High-resolution visualization of biofilm matrix development in space and time using fluorescent stains for cellulose

The establishment of microbial biofilms, communities embedded in self-produced extracellular matrices, poses growing challenges for health and antimicrobial management. Understanding biofilm formation is crucial for developing control and eradication strategies. In response to environmental cues, planktonic bacteria adopt a sessile lifestyle, coordinating growth with matrix production. We monitored cellulose biofilm formation by Pseudomonas sp. IsoF in real time using single-step fluorescent stains. Live-tracking of polysaccharide synthesis revealed dynamic matrix arrangements shaping final biofilm structure. Cellulose determined substratum adherence, cell contacts, and colony patterning in IsoF. Biofilms formed in flow-cells and at air-liquid interfaces were remarkably similar in composition, progression, and architecture. Artificial elevation of intracellular c-di-GMP levels produced cellulose-dependent biofilms distinct from the wild type and induced a secondary exopolysaccharide. Our fluorescent probes provide real-time visualization of matrix development, enabling detailed analysis of biofilm architecture and regulation in standard laboratory conditions.