.e., times series of particle concentration at a fixed location) are obtained via constant-rate releases and measurement of cell numbers at the outflow of the experimental device. In this context, BTCs reflect the advection-dispersion dynamics of bacteria in the porous matrix and can be extended by a sink term accounting for attachment. However, modeling of BTCs alone does not resolve the role of spatial organization of the porous substrate or biofilm for transport processes. Other macroscopic observables like dispersivity or deposition profiles have been proven to provide important information about the spatial distribution or the retained particles or growing communities. Microfluidics is a technology that allows studying transport in porous media by microscopy investigation9,12,16, and except a recent work10, experimental systems are typically constrained to a single length scale of resolution, that is, the pore scale or the entire fluidic device scale.
Here, we introduce a suite of combined methods to study the transport of motile and non-motile bacteria in porous landscapes at different scales. We combine observations of bacterial transport at the pore scale with information at larger scale, by means of BTC analysis. Microfluidic devices built from soft lithography using polydimethylsiloxane (PDMS) are bio-compatible, resistant to a range of chemicals, allow replicability at low costs and provide excellent optical transparency as well as low autofluorescence critical for microscopic observation. Microfluidics based on PDMS has been previously used to study the transport of microbes in simple channels17 or in more complex geometries12. However, typically microfluidics experiments focus on short-term horizons and epi-fluorescence microscopic observation of living cells is commonly restricted to genetically-modified strains (e.g., GFP-tagged strains). Here we present tools to study bacterial transport using PDMS-based microfluidic devices in combination with microscopy and larger devices fabricated from poly(methyl methacrylate) (PMMA, also known as plexiglass) in combination with flow cytometry. PDMS and PMMA differ in gas permeability and surface properties, thus providing complementary opportunities to study bacterial transport. While the microfluidic device provides a more controlled environment, the larger device allows for experiments over extended periods of time or using natural bacterial communities. Microscopy counting at high temporal resolution in a dedicated area is used to obtain BTC in the PDMS-based microfluidic device. To obtain cell counts for BTC modeling from the PMMA-based device, we introduce a self-constructed automated liquid dispenser in combination with flow cytometry. In this setup, cells pass the fluidic device and are consecutively dispensed into 96 well plates. The temporal resolution is restricted by the minimum volume that can be accurately dispensed and thus the medium flow rate through the fluidic device. Fixative in the wells prevents growth and facilitates DNA staining for downstream flow-cytometric enumeration. To prevent bacterial growth during transport experiments we use a minimal medium (termed motility buffer).
Since protocols for the preparation of fluidic devices at different scales are readily available, we only briefly introduce the techniques to produce such devices and rather focus on the experimental procedures to record BTCs. Similarly, various routines exist for the flow cytometric enumeration of microbes and users require expert knowledge to interpret results obtained by flow cytometry. We report the novel use of microfluidic devices in combination with microscopic imaging to record BTCs of fluorescently-tagged cells. At the pore scale, local velocities and trajectories are obtained by means of image processing. Further, we demonstrate the use of a PMMA-based fluidic device in combination with flow-cytometric counting to observe bacterial transport of motile and non-motile cells in porous environments colonized by a native stream biofilm.

<table>
	<tbody>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Elastomer Sylgard 184</td>
			<td>Dowsil</td>
			<td>101697</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer NovoCyte</td>
			<td>Acea</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td></td>
			<td>https://www.makeblock.com/project/xy-plotter-robot-kit</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid dispenser, XY Plotter Robot Kit</td>
			<td>makeblock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Axio Imager</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope AxioZoom v16</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides, 75 mm &#215; 25 mm</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Minipuls 3 peristaltic pump</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma bonder Corona SB</td>
			<td>BlackHole Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump New Era NE 4000</td>
			<td>New Era</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syto 13 Green Fluorescent Nucleic Acid Stain</td>
			<td>Molecular Probes, Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>Ismatec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>WF31SA universal milling machine</td>
			<td>Mikron</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To illustrate the functionality of the presented workflow, we performed experiments using genetically modified Pseudomonas putida KT2440, a gram negative motile bacterium important for bioremediation and biotechnology. Genetically modified versions of this strain that express GFP production are commercially available. A non-motile strain of P. putida KT2440 which lacks the relevant structural and regulatory genes for motility is also available. Using both, motile and non...

Watch this Scientific Journal Video about Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales at JoVE.com

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

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 ...

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