Methylene Blue Is an Effective Disclosing Agent for Identifying Bacterial Biofilms on Orthopaedic Implants

Background: Bacterial biofilms pose a challenge in treating implant-associated infections. Biofilms provide bacteria with protection against antimicrobial agents and the immune response and often are invisible to the naked eye. As a biofilm-disclosing agent, methylene blue (MB) has shown promise, but lacks rigorous in vitro evaluation. The purposes of the present study were to assess MB as a biofilm-disclosing agent in vitro for common biofilm-forming organisms and to determine performance characteristics across implant materials and healthy tissue types. Methods: Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 27853) biofilms were grown on culture for 2 days in CDC biofilm reactors on titanium, cobalt chromium, polyethylene, and polyether ether ketone (PEEK) coupons. Biofilms were stained with MB solutions of either 0.005% or 0.01% and then were washed with normal saline solution. Digital photographs were obtained to compare the visual sensitivity of the blue dye at these dilutions. Scanning electron microscopy (SEM) was performed to confirm the absence or presence of biofilm on MB-stained areas. Uninoculated controls were also assessed. Healthy adult sheep tissues were also stained to determine the staining characteristics of the host tissue. ImageJ was used to determine the relative blue intensity of stained implants and tissues compared with standard curves. Results: S. aureus and P. aeruginosa biofilms stained avidly on titanium, cobalt chromium, polyethylene, and PEEK coupons. There was visible dose-dependent staining based on dye concentration. MB was visible only where biofilms were present as confirmed by SEM. MB did not stain uninoculated controls. Articular cartilage and meniscus demonstrated appreciable staining; bone, tendon, muscle, nerve, and fat did not. Bacterial biofilms demonstrated both dose-dependent and species-specific staining. Conclusions: MB is an effective disclosing agent for S. aureus and P. aeruginosa biofilms in vitro. MB did not stain implant materials, nor did it stain most healthy tissues in vitro. MB may allow surgeons to see biofilms and may allow for enhanced debridement once visualized.