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Updated: Sep 9, 2025
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Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry
Published on: February 15, 2012
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Dynamics of bacterial biofilm development imaged using light sheet fluorescence microscopy
Lenka Šmerdová1, Tibor Füzik1, Lucie Valentová1
1Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
Biochemistry and Biophysics Reports
|September 2, 2025
Summary
This study visualizes bacterial biofilm development using microfluidics and light sheet fluorescence microscopy (LSFM). It reveals how Staphylococcus aureus and Pseudomonas aeruginosa interact, with P. aeruginosa dispersing S. aureus biofilms.
Area of Science:
- Microbiology
- Biophysics
- Biotechnology
Background:
- Bacterial biofilms are crucial in infections and industry but their complex dynamics, especially in multispecies communities, remain poorly understood.
- Understanding these dynamics is vital for developing effective strategies against biofilm-related problems.
Purpose of the Study:
- To develop and utilize a microfluidic cultivation system coupled with light sheet fluorescence microscopy (LSFM) for real-time, high-resolution imaging of bacterial biofilm development.
- To investigate the structural and dynamic interactions between the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa during co-cultivation.
Main Methods:
- A microfluidic system was integrated with light sheet fluorescence microscopy (LSFM) for continuous, multidirectional imaging.
- Three-dimensional reconstructions of biofilm structures were generated with isotropic resolution.
- The study examined single-species biofilms of S. aureus and P. aeruginosa, as well as their co-culture under varying inoculation ratios.
Main Results:
- S. aureus biofilms formed thick, mushroom-like structures (50-70 μm), while P. aeruginosa biofilms were thinner (10-15 μm) with smaller cell clusters (25 μm).
- In co-culture, P. aeruginosa invaded and dispersed S. aureus mushroom-like structures. Higher P. aeruginosa ratios led to smaller, stable S. aureus clusters overgrown by P. aeruginosa.
- Conditioned media from co-cultures induced dispersion in single-species S. aureus biofilms.
Conclusions:
- The integrated microfluidic-LSFM system provides unprecedented visualization of biofilm formation dynamics.
- P. aeruginosa actively disrupts S. aureus biofilms, suggesting interspecies competition plays a significant role in mixed-species biofilm architecture.
- This approach offers a powerful tool for studying biofilm development and evaluating the impact of compounds on biofilm structure and dispersion.
