Figure 2.

Ex-vivo burn wound model workflow. Graphical representation showing main stages of set-up and operation of the ex-vivo porcine burn wound model used in this study. (I) Sections of porcine skin are initially prepared by shaving and surface disinfection in 70% ethanol solution; (II, III) The BWAD (see Figure 1) is used to generate an array of consistent partial thickness burn injuries on porcine skin; (IV) If isolation of wounds is required, individual wounds may be excised using a punch biopsy and transferred into wells of tissue culture plates. Wound arrays may be used directly on skin sections without excision. (V–VIII) Individual wounds are inoculated with test organisms to simulate infection and wound biofilm formation. Potential treatments may be evaluated against a subset of infected wounds in the array. (IX) Infected wounds are incubated under required conditions and biofilm formation, bacterial growth, and impact on interventions tested can be evaluated as appropriate. Subsequent methods demonstrated in this study are: Recovery and enumeration of viable cells, quantification of metabolic activity via XXT reduction, the selective staining of biofilms followed by direct imaging of biofilms through CLSM and measurement of fluorescence intensity, extraction of RNA, and quantification of gene expression by qRT-PCR.