Source: Kucharíková, S., et al. Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging. J. Vis. Exp. (2015).
This video demonstrates the development of an in vivo mouse model to study the biofilm formation of Candida albicans in subcutaneous tissue. The experiment involves implanting Candida-infected devices in the animal's back region for biofilm development.
1. C. albicans Growth
2. Catheter Pieces Preparation
3. Ex vivo C. albicans Adhesion on FBS-coated Polyurethane Substrates
4. Anesthesia
5. Animal Surgery
6. Bioluminescence Imaging: Preparation of Coelenterazine (CTZ), the Substrate for G. princeps luciferase
7. Bioluminescence Imaging
Figure 1: Major steps during the animal surgery. (A) The procedure of catheter implant. (1) Place the anesthetized animal on a warm pad containing paper tissue and apply the ophthalmic ointment to the eyes. (2) Shave the lower part of the back and disinfect. (3) Create two small (approx. 0.5 cm) incisions through the skin on the left and on the right side of the back. Create a subcutaneous tunnel inside each incision and place 3 catheters inside. (4) Close the wound with sutures and place the animal on a warm pad to recover. (B) Procedure of catheter removal. (1) Place the sacrificed animal on a pad and disinfect the operated side containing catheters. Cut the wound right above the catheters. (2) Take out each catheter gently and wash twice with 1 ml of PBS. Place to the separate microcentrifuge tube.
Figure 2: Preparation of polyurethane devices. The polyurethane part of the catheter is cut into 1 cm pieces. The plastic pocket containing the catheter is open under sterile conditions and all parts, except the catheter, are removed from the package. Secondly, place a ruler under the plastic pocket and cut exactly 1 cm polyurethane pieces. Such devices are subsequently distributed to microcentrifuge tubes (max 15 pieces/tube) and submerged in 100% fetal bovine serum followed by overnight incubation at 37 °C.
The authors have nothing to disclose.
Yeast extract granulated | Merck | MERC1.03753.0500 | |
Bacteriological peptone | Oxoid | LP037B | |
Agar granulated | Difco | 214530 | |
Phosphate buffered saline | Prepared in the laboratory | for 1L of 10x PBS: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4 | |
RPMI1640 with L-glutamine and without sodium carbonate | Sigma | R6504-1L | Prepare according the protocol for Candida albicans drug susceptibility testing |
fetal bovine serum (FBS) | Sigma | F7524 | |
Polyurethane tripe-lumen intravenous catheter piece (2.4 mm diameter, Certofix Trio S730) | BBraun | CV-15703 | CV-15703 |
Dexamethasone | Fagron SAS, France | 611139 | Immunosuppressant (stock solution 10 mg/ml) |
Insulin syringes (0.3 ml) | Terumo Myjector 29G | 324826 | For injection of coelenterazine |
Electric razor | For small animals | ||
Sterile surgical tools | Scissors, 2 pairs of tweezers, scalpel | ||
Heating pad | Leica | 14042321474 | |
Skin suture | Johnson&Johnson | K890H | Surgical thread, needle |
BLI camera (IVIS Spectrum) | Perkin Elmer, Alameda | IVISSPE | |
Living Image software | Perkin Elmer, Alameda | (version 4.2) |