Microscopic approaches for bacterial collective behaviour studies

Abstract

Bacteria significantly impact our lives, from their beneficial role as probiotics to their involvement in infection environments. Their widespread presence is largely due to their ability to adapt to diverse conditions through collective behavior, which enables the devel- opment of complex strategies from the contributions of simple individual entities. However the understanding of these systems is limited by the reach of current study techniques. This work presents the development of three platforms designed to perform microscopic studies and characterise bacterial collective behaviors in situ, profiting the advantages of microfluidics over traditional culture techniques. The first platform integrates bacterial culture on solid agar directly on the microscope stage, allowing for extended observation periods of up to a week. The agar is housed within an elastomer structure sealed with glass, ensuring environmental isolation while maintaining optical accessibility. This platform was used to document the complex social strategies of Myxococcus xanthus, including motility mechanisms, predation organisation, and fruiting body formation. The second platform is an automated testing system for quantifying bacterial viability under various conditions. Using microfluidic technology, this platform streamlines and parallelise the process. It adapts the Ames genotoxicity test to a miniaturized version, using microscopy imaging as the readout. This approach reduces experimental turnaround time and minimizes the handling of hazardous substances. The third platform is a microfluidic system designed for the microscopy observation of bacteria within stabilised droplets. This approach enhances throughput and allows for the production of various types of droplets on the same chip. Bacillus subtilis bacteria were encapsulated in these droplets, and their entire biofilm formation life cycle was observed in detail. Parallel to this, custom software was developed specifically for analysing microscopy images to automatically quantify biofilm formation. Each of these platforms provides a unique perspectives in the study of bacterial collec- tive behavior to offer a comprehensive toolkit for researchers. complementing one another. This work will equip researchers with the tools to address the mysteries of bacterial col- lective behavior and opens up new possibilities for application and investigation