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

Summary

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.

Abstract

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.

Introduction

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 regim....

Protocol

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 processes described below. Consult the institutional safety officer for details.

1. Design the mask with an appropriate software (e.g.....

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 1b). The cultured bacterial solution (Figure 2) was diluted and.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Flourescent Microscope	Nikon
LB agar	Fisher	BP1425-500	suspend 40 g in 1 L of purified water
LB broth	Fisher	BP1427-500	suspend 20 g in 1 L of purified water
Biosafety hood	Microzone corporation
Petri dish	Fisher	875712	sterile 100 mm x 15 mm polystyrene Petri dish
Incubator shaker	New Brunswick Scientific		Excella E24 incubator shaker series
50 ml sterilized centrifuge tube	Corning	430828	Polypropylene RNase-/DNase-free
Tetracycline free base	MP Biomedicals	103012	50 μg/ml
SYLGARD 184 silicone	Dow Corning Corporation	68037-59-2	Elastomer Base and curing agent
Positive photoresist (AZ4620)
Plastic tube	Cole-Parmer