We thank Matt Parsek at the University of Washington (Seattle, WA) for providing P. aeruginosa and E. coli strains and Roger Nokes at the University of Canterbury (New Zealand) for providing access to Streams software. This work was supported by grant R01AI081983 from the National Institutes of Health, National Institute of Allergy and Infectious Diseases. Confocal imaging was performed at the Northwestern Biological Imaging Facility (BIF).

1. Flow Cell Setup and Inoculation
NOTE: Use a double-inlet microfluidic flow cell described in Song et al., 201414 to grow biofilms. This flow cell is able to create well-defined smooth chemical gradients. The flow cell design is shown in Figure 1 and flow cell fabrication was previously described in Song et al., 201414. Here we detail our methods by using P. aeruginosa and E. coli to form biofilms, but other species may be suitable too. We used P. aeruginosa PAO1-gfp, which constitutively expresses a green fluorescent protein (GFP), as a model organism for biofilm development. Additionally, we used E. coli DH5&#945; to form mixed-species biofilms with P. aeruginosa. P. aeruginosa and E. coli strains were grown on LB agar plates.
<ol>
	<li>Prepare the flow cell using polydimethylsiloxane (PDMS) bound to a glass coverslip via oxygen plasma treatment as described in14. The dimensions of the flow cell chamber are 23 mm &#215; 13 mm &#215; 0.24 mm (length &#215; width &#215; depth).</li>
	<li>Prepare modified FAB growth medium15. To observe biofilm growth under a nutritional gradient within the flow cell, introduce modified FAB medium15 with 0.6 &#956;M glucose in one inlet and introduce FAB medium without any carbon source through the other inlet. Filter-sterilize (pore size = 0.2 &#956;m) the glucose stock solution (60 mM) before adding it to the FAB medium. Also autoclave the FAB medium with cycle 1 (liquid, 15 min; 121 &#176;C, 17 psi) before use.</li>
	<li>Sterilize the flow system. Prior to inoculation, autoclave the entire flow path (medium bottles, tubing, bubble traps, flow cells) using cycle 1, except for the plastic three-way valves (disposable and pre-sterilized) located upstream of the flow cell. (Three-ways valves are used for injecting cell culture, fluorescent tracer and microbeads.) To avoid contamination during assembly, cover all the tubing and connector openings with aluminum foil or autoclave bags before autoclaving.</li>
	<li>Connect the flow system. Assemble the components of the flow cell system carefully (see video for flow system assembly) and deliver growth medium to the flow cell via a peristaltic pump that precisely controls the flow rate.</li>
	<li>Prepare cell culture for inoculation by transferring a colony of P. aeruginosa or E. coli from LB plates to 3 ml of LB broth and shake the culture O/N at 225 rpm and 37 &#176;C. Dilute the O/N cell cultures in 1 ml sterilized water to a final OD600 = 0.01 as the inoculum. (Culture and dilute the bacteria in a laminar flow hood to avoid contamination.)</li>
	<li>For the experiments with mixed-species biofilms, dilute the two bacterial cultures to a ratio of 1:1 in 1 ml with an equivalent OD600 = 0.01 for each bacterium.</li>
	<li>Inoculate the flow cell. Inject 1 ml of the inoculum to the flow cell inlet from the three-way valve. After the injection, pause the flow for 1 hr to allow bacterial cells to attach to the cover glass. (Make sure that the flow cell is placed with the coverslip side down to allow suspended cells to settle onto the coverslip.)</li>
	<li>After 1 hr, resume the flow and pump the growth medium to the flow cell at a constant rate of 0.03 ml/min for each inlet for 3 days.</li>
</ol>
2. Characterizing Biofilm Development in Response to Nutrient Gradients Using Confocal Microscopy

NOTE: P. aeruginosa can be imaged with constitutively-expressed GFP, but E. coli in mixed-species biofilms must be imaged by counterstaining.

