Protocols for Standard and Advanced Visualization and Quantification of Leptospira Biofilms.

The ability of Leptospira spp. to form biofilms is increasingly recognized as a key factor in their environmental persistence and possibly in host colonization. In this chapter, we present a set of protocols for the in vitro growth, quantification, and structural characterization of Leptospira biofilms. We detail standard high-throughput approaches, such as crystal violet (CV) staining in 96-well microtiter plates, as well as an alternative based on phase contrast microscopy and image analysis. To probe biofilm architecture and composition, we describe advanced imaging methods including confocal laser scanning microscopy (CLSM) with fluorescent probes targeting viability or matrix components, and scanning electron microscopy (SEM) for high-resolution surface imaging. We also introduce a method for growing Leptospira biofilms on polycarbonate membranes, facilitating downstream structural analyses without disturbing their spatial organization. Together, these protocols provide a robust and adaptable toolbox for investigating the development and complexity of Leptospira biofilms.