A protocol for the simultaneous identification of chitin-containing particles and their associated bacteria.

Chitin is the second most abundant polymer on Earth, playing a crucial role in the biogeochemical cycles. A core issue for studying its processing in aquatic systems is the identification and enumeration of chitin-containing particles and organisms, ideally in a manner that can be directly linked to bulk chitin quantification. The aim of this study was the development of such a technique. We successfully combined the methodology of bulk chitin determination using wheat germ agglutinin (FITC-WGA) for staining chitin-containing particles and organisms along with CARD-FISH staining of either chitin-containing eukaryotic cells or bacteria associated with them. Environmental chitin staining was successfully applied to natural water samples. Fungal hyphae, diatoms, and dinoflagellates, sestonic aggregates and chitin-containing structures derived from metazoa were observed. Also, hybridized bacteria attached to chitinaceous debris were clearly visualized. Finally, as proof of principle, cultured yeast cells were simultaneously-targeted by FITC-WGA and the EUK516 probe without exhibiting any interference between both stains. The presented approach appears as a powerful tool to evaluate the contribution of different size classes and organisms to chitin production and consumption, opening the possibility for application of single-cell approaches targeting the ecophysiology of chitin transformations in aquatic systems.