Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

This method can help answer key questions in the microbiology field, such as how fluid flow alters fungal biofilm growth and development. The main advantage of this technique is it allows us to quantitatively evaluate the effects of flow on biofilm growth and development in real time. Begin this experiment with assembly of the flow apparatus, as described in the text protocol.

The day of the experiment, attach the bubble trap, temperature probe, and all single use components. To assemble the filter, remove the plunger from a one-milliliter syringe to make it an adapter. Force the tubing from the filter bottle into this end and attach a 2 micron filter to the tubing leading to the growth flask.

Apply silicone vacuum grease around the barb of the slide adapter prior to connecting it, as this helps prevent air leaks into the system. Fill the attachment flask with 100 milliliters of YPD, and fill the growth flask with 200 milliliters of YPD. Ensure that the green side tubing reaches the media in each flask.

Ensure that all valves are open. Attach the bubble trap to a vacuum, and connect the pump to the green side tubing downstream of the bubble trap. Pump the fluid at a flow rate of 3.3 milliliters per minute to completely fill the green side.

Then, dispense and discard approximately one to two milliliters of the media, because the first couple of milliliters often contain dead cells or random debris. Ensure that the green side of the tubing is filled with media and has no bubbles downstream of the bubble trap before proceeding. Fill the channel slide and the reservoir with YPD, taking care not to introduce bubbles.

Connect the slide to the flow apparatus. Then pump more fluid to create a buffer of about 5 meters on the orange side. This is to prevent accidentally trapping air in the slide in the event of backflow.

Now, prepare the flow apparatus for transport to the microscope by first planting the inlet and outlet of the bubble trap closed. Then clamp the green and orange side attachment flask valves closed. Ensure that the screw caps for the pulsation dampener and filter bottle are tight, as they can loosen during autoplating.

Disconnect the pump from the tubing to make transport easier. Then move all components, including the hot plate stirrer, into a secondary container near the microscope. Attach the temperature probe to the hot plate stirrer, and begin heating the attachment flask to 37 degrees Celsius.

Stir the media at 300 RPM, and maintain this for the entire experiment. Mount the slide on the microscope, and use tape if necessary to tightly secure it. Then attach the bubble trap to a vacuum.

Now, connect the pump to the flow apparatus. Start the pump at a flow rate of 3.3 milliliters per minute, and allow it to run for approximately five to 10 seconds. Then, remove the bubble trap in let-out lid clan.

Allow the pump to continue running while the attachment flask heats up. Once the attachment flask and incubation chamber are both at 37 degrees Celsius, add enough overnight culture of the fungal cells to the attachment flask to reach one million cells per milliliter. Wait 15 minutes to allow the cells to acclimate.

Open both green and orange side attachment flask valves while closing both growth flask valves to start the flow of cells. Wait for approximately five minutes to allow cells to reach the slide. During this time, focus the microscope and adjust it to the same imaging parameters used in previous experiments.

Begin image acquisition for the attachment phase. Check back after approximately five and 10 minutes to ensure that focus has been maintained. If not, attempt to adjust the focus immediately after the next image is acquired.

Immediately after the attachment phase is finished, save the file. Then open both green and orange side growth flask valves while closing both attachment flask valves. Take care not to bump the stage if any valves are inside the incubation chamber.

Unplug the thermometer probe, and replace the attachment flask with the growth flask. Now, configure the software to acquire an image every 15 minutes over 22 hours, and begin image acquisition for the growth phase. Once the growth phase acquisition has finished and the file has been saved, open the green and orange side attachment flask valves, which may make a noise as the pressure releases on the orange side.

Pull up on the green side tubing coming from both media flasks, until they are at least several centimeters above the media. Then, run the pump at a high speed to remove all the media from the tubing, which makes cleaning much easier. Finally, quantify the videos as described in the text protocol.

This time lapse dark field microscopy video shows the attachment of wild type Candida albican cells to the substrate underflow during the attachment phase, followed by the subsequent growth and development during the growth phase leading to biofilm formation. The data from these videos can be analyzed for the rates of cell attachment and detachment and biomass over time, shown here is the total biomass within the imaging region as determined by densitometry analysis. The cumulative rate of cell attachment and the percent biomass detachment over time are also shown.

While attempting this procedure, it's important to remember to use the same imaging conditions across all experiments, as this allows for quantitative comparisons to be made between them.