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

The overall goal of this procedure is to demonstrate the formation of bacterial streamers in a microfluidic device with micro pillars. This is accomplished by first fabricating the microfluidic chip. The second step of the procedure is to culture the tested bacteria, in this case pseudomonas fluorescence.

The final steps are to assemble the experimental setup, inject the bacteria into the microfluidic chip, and collect data. Ultimately, results can show time evolution of streamers through flu fluorescence microscopy images. Visual demonstration of this metal is critical as the different steps are difficult to learn.

Because of the interdisciplinary nature of the experiment, This procedure requires silicon wafers with deep reactive ion etching. The photoresist on the wafers should be washed off. Begin with silent the master mold.

Add two or three drops of trichloroethylene to a vial and place it in a desiccate along with the mold design upward beside it. In two or three hours, the mold will be siloized. During the siloization, prepare the PDMS mix cigar 1 8 4 silicone base with a curing agent at a 10 to one ratio.

Then degas the PDMS under a vacuum for about two hours. Now transfer a siloized wafer to a holder and pour the PDMS over it, ensuring that no bubbles are formed. Allow the PDMS to cure on the wafer at 80 degrees Celsius for two hours.

This fabricates A-P-D-M-S stamp from the sized silicon mold. When the curing is completed, peel off the PDMS stamp with the aid of a cutter. Then cut the stamp into a microfluidic chip using the cutter.

Now, using a cutting core drill out holes at the inlet and outlive positions on the stamp. The next step is to bond the stamp to glass. Expose a 24 millimeter cover slip and the PDMS stamp to oxygen plasma for 30 seconds.

After the exposure, attach the cover slip to the bottom side of the PDMS stamp and a bond will form. Place the assembly in a 70 degree Celsius oven for 10 minutes. To complete the process for this protocol, prepare LB plates made with ultrapure water and dosed with 50 micrograms per milliliter of tetracycline added at 50 to 55 degrees Celsius.

When pouring the plates, bubbles can be popped by flaming them. The cooled and solidified plates should be dated and stored at four degrees Celsius in a tinfoil wrap. Also have prepared LB broth made with ultra pure water and dosed with 50 micrograms per milliliter of tetracycline.

Store the broth at four degrees Celsius and also wrapped in foil. Now culture stocks of pseudomonas fluorescence stored at negative 80 degrees Celsius on the LB plates, streak the plates in a zigzag pattern and incubate them overnight at 30 degrees Celsius in the dark. The next day, pick a colony from the plate and inoculate a flask of freshly prepared S one incubated overnight at 30 degrees Celsius with 150 RPM of shaking.

Measure the optical density of the culture after the incubation. Then make the S two solution. Add five milliliters of LB to a plastic tube, and then add enough of the S one solution for an OD at 600 nanometers of 0.1, which is typical for a biofilm experiment.

First, set up the experiment using tweezers Connect plastic tubes with an inner diameter of 0.20 inches to the inlets and outlets of the prepared chip. The tubes must be flexible and sufficiently long. Here they are about 20 inches.

Then fill syringes with the S two solution. Attach 30 gauge blunted needles and remove bubbles. Preventing air bubbles from entering the channel is a critical step, especially due to line duration of the experiment.

Bubbles can damage the soft structures formed by the bacteria. Then connect the syringes to the inlet tubes and connect the outlet tubes to waste containers. Now position the syringes into a syringe pump and set the pump to the desired flow rate, such as eight microliters per hour on a microscope outfitted with a cell chamber set to the incubation temperature.

Use a 40 x objective perhaps to view the microfluidic chip. Now, start the pump. When the bacteria are introduced to the chamber, biofilm formation is also initiated.

The biofilm will form and mature for hours, days, take images to track its progress. Using SEMA fabricated microfluidic chip was imaged. The fork like entrance is created to equalize pressure head across the device.

It can also be seen that the pillar walls are almost vertical. To examine biofilm formation pf fluorescence were injected into the device at eight microliters per hour. Biofilm formation started after a few minutes of infusion of the diluted bacterial culture.

However, after a few hours appearance of filamentous structures extending between micro pillars was observed near the midsection. The dashed ellipse demarcates a forming streamer streamers formed tethered at one end to the pole region of one of the micro pillars. Streamers were also seen along the diagonal between two micro pillars.

The thickness of streamers increased with time due to cell division, as well as incorporation of planktonic bacteria. They also proliferated with time streamers occupying a large volume in the device led to formation of mature biofilm, which could clog the device. A mechanical simulation of flow through the device shows that streamers initially orient themselves along fluid streamlines.

Moreover, in the initial phase, streamers originate at locations of highest flow velocity. After watching this video, you should have a good understanding of how biofilm streamers form and evolve in a microfluidic environment. Don't forget that working with bacteria can be extremely hazardous, and precautions such as biosafety protocols should always be taken while performing this procedure.