The authors would like to thank Professor Howard Ceri from the Biological Sciences Department of the University of Calgary for providing bacterial strains. A. Kumar acknowledges support from NSERC. T. Thundat acknowledges financial support from the Canada Excellence Research Chair (CERC) program. The authors would also like to acknowledge help from Ms. Zahra Nikakhtari for help with videography.

Perform the experimental protocols here in the order described below. Microfabrication protocols for creating the microfluidic platform are discussed in Step 1. Step 2 describes the bacterial culture protocol (Figure 2), and Step 3 pertains to assembly of the experimental setup (Figure 3). Finally, the actual experimental step is described in Step 4.
1. Chip Fabrication Procedure
NOTE: Proper safety procedures must be followed for the...

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The authors have nothing to disclose.

We demonstrated a simple microfluidic device that mimics porous media for studying biofilm development in complex habitats. There are several critical steps that dictate the outcome of the experiments. They include device geometry. While the post geometry can vary, adequate pore-space for streamers to form is necessary. Moreover, Valiei et al.1 have demonstrated that streamer formation occurs only in a certain flow rate range. At flow rates lower than a threshold value, deformation of biofilms into st...

Recently, we demonstrated bacterial biofilm formation dynamics in a microfluidic device that mimics porous media1. Bacterial biofilms are essentially colonies of surface aggregated bacteria that are encased by extracellular polymeric substances (EPS)2-4. These thin films of bacteria can form in almost every conceivable niche ranging from smooth surfaces to the much more complex habitat of porous media. Valiei et al.1 used a microfluidic device with an array of micro-pillars to simulate a porous media structure and studied biofilm formation in this device as a function of fluid flow rate. They found that in a certain flow regime, filamentous biofilms known as streamers began to emerge between different pillars. Streamers can be tethered at one or both ends to solid surfaces, but the rest of the structure is suspended in liquid. Streamer formation typically starts after an initial layer of biofilm has formed and its formation can dictate the long-term evolution of biofilm in such complex habitats. Recently, several researchers have investigated the dynamics of streamer formation. Yazdi et al.5 showed that the streamers can form in vortical flows originating from an oscillating bubble. In another experiment, Rusconi et al.6 investigated the effect of channel curvature and channel geometry on the formation of streamers. They found that the streamers can form in curved sections of microchannels, and streamer morphology is related to motility. Recent research has demonstrated that streamers can have wide repercussions in various natural and artificial scenarios as they can act as precursors to the formation of mature structures in porous interfaces, lead to rapid and catastrophic biofilm proliferation in a biomedical systems, and also cause substantial flow-structure interactions, etc1,7-9.
Biofilm streamers often form in complex habitats such as porous media. Understanding biofilm growth in porous media environment is relevant to several environmental and industrial processes such as biological wastewater treatment10, maintaining well-bore integrity in situations such as CO2 capture11 and plugging of pores in soil12. Observing biofilm formation in such complex habitats can often be challenging due to the opacity of porous media. In such situations, microfluidics based porous media platforms can prove extremely advantageous as they allow real-time and in&#160;situ monitoring. Another advantage of microfluidics is the ability to build multiple bioreactors on a single bio-microfluidic platform and simultaneously allow for online monitoring and/or incorporation of sensors. The flexibility to implement multiple laboratory experiments in one device and the ability to collect significant pertinent data for accurate statistical analysis is an important advantage of microfluidic systems13,14.
In the context of the above discussion, understanding streamer formation dynamics in a porous media environment would be beneficial to several applications. In this study, we develop the protocol for investigating streamer formation in a device that mimics porous media. Fabrication of the microfluidic platform, necessary steps for cell culture and experimentation are described. In our experiments, the wild type bacterial strain of Pseudomonas fluorescens was employed. P. fluorescens, found naturally in soil, plays a key role in maintaining soil ecology15. The bacterial strain employed had been genetically engineered to express green fluorescent protein (GFP) constitutively.

<table border="0" cellpadding="0" cellspacing="0" width="1038">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Name of Material/ Equipment</td>
			<td style="width:255px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:339px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Flourescent Microscope</td>
			<td style="width:255px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">LB agar</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">BP1425-500</td>
			<td>suspend 40 g in 1 L of purified water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">LB broth</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">BP1427-500</td>
			<td>suspend 20 g in 1 L of purified water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Biosafety hood</td>
			<td style="width:255px;">Microzone corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Petri-dish</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">875712</td>
			<td>sterile 100mmx15mm polystyrene petri dish</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:325px;">Incubator shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:339px;">Excella E24incubator shaker series</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL sterilized centrifuge tube</td>
			<td style="width:255px;">Corning</td>
			<td style="width:119px;">430828</td>
			<td>Polypropylene Rnase-/Dnase-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Tetracycline free base</td>
			<td style="width:255px;">MP Biomedicals</td>
			<td style="width:119px;">103012</td>
			<td>50 ug/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">SYLGARD 184 silicone</td>
			<td style="width:255px;">Dow Corning Corporation</td>
			<td style="width:119px;">68037-59-2</td>
			<td>Elastomer Base and curing agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Positive photoresist (AZ4620)</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Plastic tube</td>
			<td style="width:255px;">Cole- Parmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the above mentioned microfabrication protocol, a PDMS based microfluidic device was constructed. Figure 1 shows the scanning electron microscope (SEM) images of the PDMS device. Figure 1a shows the entrance section of the device. A fork-like entrance is created to equalize pressure head across the device. Further SEM imaging also showed that the pillar walls are almost vertical (Figure&#160;1b). The cultured bacterial solution (Figure&#160;2) was d...

Watch this Scientific Journal Video about Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars at JoVE.com

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

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 ...

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12.1K vistas.  University of Alberta. 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. 