The overall goal of the following experiment is to validate the use of a luciferase expressing strain for the noninvasive monitoring of in vivo biofilm formation by candida albican cells. This is achieved by implanting catheter fragments colonized with a luciferase expressing strain of candida albicans into the back of a mouse. During the second step, the yeast is allowed to grow into a thick layer of cells embedded within an extracellular matrix, generating a mature biofilm on the inside of the catheters.
Next, a substrate is added that will be converted by the luciferase enzyme. This reaction produces measurable light Bioluminescent imaging or BLI is then used to record and quantify the formation of the biofilm within the living host. The main advantage of this technique over other existing techniques, such as the central Venmo system, is that we can test up to six biofilms inside one animal, thereby greatly reducing the number of animals that we require for statistical analysis.
The implications of this technique extend towards the therapy of candida albicans biofilm formation as our method allows the easy testing of existing and novel antifungal components. Also, this method can provide insight into candida albicans, fungal biofilms. It can also be applied to other microbes like bacteria or other fun species.
The proposed procedures are easy to implement in any laboratory that has access to an animal facility 24 hours prior to the surgical procedure opened the package containing the triple lumen catheters within a biological safety cabinet. Use sterile tweezers to remove all of the unnecessary parts and a sterile scalpel to cut the part attached to the catheter. Then place a ruler under the plastic package and cut the polyurethane catheter into one centimeter long pieces.
Next, fill each catheter with approximately 1.8 milliliters of 100%fetal bovine serum and incubate the pieces at 37 degrees Celsius overnight. The next morning, transfer each serum coated catheter piece into individual 1.5 milliliter micro centrifuge tubes, and scrape C albicans from A YPD plate into one milliliter of PBS in a separate micro centrifuge tube at a one to 100 dilution. After counting, dilute the cell suspension to a final concentration of five times 10 to the fourth cells per milliliter.
And add one milliliter of cells to each of the serum coated catheter pieces. Allow the cells to adhere to the catheters for 90 minutes at 37 degrees Celsius. Then holding the tubes in a vertical position with sterile tweezers.
Gently flush each piece through the lumen twice with one milliliter of PBS to place the catheters. Begin by shaving the lower back of an eight week old female anesthetized mouse positioned on a heating pad. Then disinfect the skin and make one small 0.5 to one centimeter incision on each side of the animal.
Next, dissect the subcu with scissors to create two subcutaneous tunnels about 1.5 centimeters long and one centimeter wide. Insert three catheter pieces into each tunnel ensuring that the pieces lie next to each other in a horizontal arrangement without overlapping. Close the incisions with sutures and then clean the wounds very gently with a disinfectant.
Then apply a local anesthetic directly onto the incisions and administer an anesthesia reversing agent monitoring the animals until they are fully recovered. To image the biofilm formation at the appropriate experimental time point, inject 100 microliters of freshly prepared coil and terezin subcutaneously into each area containing the catheters in an anesthetized animal. Then acquire consecutive scans with acquisition times ranging from 20 to 60 seconds until the maximum signal intensity is reached.
During the acquisition of the next frame, place a region of interest over each trio of catheters to measure the bioluminescent imaging or BLI signal intensity of the previously acquired frames. Place a rectangular region of interest of fixed size on a control region as well to measure the background signal in living image software. Measure the photon flux through the regions of interest for each catheter trio as well as for the background.
After repeating these measurements for every animal and at different time points during biofilm formation report, the BLI signal intensity of each catheter trio as photon flux per second. The data can be copied and pasted directly into a spreadsheet program. At the end of the longitudinal experiment, sacrifice each animal cut through the subcutaneous tissue and use sterile tweezers to remove the catheter fragments one by one.
Wash the catheters two times in one milliliter of PBS as just demonstrated. Then sonicate the pieces in a new micro centrifuge tube in one milliliter of fresh PBS at 40, 000 hertz in a water bath. Sonicate after 10 minutes, place the tubes containing the catheters on ice and plate 100 microliters of the original samples at one to 10 and one to 100 dilutions on YPD agar plates.
In duplicate. Finally plot the mean and standard deviation of the colony forming units. And BLI signal for each animal on a logarithmic scale.
In this image, a representative animal displaying the bioluminescence signal to together with a regions of interest denoted six days after biofilm development is shown. Note the lack of signaling in the control background area confirming the limitation of the biofilm to the areas immediately adjacent to the candida coated catheters. In this representative experiment, a clear BLI signal produced by the luciferase expressing biofilms can be observed, whereas only background signal appears to be produced by the wild type strain.
This increase in signal follows the same trend as the increase in colony forming units per biofilm and the photon flux measurements for each group of animals taken together. These data illustrate that BLI is a powerful technique for monitoring and quantifying in vivo mature C albicans biofilm formation in a subcutaneous mouse model. Once mastered infection of devices with candida cells may take two hours, whereas the catheter implant can be done in 10 minutes per mouse.
BLI imaging can be performed in five minutes per mouse. The big advantage of using BLI is that you can monitor biomass and biofilm development repeatedly in the same animal. Over time Following this procedure, other methods like MRI or CT can provide information on the exact cathedral position.
This information can then be used for quantification in more complex models like the deep vein Cathedral model. After watching this video, you should have a very good understanding of how to perform in vivo, subcutaneous biofilm, ss in mice, and this may form the basis for further in-depth analysis of host pathogen interactions.
