An Integrated Workflow for the Purification and Fluorescence Imaging of Clostridioides difficile Spores Using...

We present a robust protocol for both the purification and imaging of C. difficile spores. First, we describe a simplified, one-step isopycnic centrifugation method using Percoll that enables efficient separation of spores from vegetative cells. Next, we introduce a fluorescence-based imaging method using lanthanide beta-diketone complexes (primarily Eu[TTA]₃) that allows for the differential visualization of dormant and germinated spores, as well as sporulating cells. These complexes exploit the unique photophysical properties of lanthanides, including sharp emission spectra and resistance to photobleaching, to enhance spore imaging under USB-powered, wide-field, and confocal microscopy. Staining is instantaneous, and no permeabilization or fixation steps are required. We further demonstrate co-staining with dyes such as DAPI and FITC-dextran to reveal spore compartmentalization and confirm germination status. This integrated approach enables detailed characterization of C. difficile spore morphology and offers a versatile way to study spore germination, antibiotic response and pathogen persistence at single-spore resolution.