<ol>
	<li>Observe the 3-day biofilms using confocal microscopy. For the representative results, observe biofilms using a confocal microscope with a 63X objective. Prior to imaging, mark the double-inlet flow cell with a grid on the cover glass side. (The purpose of this grid is to allow the experimenter to locate the imaging regions within the flow cell chamber.)</li>
	<li>To counterstain, dilute 10 &#956;l of cell-permeant red fluorescent nucleic acid stain, such as green fluorescent nucleic acid stain such as STYO 62, stock solution (1 mM) in 990 &#956;l sterilized water and then slowly inject the diluted stain using a syringe into the flow cell chamber from the upstream three-way valve. Keep the flow cell stagnant and in the dark for 30 min, then resume the flow to wash out the unbound stain for 15 min.</li>
	<li>Perform confocal imaging. Activate the 488 nm Argon laser for excitation and the emission collecting channels for GFP (500-535 nm) and nucleic acid stain (for green fluorescent nucleic acid stain, 650-750 nm). Adjust gains and offsets for both channels to have bright and clean images. Select xyz and simultaneous imaging mode. Use the z-control knob to identify the bottom and top of the biofilm in the field. Set the z-step to 0.5 &#956;m for acquiring a 3-D image stack. (To ensure high image quality, use a line average of four for image acquisition.)</li>
	<li>To map the spatial patterns of biofilm development within the flow cell, image biofilms at three or more longitudinal (x) distances along the flow cell inlet, and at three transverse (y) positions relative to the imposed nutritional gradient, as shown in Figure 1.</li>
</ol>
3. Characterizing Internal Solute Transport by Injections of Conservative Fluorescent Tracer
NOTE: A conservative fluorescent tracer, such as Cy5, can be used to characterize solute transport patterns within the biofilm.
<ol>
	<li>Dissolve Cy5 (Mono-Reactive NHS Ester) in water to a final concentration of 10 mg/ml as a stock solution. Store the Cy5 stock solution in a -20 &#176;C freezer. 
	NOTE: As Cy5 is light-sensitive, store, dilute and inject Cy5 solutions in the dark.</li>
	<li>Prior to the injections of the fluorescent tracer, take a 3-D image stack of the 3-day biofilm with confocal microscopy (with a 63X objective). (Wide regions of biofilm colonies are ideal for time-series observations of dye transport.) Also choose a z-plane with well-separated colonies for imaging (as shown in Figure 5A).</li>
	<li>For each Cy5 injection experiment, dilute 2 &#956;l of the Cy5 stock solution in 998 &#956;l sterilized water to yield an injection solution having a Cy5 concentration of 20 &#956;g/ml.</li>
	<li>Stop the pump to pause the flow and inject the Cy5 solution into the upstream of the flow cell using a syringe via the 3-way valve. (It is important to prevent air bubbles from entering the flow path while injecting. Bubble traps should be kept open during the back injection.)</li>
	<li>After injecting the Cy5 solution, close the bubble traps, adjust the 3-way valve, and restart the pump to deliver the injected Cy5 solution into the flow cell.</li>
	<li>Switch confocal imaging mode to xyt. Activate Cy5 and GFP channels under simultaneous imaging mode. Adjust confocal settings on Cy5 intensity (laser intensity, gain and offset) to avoid saturating signals, which is critical for quantification. (High temporal resolution is preferred for visualizing dye penetration. However, fast scanning decreases image quality.) Choose proper scanning speed and line average to balance the time resolution of imaging and the image quality. For this protocol use a line average of four and a frame rate of 0.15 Hz.</li>
	<li>Resume the flow to deliver Cy5 into the flow cell (still at a flow rate of 0.03 ml/min). Simultaneously start time-series confocal imaging.</li>
