A non-invasive method for time-lapse imaging of microbial interactions and biofilm dynamics

Complex interactions between microbial populations can greatly affect the overall properties of a microbial community, sometimes leading to cooperation and mutually beneficial coexistence, or to competition and the death or displacement of particular organisms or sub-populations. Interactions between different biofilm populations are highly relevant in diverse scientific areas, from antimicrobial resistance to microbial ecology. The utilization of modern microscopic techniques has provided new and interesting insight into how bacteria interact at the cellular level to form and maintain microbial biofilms. However, our ability to follow complex intra- and inter-species interactions in vivo at the microscopic level has remained somewhat limited. Here, we detail BacLive, a novel non-invasive method for tracking bacterial growth and biofilm dynamics using high resolution fluorescence microscopy and an associated ImageJ processing macro (https://github.com/BacLive) for easier data handling and image analysis. Finally, we provide examples of how BacLive can be used in the analysis of complex bacterial communities. ImportanceCommunication and interactions between single cells are continuously defining the structure and composition of microbial communities timely and spatially. Methods routinely used to the study of these communities at cellular level rely on sample manipulation what deprives from microscopic time lapse experiments on a given sample. BacLive is conceived as a method for the non-invasive study of the formation and development of bacterial communities, such as biofilms, and the dynamics of formation of specialized subpopulations in time-lapse experiments. In addition we prove that BacLive largely simplifies the analysis of the data generated.