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

Take a pre-sterilized skin biopsy section obtained from a lamb's forehead in an antibiotic-free medium.

The section comprises an outer epidermis and an underlying dermis layer.

Remove the medium and use a buffer to wash the section.

Make a circular cut in the section and remove the top layer. This breaks the barrier, generating a wound bed in the skin.

Transfer the wounded sections onto a permeable membrane insert in a multiwell plate containing an antibiotic-free medium.

Introduce a suspension of pathogenic bacteria onto the wound bed and incubate.

During incubation, the wound bed provides a tissue surface for bacterial attachment and the nutrients to support bacterial growth.

This allows the multiplication and spreading of the bacteria across the wound bed, establishing an infection.

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 sk