	<li>Quantify Cy5 penetration by radially averaging Cy5 intensity within a 2-D circular biofilm colony. To average, first identify the edge of a biofilm cluster, then generate a distance map for a 2-D section of the cluster, and finally average radial patterns of Cy5 intensities to generate a penetration curve (see Figure&#160;6).
	<ol>
		<li>To computationally realize step 3.8, use BioSPA (Biofilm Spatial Pattern Analysis) software package (unpublished, software package and manual available upon request) or other image analysis programs.</li>
		<li>To use BioSPA, first import an image set into BioSPA. Then select proper threshold for GFP and Cy5 channels to make binary images. In the analysis/calculation menu, select Advanced Analysis and then Diffusion Analysis. Use the GFP channel to define the edge of a cluster and use the polygon tool to select one cluster as the region of interest (ROI).</li>
		<li>Double click the selected biofilm cluster to perform diffusion analysis. Save the diffusion analysis results and calibrate Cy5 intensity to Cy5 concentration using a calibration curve.
		<ol>
			<li>To generate Cy5 concentration calibration curve, measure Cy5 intensities over a Cy5 concentration gradient under confocal microscopy. (Cy5 intensity and concentration have a linear relationship.)</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Characterizing the Surrounding Flow Field by Tracking Fluorescent Particles
NOTE: Fluorescent particles, such as fluorescent microbeads, can be used to characterize the flow field around biofilms. Flow field around biofilms can be calculated from the time-series observations of particle positions using particle tracking velocimetry software. The results shown here were obtained with the Streams 2.02 software package16 (software download and user&#8217;s guide available at <a href="http://www.civil.canterbury.ac.nz/streams.shtml">http://www.civil.canterbury.ac.nz/streams.shtml</a>).
<ol>
	<li>Dilute the fluorescent microbeads (diameter ~1 &#956;m) in sterilized water to a final solid concentration of 0.2% and a final volume of 0.5 ml. Vortex to make sure that the microbeads are well dispersed.</li>
	<li>Inject and deliver the diluted fluorescent microbeads into the flow cell following procedures from steps 3.3 to 3.5. To allow an accurate tracking of the particle path, use a relatively low flow rate (0.01 ml/hr for the particle tracking results in this paper).</li>
	<li>Switch confocal settings to xyt mode to track particle movement at one z-slice and take time-series images. (A frame rate of 1 Hz was used for the representative results shown in this paper.) Repeat particle injection and image at different z locations to generate 3-D flow field.</li>
	<li>Pre-process the time-series confocal images by subtracting the background fluorescence signal (the signal in the first acquired image of the image set) using ImageJ (software download and manual available on <a href="http://imagej.nih.gov/ij/">http://imagej.nih.gov/ij/</a>) or other programs.</li>
	<li>In ImageJ, open the first image of an image set and then import the image set. Under Process / Image Calculator, subtract the first image from the image set. Save the pre-processed image set.</li>
	<li>To calculate flow vectors, import time-series confocal images in Streams 2.02. Under &#8220;Open process view&#8221;, select and execute &#8220;Identify particles&#8221;. Use proper threshold to identify particles. Use 0.5 and 5 &#956;m as minimum and maximum diameter thresholds for 1 &#956;m particles.</li>
	<li>Then under &#8220;Open process view&#8221;, select and execute &#8220;PTV analysis pipeline&#8221; to generate particle paths. Check the particle paths to see if they represent particle movement. Finally, select and execute &#8220;Create velocity field&#8221; to generate a flow vector map.</li>
</ol>

