Generating an Ex Vivo Ovine Wounded Skin Model Infected with a Bacterial Pathogen

To infect the skin sample, prepare a fresh 24-well plate with 400 microliters of pre-warmed antibiotic-free media, and add the 24-well inserts using sterile forceps. Remove the media from the skin samples, wash them with 500 microliters of sterile PBS, and remove the wash.

Use sterile forceps to gently hold the sample to the bottom of the well. Use a 4-millimeter punch biopsy to pierce through the skin sample to a rough depth of 1 to 2 millimeters, and make a central wound flap. Then, use a number 15 blade scalpel and sterile-toothed Allis tissue forceps to remove the top layer of the wound flap.

Once all the samples have been wounded, transfer them to the 24-well inserts using sterile forceps. Pipette 15 microliters of the bacterial inoculum into the wound bed before incubating for 24 hours at 37 degrees Celsius in a humidified tissue incubator. For longer incubation periods, remove the media, replace it with fresh media every 24 hours, and incubate the plates under the same conditions.