Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Hello, I'm Matt Ma Niton from the Technical University of Denmark. Hi, and I'm Reba from the University of Copenhagen. We work on yeast and bacterial biofilms in flow cell systems And in this movie we are gonna show you how to set up a flow cell system and visualized biofilm by convertible micr.

Should we do that now? Yeah, sure. Let's do it.

Gluing of the flow cells. The flow cells are located between the bubble trap and waste bottle. Now we'll show you how to attach.

This is stratum to the flow cell. This is done by the several components that you see on the table. A 200 microliter tip is cut into two pieces.

The thin part is now inserted into a syringe, which will become the perfect tool for applying thin lines of silicon. In order to glue the glass of stratum to the flow cell, push the thin tip out of the syringe. Now, in order to make an easier application of the silicon to the base of the flow cell, cut the outmost part of the tip off, take out the piston and apply an appropriate amount of silicon into the syringe.

Put the piston back into the syringe and push a little bit of silicon out in order to make sure that all the bubbles are gone. Now start applying thin lines of silicon to the flow cell. Try and make smooth and undisrupted lines of silicon to the flow cell.

This will ensure that the silicon will attach evenly to the substratum When applying silicon in The opposite direction. Make sure not to scratch out the already made lines, but try and gently lift the tip as you go across, and this way you'll make sure that no openings in the silicon lines will lead to leakages between the chambers or from out the flow cell. Take a cover glass and gently place it on top of the silicon lines on the flow cell.

As seen here, the cover glass will easily stick to the silicon glue. Turn the flow cell over and gently push down on the back of the flow cell with your fingers. This will ensure that the cover glass will attach in a flat manner according to the flat surface of the table.

Now to make a perfect seal or to try and correct for unsealing areas, gently push down with an instrument As a piston from a syringe. Do this very carefully. As a cover glass can easily break.

By doing this, you'll be able to make an even better seal and you will probably be able to correct eventually troublesome zones to avoid leakages inspecting the flow cells. In order to have a successful experiment, it's important to inspect your flow cells after the silicon has solidified. Here we show a flow cell with a non-perfect seal, which undoubtedly will lead to leakages assembly of the bubble traps and attachment of the tubings.

The bubble traps are located between the pump and the flow cells. Here we show the different components for the use of the DTU bio bubble traps. They're fabricated in a way that will ensure that the bubbles are trapped in the system and only media without bubbles will enter the flow cells.

Five milliliter syringes are placed on top of each of the special nozzles. On the bubble trap, a tight seal is insured by the old rings. On each nozzle, place a cap on each of the syringes.

Now, as this will be the Connection for the flow cells, be sure to attach the long tubings for the flow cell to be able to reach a confocal microscope. These tubings should be attached to the bubble traps where the lower ledge of the nozzle has its outlet. As the entry ledge in the bubble trap is higher than the exit ledge, the bubbles are not able to leave the syringe and enter the flow cell attachment of the flow cells to the bubble traps.

The flow cells are located between the bubble traps and the waste bottle. Here it is required to have long tubings for the flow cell to reach the confocal microscope. Again, be sure to connect the flowal to the correct side of the bubble trap.Here.

Notice the Long tubings leading to the flowal from the bubble trap. This can of course vary on the construction of one setup. Now firmly attach all the tubings to the flow cell Assembly of the system Bubble traps to the pump and the T connectors.

For the media feeding tubes. For the peristaltic pump, we use the RIN tubings. This specific kind of tubings are able to withstand the mechanical pressure from the pump.

Set up a tube for each of the chambers in your flow cells. Here we'll now show how the tubings going from the media bottle to the pump are assembled. Cut the silicon tubing into appropriate size pieces and connect them with the T connectors.

Assemble the tubings so that there will be one connection for each of the mar brain tubings in the pump. Be sure to make a tube for each of the chambers in the flow cell. Try to make your tubes as short as possible to have an easier overview of your connections.

Now from the center feeding tube attached onto the T connectors short tubes that can be attached to the ING tubings in the Pump. Be sure that the silicone tubes are tightly attached to the connectors. Now with the media feeding Tubes on the left side of the pump, we'll now attach the rest of the system to the pump.

Here are the tubings going to the bubble trays are attached to the rine tubings on the pump. Again, here you will lighten the overview of your experiment. If you know which tubes goes to which connection in the pump, the length of these tubings are not as crucial as the tubings on the other side of the bubble trap.

Leading media to the flow cells, We have now shown You how to successfully assemble a system with one bubble trap and one flow cell On the exit point of the flow cell. We have connected tubings that are long enough to go into a waste container sterilization of the system in order to sterilize the system, make a 0.5 solution of sodium hydrochloride. Place the feeding tubes of the pump into the hydrochloride solution.