<ol>
	<li>Hall-Stoodley, L., Costerton, J. W., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+From+the+natural+environment+to+infectious+diseases.">Bacterial biofilms: From the natural environment to infectious diseases.</a> Nat Rev Microbiol. 2 (2), 95-108 (2004).</li><li>Stewart, P. S., Franklin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+heterogeneity+in+biofilms.">Physiological heterogeneity in biofilms.</a> Nat Rev Microbiol. 6 (3), 199-210 (2008).</li><li>Xu, K. D., Stewart, P. S., Xia, F., Huang, C. T., McFeters, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+physiological+heterogeneity+in+Pseudomonas+aeruginosa+biofilm+is+determined+by+oxygen+availability.">Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability.</a> Appl Environ Microb. 64 (10), 4035-4039 (1998).</li><li>Costerton, J. W., 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=Bacterial+Biofilms+in+Nature+and+Disease.">Bacterial Biofilms in Nature and Disease.</a> Annu Rev Microbiol. 41, 435-464 (1987).</li><li>Battin, T. J., Kaplan, L. A., Newbold, J. D., Hansen, C. M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+microbial+biofilms+to+ecosystem+processes+in+stream+mesocosms.">Contributions of microbial biofilms to ecosystem processes in stream mesocosms.</a> Nature. 426 (6965), 439-442 (2003).</li><li>Costerton, J. W., Stewart, P. S., Greenberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+A+common+cause+of+persistent+infections.">Bacterial biofilms: A common cause of persistent infections.</a> Science. 284 (5418), 1318-1322 (1999).</li><li>Stoodley, P., Dodds, I., Boyle, J. D., Lappin-Scott, H. M. <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+hydrodynamics+and+nutrients+on+biofilm+structure.">Influence of hydrodynamics and nutrients on biofilm structure.</a> J Appl Microbiol. 85, 19S-28S (1999).</li><li>Stoodley, P., Lewandowski, Z., Boyle, J. D., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+deformation+of+bacterial+biofilms+caused+by+short-term+fluctuations+in+fluid+shear:+An+in+situ+investigation+of+biofilm+rheology.">Structural deformation of bacterial biofilms caused by short-term fluctuations in fluid shear: An in situ investigation of biofilm rheology.</a> Biotechnol Bioeng. 65 (1), 83-92 (1999).</li><li>Wasche, S., Horn, H., Hempel, D. C. <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+growth+conditions+on+biofilm+development+and+mass+transfer+at+the+bulk/biofilm+interface.">Influence of growth conditions on biofilm development and mass transfer at the bulk/biofilm interface.</a> Water Res. 36 (19), 4775-4784 (2002).</li><li>Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-review:+Convection+around+biofilms.">Mini-review: Convection around biofilms.</a> Biofouling: The Journal of Bioadhesion and Biofilm Research. 28 (2), 187-198 (2012).</li><li>Flemming, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorption+sites+in+biofilms.">Sorption sites in biofilms.</a> Water Sci Technol. 32 (8), 27-33 (1995).</li><li>Debeer, D., Stoodley, P., Lewandowski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Flow+in+Heterogeneous+Biofilms.">Liquid Flow in Heterogeneous Biofilms.</a> Biotechnol Bioeng. 44 (5), 636-641 (1994).</li><li>Schultz, M. P., Swain, G. W. <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+biofilms+on+turbulent+boundary+layers.">The effect of biofilms on turbulent boundary layers.</a> J Fluid Eng-T Asme. 121 (1), 44-51 (1999).</li><li>Song, J. S. L., Au, K. H., Huynh, K. T., Packman, 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=Biofilm+Responses+to+Smooth+Flow+Fields+and+Chemical+Gradients+in+Novel+Microfluidic+Flow+Cells.">Biofilm Responses to Smooth Flow Fields and Chemical Gradients in Novel Microfluidic Flow Cells.</a> Biotechnol Bioeng. 111 (3), 597-607 (2014).</li><li>Shrout, J. D., 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=The+impact+of+quorum+sensing+and+swarming+motility+on+Pseudomonas+aeruginosa+biofilm+formation+is+nutritionally+conditional.">The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional.</a> Mol Microbiol. 62 (5), 1264-1277 (2006).</li><li>Maxworthy, T., Nokes, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+gravity+currents+propagating+down+slopes.+Part+1.+The+release+of+a+fixed+volume+of+heavy+fluid+from+an+enclosed+lock+into+an+open+channel.">Experiments on gravity currents propagating down slopes. Part 1. The release of a fixed volume of heavy fluid from an enclosed lock into an open channel.</a> J Fluid Mech. 584, 433-453 (2007).</li><li>Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+experimental+measurements+of+effective+diffusive+permeabilities+and+effective+diffusion+coefficients+in+biofilms.">A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms.</a> Biotechnol Bioeng. 59 (3), 261-272 (1998).</li><li>Schramm, A., De Beer, D., Gieseke, A., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironments+and+distribution+of+nitrifying+bacteria+in+a+membrane-bound+biofilm.">Microenvironments and distribution of nitrifying bacteria in a membrane-bound biofilm.</a> Environ Microbiol. 2 (6), 680-686 (2000).</li><li>Santegoeds, C. M., Schramm, A., de Beer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsensors+as+a+tool+to+determine+chemical+microgradients+and+bacterial+activity+in+wastewater+biofilms+and+flocs.">Microsensors as a tool to determine chemical microgradients and bacterial activity in wastewater biofilms and flocs.</a> Biodegradation. 9 (3-4), 159-168 (1998).</li><li>Debeer, D., Stoodley, P., Roe, F., Lewandowski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Biofilm+Structures+on+Oxygen+Distribution+and+Mass-Transport.">Effects of Biofilm Structures on Oxygen Distribution and Mass-Transport.</a> Biotechnol Bioeng. 43 (11), 1131-1138 (1994).</li><li>Liu, Y., Tay, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+role+of+hydrodynamic+shear+force+in+the+formation+of+biofilm+and+granular+sludge.">The essential role of hydrodynamic shear force in the formation of biofilm and granular sludge.</a> Water Res. 36 (7), 1653-1665 (2002).</li><li>Zhang, W., 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=A+Novel+Planar+Flow+Cell+for+Studies+of+Biofilm+Heterogeneity+and+Flow-Biofilm+Interactions.">A Novel Planar Flow Cell for Studies of Biofilm Heterogeneity and Flow-Biofilm Interactions.</a> Biotechnol Bioeng. 108 (11), 2571-2582 (2011).</li><li>Tseng, B. S., 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=The+extracellular+matrix+protects+Pseudomonas+aeruginosa+biofilms+by+limiting+the+penetration+of+tobramycin.">The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin.</a> Environ Microbiol. 15 (10), 2865-2878 (2013).</li><li>Debeer, D., Srinivasan, R., Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Measurement+of+Chlorine+Penetration+into+Biofilms+during+Disinfection.">Direct Measurement of Chlorine Penetration into Biofilms during Disinfection.</a> Appl Environ Microb. 60 (12), 4339-4344 (1994).</li></ol>

