Dual Fluorescent Labeling for Bacterial Aggregation Assays.

Clostridioides difficile is an anaerobic, spore-forming pathogen that causes severe gastrointestinal disease. During infection, C. difficile interacts with other gut microbes within the intestinal mucus layer, which potentially influences colonization and disease progression. Here, we present a fluorescence-based protocol to examine bacterial adhesion and aggregation between C. difficile and other gut bacteria, such as Fusobacterium nucleatum. In this method, C. difficile is labeled with the green fluorescent dye CFDA-SE (carboxyfluorescein diacetate succinimidyl ester) and F. nucleatum or other bacterial partners are labeled with the red fluorescent dye CellTracker™ Orange CMRA (2,6-Bis-(4-chloro-phenyl)-isonicotinic acid 4-(1-carboxy-3-methyl-butoxy)-phenyl ester.) Following labeling, bacterial cultures are co-incubated in aggregation buffer, and aggregation is assessed by fluorescence microscopy. Alternatively, the labeled bacteria can be applied to cultured epithelial cells to evaluate both aggregation and host adhesion. This approach enables direct visualization and quantification of interspecies interactions, providing valuable insights into how pathobionts exploit interbacterial adhesion to persist and thrive within polymicrobial intestinal communities.