The overall goal of this protocol is to visualize the biofilm formation of Candida albicans in real time under host physiological conditions. This method enables the observation of biofilm formation as it develops in real time and can be used to test the effectiveness of antimicrobial agents or to assess genetic mutants. This technique can be used to screen potential therapeutics for activity against biofilms in a high throughput manner and their effectiveness can be measured, visualized, and compared to current treatments.
The main advantage of this technique is that it can be used to assay biofilm formation under several different conditions that are of interest to the researchers. Though this method was developed specifically to study Candida albicans biofilm formation, it can also be applied to other biofilm-forming microorganisms including other fungal species as well as bacteria. To begin, prepare single colony cultures of different C.albicans strains from cells plated on yeast extract peptone dextrose or YPD medium agar following standard microbiological practices.
Next, turn on the microfluidic system which includes the controller, the two power supply units, the microscope, the camera, and computer attached to the system. Once the computer loads, open the software that controls the system. Locate the barcode number of the plate in use and input the number when prompted by the software.
Then in the control module, set the microfluidic system temperature to 37 degrees Celsius. Next, click on the inlet wells I1, I7, I13, and I19 and flow sterile air through the interface plate tubes for five minutes to remove any condensation. When all the condensation evaporates, place the 48-well 0-20 dyne microfluidic plate on the microscope stage.
For a 12-hour biofilm formation experiment, start by adding 600 microliters of pre-warmed medium to the inlet wells. Slowly lower the cleaned interface plate on top of the 48-well microfluidic plate. Then slide the interface plate slightly to the left until the tubes are positioned on top of the inlet and outlet wells and are locked into position.
In the shear control section of the software, set the flow mode to constant and set the max shear to one dyne per centimeter squared. Then click on the inlet wells with media to begin the flow of media from inlet to outlet wells in order to prime the channel. After five minutes of flow, click on the stop button in the well plate control section of the software.
Then remove the interface plate and observe the microfluidic plate wells. Only a small drop of media should be visible in the outlet wells. To test wild-type and mutant C.albicans strains, add 50 microliters of wild-type SC5314 cell culture in spider media to two outlet wells, add the bcr1 mutant in spider media to two outlet wells and add the EFG1 mutant in spider media to two outlet wells.
Place the interface plate back onto the microfluidic plate and lock the lid into place. Back in the shear control section of the software, set the flow mode to constant and set the max shear to two dyne per centimeter squared. Click on the outlet wells with cells to begin the flow of media from the outlet to the inlet wells to begin seeding of C.albicans.
After three seconds, click the stop button in the well plate control section to prevent cells from entering the inlet wells. Allow the cells to remain stationary with no flow for 10-20 minutes in the viewing channel for initial cell adherence. In the imaging module window, open the multidimensional acquisition module and click on the stage tab in the menu on the left-hand side.
In the stage section, load the master stage list for the 48-well 0-20 dyne microfluidic plate. Next, open the sample reload adjustment and move stage to absolute position modules. In the sample reload adjustment module, press the live button to see an image preview with crosshairs.
Using the joystick, position the well plate so the tip of the arrow adjacent to viewing channel four aligns with the crosshairs on the software. Then go to the move stage to absolute position menu and press the set origin button. Next, go back to the sample reload adjustment module, click on the initial reference point section in the left-hand menu and load the file for the 48-well 0-20 dyne microfluidic plate.
On the stage section of the multidimensional acquisition module, double click on stage position 22.2 to bring the microscope viewing stage and the camera focus into close proximity to the arrow marker by viewing channel 22. Then use the joystick to position the well plate such that the tip of the arrow adjacent to viewing channel 22 aligns with the crosshairs. Copy the Y-coordinate in the multidimensional acquisition module into the Y-position of.
2 in the update reference points tab of the sample reload adjustment module. Finally, in the update stage positions tab of the sample reload adjustment module, click on apply to MDA stage list. In the imaging module window, use the multidimensional acquisition module and click on the save tab in the left-hand menu.
Then select the filename for the experiment and the folder to store the acquired images on the computer. Next, click on the timelapse tab and set it to acquire 145 total images five minutes between each image. Then in the wavelengths tab, set the number of wavelengths to one and capture at 50%Brightfield and 50%camera with an exposure time of 12-20 milliseconds.
Set the gain to 0.6 and the digitizer to 20 milliHertz. After setting the focus as described in the accompanying text protocol, select acquire to begin capturing pre-flush images of adhered cells for all substages. In the shear control section, set the flow mode to constant and the max shear to one dyne per centimeter squared.
Then click on the inlet wells to begin the flow of media from inlet to outlet wells in order to remove any nonadhered cells. After five minutes, click on the stop button in the well plate control section. Allow for a second round of images to be acquired and then in the shear control section, adjust the max shear flow to 0.5 dyne per centimeter squared and confirm the change.
In the imaging module window, select the analysis tools tab and click review multidimensional data from the dropdown menu. Then click on select base file. This will open the multidimensional data set utilities window.
Next, click on the select directory button and choose the folder which was used to save the experiment. In the data sets list, select the experiment file log. Once the log is loaded, click on the view button.
Select the wavelength by clicking on the options in the left-hand wavelengths menu and choose the stage position to view from a dropdown menu. Choose the image to load from the numbers on the top of the module by right clicking the image number. Then select all images in order to make a timelapse video.
The selected images will open as a video in a new window. Click on the file tab in the imaging module window and choose save as from the dropdown menu. Save the resulting video as the default file format.
Finally, open and load the saved video file into image J.In image J, click on the file tab and then click on save as. Choose AVI from the dropdown menu and convert the file to an AVI file using the JPEG conversion set to 10 frames per second. Under wild-type conditions in RPMI 1640 media, several cells strongly adhered to the channel.
They form hyphae and a thick biofilm can be observed with intercalating hyphae and yeast cells. Towards the end of the microfluidic experiment, the wild-type biofilm completely fills this viewing channel. The presence of amphotericin B, however, has a pronounced effect on reducing the biofilm formation.
Specifically, the adherence of cells is diminished and several cells can be seen flowing away with the shear flow of the media. The wild-type control cells, this time grown in spider media, formed a biofilm similar to the one just shown growing in RPMI. Also grown in this media are two biofilm-defective mutants.
Both strains show severely reduced biofilm formation compared to the control. In this cell population, adhered cells do not form hyphae and the resulting biofilm is severely defective. This other strain is both defective in biofilm formation and in its ability to adhere to the channel.
Once mastered, this experiment can be set up in less than an hour. Using this method, one can learn a lot about a number of different aspects of biofilm formation including initial cell adherence, biofilm maturation, and cell dispersal. This protocol is highly customizable and versatile.
It can be adjusted to assess different microbial species across kingdoms. Flow rates, temperatures, incubation times, and media can also be used to study mixed species biofilms. We highly recommend this microfluidic biofilm assay as an in vitro assay to be utilized before in vivo animal testing.
In our experience, the results from this assay correlate very well with existing in vivo biofilm assays.