The authors have nothing to disclose.

We demonstrated a suite of methods to characterize three important biofilm-environment interactions: biofilm response to chemical gradients, effects of biofilm growth on the surrounding flow microenvironment, and biofilm heterogeneity resulting from internal transport limitations.
We first showed the use of a novel microfluidic flow cell to impose a well-defined chemical gradient for biofilm development. To generate a well-defined chemical gradient within the flow cell, it is important to main...

Microorganisms attach to surfaces and form biofilms &#8212; cell aggregates enclosed in an extracellular-polymer matrix1. Biofilms behave very differently from individual microbial cells, because biofilms have dramatic spatial heterogeneity resulting from a combination of internal solute transport limitations and spatial variations in cellular metabolism2,3. Oxygen and nutrient concentrations drastically decrease at the interface between biofilm and surrounding fluid and get further depleted within in the biofilm2. Spatial variations in biofilm respiration and protein synthesis can also occur as a response to localized oxygen and nutrient availability2.
In aquatic and soil environments, most bacteria dwell in biofilms. Natural biofilms carry out important biogeochemical processes including cycling carbon and nitrogen and reducing metals4,5. Clinically, biofilm formation is responsible for prolonged pulmonary and urinary infections6. Biofilm-associated infections are highly problematic because cells in biofilms have extremely high resistance to antimicrobials compared to their planktonic counterparts6. Because biofilms are important in diverse settings, a substantial amount of research has been focused on understanding the environmental factors that control biofilm activities and the spatial heterogeneity in biofilms and the surrounding microenvironment.
Previous studies have found that biofilm development is strongly regulated by a number of environmental factors: biofilms develop different morphologies under various flow conditions; oxygen and nutrient availability influence biofilm morphology; and hydrodynamic shear stress affects the attachment of planktonic cells to surfaces and the detachment of cells from biofilms7-9. Furthermore, external flow condition influences the delivery of substrates into and within biofilms10. The growth of biofilms also alters surrounding physical and chemical conditions. For example, biofilm growth leads to local depletion of oxygen and nutrients2; biofilms accumulate inorganic and organic compounds from the surrounding environment11; and biofilm clusters divert flow and increase surface friction12,13. Because biofilms interact with their surrounding environment in very complex ways, it is critical to simultaneously obtain information on biofilm properties and environmental conditions, and multi-disciplinary approaches need to be used to comprehensively characterize biofilm-environment interactions.
Here we present a series of integrated methods to characterize spatial patterns in microbial growth within mono-species and dual-species biofilms under an imposed nutritional gradient, and to observe the resulting modification of the local chemical and fluid microenvironment. We first describe the use of a recently developed double-inlet microfluidic flow cell to observe biofilm growth under well-defined chemical gradients. We then demonstrate the use of this microfluidic flow cell to observe the growth of two species of bacteria, Pseudomonas aeruginosa and Escherichia coli, in biofilms under a range of nutritional conditions. We show how in situ visualization of fluorescent tracer propagation into biofilm colonies can be used to quantitatively assess patterns of solute transport in biofilms. Finally, we show how microscale particle tracking velocimetry, performed under confocal microscopy, can be used to obtain local flow field around the growing biofilms.

