Candida albicans Biofilm Formation in an In Vivo Mouse Model
Transcrição
Twenty-four hours prior to the surgical procedure, open 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 microcentrifuge tubes and scrape C. albicans from a YPD plate into 1 milliliter of PBS in a separate microcentrifuge tube at a 1 to 100 dilution. After counting, dilute the cell suspension to a final concentration of 5 times 10 to the fourth cells per milliliter, and add 1 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 1 milliliter of PBS.
To place the catheters, begin by shaving the lower back of an 8-week-old female anesthetized mouse, positioned on a heating pad. Then, disinfect the skin and make one small 0.5 to 1-centimeter incision on each side of the animal. Next, dissect the subcutis with scissors to create two subcutaneous tunnels about 1.5 centimeters long and 1 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.
