Real-Time Visualization of Nutrient Media Impact on Pseudomonas aeruginosa Biofilm Development Using a Microfluidic System.

The pathogenic bacterium Pseudomonas aeruginosa is a major cause of numerous nosocomial infections, and its growing antimicrobial resistance has led to it posing a significant public health threat. This article presents a comprehensive protocol detailing the use of a microfluidic system for the real-time visualization and quantification of biofilm development in two key P. aeruginosa strains, PAO1 and PA14. The method employs optically transparent, multichannel microchannel plates to subject bacterial cultures to a continuous, steady flow of media, including tryptic soy broth (TSB) or modified minimal fastidious anaerobic broth (FAB) with varying carbon source concentrations, thereby mimicking conditions found in the clinical environments. Over a 24-h period, automated real-time imaging captures the growth and maturation of biofilms in the form of biofilm surface area coverage, thickness, and surface roughness in a highly reproducible manner. The experimental objective is to use the results to demonstrate that biofilm formation for both strains is significantly impacted by changes in nutrient media composition. The goal of this visualized protocol is to provide a method for researchers to study biofilm dynamics under steady laminar flow conditions, and the insights gained could be leveraged to develop alternative, non-antimicrobial strategies for eradicating early-stage P. aeruginosa biofilms in nosocomial settings.