<table border="0" cellpadding="0" cellspacing="0" width="586">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:89px;">Company</td>
			<td style="width:104px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">Peristaltic Pump</td>
			<td style="width:89px;">Gilson</td>
			<td style="width:104px;">Miniplus 3</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">PUMP TUBING 0.50MM OVC, Orange/Yellow</td>
			<td style="width:89px;">Gilson</td>
			<td style="width:104px;">F117934</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Three-way Stopcock w/ Swivel male Luer lock</td>
			<td style="width:89px;">Smiths Medical&#160;</td>
			<td style="width:104px;">MX9311L</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Sylgard 184 Solar Cell Encapsulation for Making Solar Panels</td>
			<td style="width:89px;">ML Solar LLC</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Pyrex Medium Bottle, 1L, GL45</td>
			<td style="width:89px;">VWR</td>
			<td>16157-191</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">C-FLEX Tubing</td>
			<td style="width:89px;">Cole-Parmer</td>
			<td style="width:104px;">06422-02</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">1 mL TB Syringe</td>
			<td style="width:89px;">BD</td>
			<td style="width:104px;">309659</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Polymer Tubing</td>
			<td style="width:89px;">IDEX</td>
			<td style="width:104px;">1520G</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Sterile Intramedic Luer Stub Adapter</td>
			<td style="width:89px;">Clay Adams</td>
			<td style="width:104px;">427564</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">PrecisionGlide Needle</td>
			<td style="width:89px;">BD</td>
			<td style="width:104px;">305195</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Spectrophotometer</td>
			<td style="width:89px;">HACH</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Syringe filters- sterile (0.2 &#956;m)</td>
			<td style="width:89px;">Fisherbrand</td>
			<td style="width:104px;">09-719A</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">MAXQ Shaker</td>
			<td style="width:89px;">Thermo Scientific</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Ammonium sulfate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">A4418</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium phosphate dibasic anhydrous</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">RES20908-A7</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Monobasic potassium phosphate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">P5655</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">S7653</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Magnisium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">M8266</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Calcium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C5670</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Calcium sulfate dihydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C3771</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Iron(II) sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">215422</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Manganese(II) sulfate monohydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">M7634</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Copper(II) sulfate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">451657</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Zinc sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">Z0251</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Cobalt(II) sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C6768</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium molybdate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">243655</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Boric acid</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">B6768</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Dextrose</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">D9434</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Luria Bertani Broth</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">L3022</td>
			<td>Growth media</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">TCS SP2 Confocal Microscopy</td>
			<td style="width:89px;">Leica</td>
			<td style="width:104px;"></td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:227px;">SYTO 62</td>
			<td style="width:89px;">Life Technology</td>
			<td style="width:104px;">S11344</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:227px;">Cy5</td>
			<td style="width:89px;">GE Healthcare Life Sciences</td>
			<td style="width:104px;">PA15100</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Red Fluorescent (580/605) FluoSphere</td>
			<td style="width:89px;">Life Technology</td>
			<td style="width:104px;">F-8801</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">BioSPA</td>
			<td style="width:89px;">Packman Lab</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">ImageJ</td>
			<td style="width:89px;">NIH</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">Volocity</td>
			<td style="width:89px;">PerkinElmer</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Streams 2.02</td>
			<td style="width:89px;">University of Cantebury</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
	</tbody>
</table>

methods for characterizing the co-development of biofilm and habitat heterogeneity

Les biofilms sont des communautés microbiennes attachée surface qui ont des structures complexes et produisent hétérogénéités spatiales importantes. le développement du biofilm est fortement réglementé par le flux environnante et l&#39;environnement nutritionnel. la croissance du biofilm augmente également l&#39;hétérogénéité du microenvironnement local en générant des champs d&#39;écoulement complexes et des structures de transport de solutés. Pour étudier le développement de l&#39;hétérogénéité dans les biofilms et les interactions entre les biofilms et leur micro-habitat local, nous avons grandi biofilms mono-espèces de Pseudomonas aeruginosa et les biofilms double-espèces de P. aeruginosa et Escherichia coli sous gradients nutritionnels dans une cellule d&#39;écoulement microfluidique. Nous fournissons des protocoles détaillés pour la création de gradients de nutriments dans la cellule d&#39;écoulement et pour la culture et la visualisation de développement du biofilm dans ces conditions. Nous avons également des protocoles actuels pour une série de méthodes optiques de quantifier la répartition spatiale de la structure du biofilm, distri circulerbutions sur les biofilms, et le transport de masse autour et à l&#39;intérieur des colonies de biofilm. Ces méthodes prennent en charge des enquêtes approfondies de la co-développement du biofilm et l&#39;habitat hétérogénéité.

The double-inlet microfluidic flow cell allows observation of biofilm growth under a well-defined chemical gradient formed by mixing of two solutions within the flow chamber. The resulting chemical gradient was formerly observed by dye injection and characterized in detail by Song et al.14. Smooth concentration gradients were formed in the transverse direction, as shown in Figure 1. The concentration profile was steep near the inlet and got relaxed downstream due to diffusion (<strong...

Watch this Scientific Journal Video about Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity at JoVE.com

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Méthodes de caractérisation de la Co-développement de biofilms et Habitat Hétérogénéité

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

methods-for-characterizing-co-development-biofilm-habitat

Research

JoVE Journal

Bioengineering

9.8K vues.  Northwestern University. Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies. 