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

Abstract

Source: Regan, H. C., et al. A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics. J. Vis. Exp. (2022).

The video demonstrates the generation of an ex vivo ovine skin model infected with Staphylococcus aureus. This entails treating a lamb's forehead skin biopsy section with antibiotics, establishing a wound bed, positioning it on a permeable membrane in a multiwell plate with antibiotic-free medium, and establishing bacterial infection in the skin wound.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Sterilization

1. Disinfect forceps prior to collection of the heads by taking clean forceps and performing dry heat sterilization in an oven at 200 °C for 1 h. Autoclave all glassware at 121 °C for 15 min before use.
2. Carry out all described work within a microbiology class 2 cabinet. Prepare all reagents as per the manufacturer's instructions.

2. Sample collection

1. In the abattoir, Swaled....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24 Well Companion Plate	SLS	353504
4 mm Biopsy Punch	Williams Medical	D7484
50 ml centrifuge tubes	Fisher Scientific	10788561
8 mm Biopsy Punch	Williams Medical	D7488
Amphotericin B solution, sterile	Sigma	A2942
Colour Pro Style Cordless Hair Clipper	Wahl	9639-2117X	Hair Clippers
Dual Oven Incubator	SLS	OVe1020	Sterilising oven
Epidermal growth factor	SLS	E5036-200UG
Ethanol	Honeywell	458600-2.5L
F12 HAM	Sigma	N4888
Foetal bovine serum	Labtech International	CA-115/500
Forceps	Fisher Scientific	15307805
Hair Removal Cream	Veet	Not applicable
Heracell VIOS 160i	Thermo Scientific	15373212	Tissue culture incubator
Heraeus Megafuge 16R	VWR	521-2242	Centrifuge
Insert Individual 24 well 0.4um membrane	VWR International	353095
Insulin, recombinant Human	SLS	91077C-1G
Medium 199 (MK media)	Sigma	M0393
Microplate, cell culture Costar 96 well	Fisher Scientific	10687551
Multitron	Infors	Not applicable	Bacterial incubator
PBS tablets	Sigma	P4417-100TAB
Penicillin-Streptomycin	SLS	P0781
Plate seals	Fisher Scientific	ESI-B-100
Safe 2020	Fisher Scientific	1284804	Class II microbiology safety cabinet
Scalpel blade number 15	Fisher Scientific	O305
Scalpel Swann Morton	Fisher Scientific	11849002
Sodium bicarbonate	Sigma	S5761-1KG
Toothed Allis Tissue Forceps	Rocialle	RSPU500-322
Tryptic Soy Broth	Merck Life Science UK Limited	41298-500G-F
Vimoba Tablets	Quip Labs	VMTAB75BX