Myxococcus xanthus colonies develop different strategies to adapt to their environment, leading to the formation of macroscopic patterns from microscopic entities. (Image by the Authors of the manuscript.)Tutorial for the growth and development of Myxococcus xanthus as a Model System at the Intersection of Biology and Physics
Jesus Manuel Antúnez Domínguez, Laura Pérez García, Natsuko Rivera-Yoshida, Jasmin Di Franco, David Steiner, Alejandro V. Arzola, Mariana Benítez, Charlotte Hamngren Blomqvist, Roberto Cerbino, Caroline Beck Adiels, Giovanni Volpe
Soft Matter 21, 8602-8623 (2025)
arXiv: 2407.18714
doi: 10.1063/5.0235449
Myxococcus xanthus is a unicellular organism known for its capacity to move and communicate, giving rise to complex collective properties, structures and behaviors. These characteristics have contributed to position M. xanthus as a valuable model organism for exploring emergent collective phenomena at the interface of biology and physics, particularly within the growing domain of active matter research. Yet, researchers frequently encounter difficulties in establishing reproducible and reliable culturing protocols. This tutorial provides a detailed and accessible guide to the culture, growth, development, and experimental sample preparation of M. xanthus. In addition, it presents several exemplary experiments that can be conducted using these samples, including motility assays, fruiting body formation, predation, and elasticotaxis—phenomena of direct relevance for active matter studies.
Automated segnmentation of bacterial structures within a droplet. The image shows a bright-field microscopy view where a large biofilm region (green, outlined in blue) has been segmented from surrounding features. Small aggregates (yellow contours) are also highlighted. This segmentation enables structural differentiation of biofilm components for downstream quantitative analysis. (Image by D. Pérez Guerrero.)Latent Space-Driven Quantification of Biofilm Formation using Time Resolved Droplet Microfluidics
Daniela Pérez Guerrero, Jesús Manuel Antúnez Domínguez, Aurélie Vigne, Daniel Midtvedt, Wylie Ahmed, Lisa D. Muiznieks, Giovanni Volpe, Caroline Beck Adiels
arXiv: 2507.07632
Bacterial biofilms play a significant role in various fields that impact our daily lives, from detrimental public health hazards to beneficial applications in bioremediation, biodegradation, and wastewater treatment. However, high-resolution tools for studying their dynamic responses to environmental changes and collective cellular behavior remain scarce. To characterize and quantify biofilm development, we present a droplet-based microfluidic platform combined with an image analysis tool for in-situ studies. In this setup, Bacillus subtilis was inoculated in liquid Lysogeny Broth microdroplets, and biofilm formation was examined within emulsions at the water-oil interface. Bacteria were encapsulated in droplets, which were then trapped in compartments, allowing continuous optical access throughout biofilm formation. Droplets, each forming a distinct microenvironment, were generated at high throughput using flow-controlled pressure pumps, ensuring monodispersity. A microfluidic multi-injection valve enabled rapid switching of encapsulation conditions without disrupting droplet generation, allowing side-by-side comparison. Our platform supports fluorescence microscopy imaging and quantitative analysis of droplet content, along with time-lapse bright-field microscopy for dynamic observations. To process high-throughput, complex data, we integrated an automated, unsupervised image analysis tool based on a Variational Autoencoder (VAE). This AI-driven approach efficiently captured biofilm structures in a latent space, enabling detailed pattern recognition and analysis. Our results demonstrate the accurate detection and quantification of biofilms using thresholding and masking applied to latent space representations, enabling the precise measurement of biofilm and aggregate areas.
The three platforms developed to observe and characterise bacterial collective behaviour in different conditions. (Image by J. Dominguez.)Jesús Manuel Antúnez Domínguez defended his PhD thesis on 6 September 2024. Congrats!
The defense took place in PJ, Institutionen för fysik, Origovägen 6b, Göteborg.
Title: 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 development 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 collective 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 collective behavior and opens up new possibilities for application and investigation.