At this point, it is extremely important to fill the whole system with the hydrochloride solution. Allows syringes of the bubble traps to fill totally with hydrochloride when they're filled, put the caps back on, which in the meantime should have been placed in ethanol. An important step in the sterilization of the system is the removal of bubbles from the flow cells.

Such bubbles can easily be seen in the flow cells shown here, but the high success of bubble removal turn the pump to its highest rounds per minute. This will lead to a higher liquid flow in the flow cells, which in time will help to get rid of the bubbles, lift the flow cell and lift the tubings leading to the flow cell and firmly tap the flow cell on the table to loosen the bubbles in order to get them flushed out. With the liquid flow, this procedure will sometimes take a long time, but you will in this way be able to get rid of the bubbles.

If you're a patient, now, leave the flow cells sterilized for three to four hours at a flow of approximate 0.2 millimeters per second. Washing the system first into the system by allowing air to push the hydrochloride out of the entire System. As the system now Has been emptied up the hydrochloride, place the central feeding tube into a sterile bottle of water and cover the top.

The system now needs To be washed two to three times by filling an empty in the system with sterile water, connecting the medium and saturation of tubings overnight. When connecting the media bubble to the system, the system has to be emptied of water. As with the hypo chlorate, fill the bubble trash with media before putting the caps back on.

When connecting the media, be very careful to keep everything sterile. Have ethanol ready at the side at the connection point. Place the two tubes in the ethanol just before connecting the tubes.

Now with the media attached to the system, it is now ready for overnight saturation or inoculation. Depending on the choice of media inoculating the flow cell. In the DTU bio flow cells, you have three inoculation sites for the three individual growth chambers.

The first thing to do is to sterilize the inoculation site from an overnight culture. Make it allusion to reach your desired optical density. Use 500 microliter syringe with a 27 G needle to inoculate the sample.

An inoculum of 250 microliters is sufficient for the DTU bio flow cells. Now the inoculation site is wiped off with the net ethanol swipe and the inoculation side is closed off with silicon glue. Now turn the flow cell upside down and leave it in this position for an hour without any media flow.

After the flow cell has been upside down for an hour, take the clam back off and start the media flow and place a flow cell in an upside position again. Now start the incubation of the system. The incubation time and choice of temperature vary among choices, strains and experimental setups, staining of a yeast biofilm, and visualization by confocal laser scanning microscopy.

In order to visualize the yeast biofilm with a confocal microscope, we stain it with the green fluorescent nucleic acid stain CY nine. Here we use a dilution of one to thousand. First, the tubings leading back to the bubble traps from the flow cells are clammed off.

This will prevent backflow from the flowal during the staining procedure. Now stop the media flow to the bubble trap. Use a 500 microliter syringe as for the inoculation to inject the cyto nine stain into the flow chamber.

Again, use an ethanol wipe to sterilize the inoculation site. Inject 250 microliters into the chamber here. Be very careful and gentle.

When inoculating inoculation speed should be very low in order not to disrupt the biofilm. Close off the inoculation hole with silicon glue. Allow the staining to settle for 15 minutes without flow.

Take off the clamp and start the flow again. You're now ready for analyzing the stained biofilm. With confocal laser scanning microscopy, the flow cells has special ledges which enable it to be placed in the holders on the microscope stage.

When the flow cell is placed in the microscope stage, a drop of oil is placed on the glass of stratum. In order to use the oil immersion objective, turn on the white light and lower the objective for it to get in contact with the oil and locate the cells in the chamber. Be careful if using a glass of stratum as it can easily break.

Here we show a yeast biofilm as seen with the white light in the microscope. As the fluorescent light is turned on, one can easily see the cyto nine staining of the cells. The cyto nine stain will now enable scanning of the biofilm with a confocal microscope.

For further analysis with the Amer software, locate the position of interest in the flow chamber with the microscope. Remember to take into account for the different environments that is generated over time within the flow chamber. When choosing a location, now switch over the microscope from ocular visualization to scanning laser visualization.

With the size LM software, now start applying the decide settings for scanning your samples. A fast scanning of your samples will give you indication of the structural composition of the biofilm in the growth chambers. Before starting the more time consuming high resolution scanning here we said What is the first and what is the last position of the scanning, and thereby how big the stack will be.

We're able to scan the several frequencies due to the different lasers installed in the microscope. This will enable investigation of numerous different subpopulations. Here we see the green shadow nine stain yeast biofilm presented in the window from the specific wavelengths used.

Each of the scan stacks can now be analyzed in software programs as errors.