$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Inoculation with Pseudomonas aeruginosa
- Dilute overnight PAO1:lux culture to OD600 = 0.05 in 75 mL of Lysogeny broth (LB) media containing 100 µg/mL carbenicillin and grow the bacteria until the culture is in early exponential phase (OD600≈0.3). This should take approximately 2–3 h.
- Dilute PAO1:lux in phosphate buffered saline (PBS) to a concentration of (7.5 ± 2.5) x 102 CFU/mL. Be sure to prepare excess inoculum to ensure sufficient volume and to allow for plating after the experiment. If transporting between facilities , use double containment in a leak proof box clearly marked Biohazard.
- Perform all work with P. aeruginosa and mice using approved personal protective equipment in an Animal Biosafety Level 2 (ABSL-2) approved biological safety cabinet (BSC). Reusable equipment such as the weighing scale should be covered with cling wrap to prevent contamination.
- Anesthetize the mouse using 3% isoflurane. Weigh the mouse and record the weight. Inject the mouse subcutaneously with pre-warmed sterile 0.9% sodium chloride, 250 µL at each flank for a total of 500 µL.
- If the mouse's transparent film dressing has come off overnight, remove any resulting scab carefully and put on a new dressing.
- Use a 500 µL tuberculin 27 G safety cap syringe to inject 40 µL of the PAO1:lux suspension through the transparent film dressing into each wound. Different mice should be used for non-inoculated/PBS wound controls in order to prevent cross-contamination from the contralateral side.
- Place the mouse back in its cage on a heating pad and monitor until it wakes up. All mice should be housed individually in separate cages to prevent cross-contamination.
- Provide high calorie nutritional supplement paste sandwiched between food pellets on the floor of the cage.