Bacterial Size Analysis Using High-Content Imaging: Application to Leptospira Strains

Leptospires are thin, motile spirochetes whose morphology and size can vary among species and strains. Accurate measurement of bacterial size is essential for phenotypic characterization and comparative analyses. Here, we describe a standardized protocol for labeling and measuring the size of Leptospira using carboxyfluorescein succinimidyl ester (CFSE), and high-content fluorescence microscopy. Leptospiral strains were cultured until the exponential phase, stained with CFSE, and plated prior to imaging. Imaging was performed using the OPERA Phenix system (Revity). ImageJ with the MicrobeJ plugin was used to analyze the images, allowing for automated detection, and measurement of individual bacteria. This method enables accurate and reproducible measurement of leptospiral size across different species. It can also be adapted to other bacterial species.