CLASI-FISH Imaging of Fusobacterium in Oral Microbial Communities

The human oral microbiome comprises a complex community of over 700 species of bacteria and other microbes with defined structure-function relationships, especially in dental plaque biofilms. Fusobacterium nucleatum is a ubiquitous organism in dental plaque known to coaggregate with multiple different species of oral microbes and hypothesized to play a crucial structural role in bridging early and late colonizing species in dental plaque. Microbial fluorescence in situ hybridization allows the mapping of bacteria in dense polymicrobial communities with exquisite taxonomic specificity. Here we provide an experimental procedure to label and visualize Fusobacterium nucleatum within dental plaque biofilms using combinatorial labeling and spectral imaging fluorescence in situ hybridization (CLASI-FISH). CLASI-FISH allows mapping of dozens of different microbial taxa in a single experiment. This streamlined protocol enhances accessibility for studying oral microbiomes. With rational probe design, this technique could be applied to map any organism of interest in the context of dozens of other microbial community members in diverse microbiomes.