Evaluation of histochemical staining techniques for visualization of bacterial biofilm-associated extracellular matrix in skin

Biofilms play a significant role in histopathology and are complex structures consisting of bacterial cells embedded in an extracellular matrix that contains polysaccharides, proteins, and deoxyribonucleic acid (DNA). The biofilm matrix limits the effectiveness of topical antibiotic treatment in infected wounds and impedes wound healing and immune responses. The purpose of this study was to visualize the biofilm-associated extracellular matrix using standard histological techniques. The commercially available MatTek epidermal full-thickness skin tissue model (EFT-400) was injured and infected for 24 h with biofilm-forming Staphylococcus aureus. Tissue for paraffin sections was fixed in formalin, microwave-processed, and embedded in paraffin. Serial sections cut to 5 microns were stained with Periodic acid-Schiff reagent, Calcofluor, modified Congo Red/Ziehl carbol fuchsin stain and Feulgen reaction. Stained tissues were evaluated using light and fluorescent microscopy. A detailed analysis of the application of the different staining techniques in demonstration of biofilm-associated extracellular matrix revealed that both carbohydrates and DNA were present. Discussion of the value of each staining technique is presented.