Improved Visualization of Oral Microbial Consortia.

Bacteria on the tongue dorsum (TD) form consortia tens to hundreds of microns in diameter organized around a core of epithelial cells. Whole-mount preparations have been instrumental in revealing their organization and specific microbial associations. However, their thickness and intricate 3-dimensional complexity present challenges for a comprehensive spatial analysis. To overcome these challenges, we employed a complementary approach: embedding in hydrophilic plastic followed by sectioning and postsectioning labeling. Samples were labeled by hybridization with multiplexed fluorescent oligonucleotide probes and visualized by spectral imaging and linear unmixing. Application of this strategy to TD biofilms improved the visualization of bacteria that were difficult to resolve in whole-mount imaging. Actinomyces, previously detected as patches, became resolved at the single-cell level. The filamentous taxa Leptotrichia and Lachnospiraceae, located at the core of the consortium, were regularly visualized whereas previously they were rarely detected when using whole mounts. Streptococcus salivarius, heterogeneously detected in whole mounts, were regularly and homogenously observed. Two-dimensional images provide valuable information about the organization of bacterial biofilms. However, they offer only a single plane of view for objects that can extend to hundreds of microns in thickness, and information obtained from such images may not always reflect the complexity of a 3-dimensional object. We combined serial physical sectioning with optical sectioning to facilitate the 3-dimensional reconstruction of consortia, spanning over 100 µm in thickness. Our work showcases the use of hydrophilic plastic embedding and sectioning for examining the structure of TD biofilms through spectral imaging fluorescence in situ hybridization. The result was improved visualization of important members of the human oral microbiome. This technique serves as a complementary method to the previously employed whole-mount analysis, offering its own set of advantages and limitations. Addressing the spatial complexity of bacterial consortia demands a multifaceted approach for a comprehensive and effective analysis.