Abstract
Biofilms are a ubiquitous form of growth for bacteria on surfaces in most environments, natural or manmade. Here we present a protocol using the Bioflux microfluidic system to investigate the organized structure and development of these multicellular communities. Microfluidic systems present an opportunity to grow biofilms in a stable, physiologically maintained environment that is readily observable via time-lapse microscopy.
Keywords: Biofilms, Microfluidics, Bioflux, Microscopy, Pseudomonas aeruginosa, Staphylococcus aureus, eDNA, EPS, Matrix, Expolysaccharides, Extracellular DNA
Background
The Bioflux microfluidic system is a commercially available system that consists of a series of input and output wells linked by microfluidic channels set in a standard microplate format. Growth medium is flowed through the channel by application of controllable pneumatic pressure. The system allows for high throughput, reproducible growth of biofilms whilst avoiding the lengthy preparation required for ‘home constructed’ microfluidic set-ups.The complex and heterogenous biofilms formed by bacteria have long proved challenging to cultivate and visualize. Traditional methods of biofilm growth, such as the use of abiotic surfaces placed in a bacterial growth medium suffer from myriad artifacts such as nutrient depletion and disturbance of the biofilm. Home-made flow systems alleviate many of these issues but remain labor intensive and unwieldy. The nature of the traditional flow systems renders them difficult to use in combination with time lapse microscopy, particularly as the large channel sizes means the continuous use of stains is costly. As a result, the structure and architecture of bacterial biofilms has so far been probed using ‘snapshots’ at arbitrary time points.Here, we describe a simple protocol for the effective use of the Bioflux microfluidic systems, with a focus on the use of fluorescent probes to examine changes in the structure and architecture of the model biofilm-forming bacteria, Pseudomonas aeruginosa and Staphylococcus aureus.This protocol can be easily adapted to different fluorescent stains, bacterial species, including complex multispecies biofilms such as oral biofilms. Data obtained are reproducible and biofilms are in line with the architectural features and dimensions reported in the literature.
Materials and Reagents
Equipment
Software
Procedure
This protocol is for the growth of either P. aeruginosa or S. aureus in the Bioflux system. Where differences in the protocols are used, those for S. aureus are listed in square brackets.
Notes
Recipes
Acknowledgments
This work has been funded by the EMPIR program co-financed by the Participating States and from the European Union’s Horizon 2020 research and innovation program (grant reference 15HLT01 MetVBadBugs). This protocol was adapted from the manufacturer’s instructions (Fluxion Bioscience).
Competing interests
A small number of Bioflux plates were provided by Fluxion Bioscience.
References
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