Fluidic resistance control enables high-throughput establishment of mixed-species biofilms

Bacteria often live in communities of mixed species embedded in a self-produced extracellular matrix of polysaccharides, proteins and DNA, termed biofilms. The BioFlux microfluidic flow system is useful for studying biofilm formation in different media under flow. However, analyzing the architecture and maturation of biofilms under flow requires a proper seeding, which can prove difficult when working with bacteria of different sizes, motile bacteria or aiming for a high number of replicates. Here we developed an efficient protocol that exploits viscosity tuning and seeding indicator dyes to improve seeding and allow for high-throughput examination and visualization of consistent mono- and mixed-species biofilm developments under flow. METHOD SUMMARY An improved protocol for seeding bacteria in the BioFlux microfluidic flow system was developed using glycerol and Nigrosin to increase the viscosity and stain the media, respectively. This new protocol does not alter biofilm development and allows consistent mixed-species biofilm formation in a copious numbers of replicates.