RNA fluorescence in situ hybridization (FISH) to visualize microbial colonization and infection in the Caenorhabditis elegans intestines

Abstract

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. This protocol introduces RNA fluorescence in situ hybridization (FISH) as a tool used for the identification, visualization, and quantification of the microbes within the intestines of C. elegans. FISH probes that label the highly abundant small subunit ribosomal RNA can produce a bright signal for bacteria and microsporidian cells, and similar probes can be used to label viral RNA. FISH probes can be ordered from a commercial source as single-stranded DNA end-labeled with fluorophores. One limitation is that FISH may not provide robust signal against low copy targets, although signal can be boosted by using multiple probes (so-called single-molecule FISH). FISH staining involves collecting colonized or infected animals, washing to eliminate external contamination, followed by fixation in either paraformaldehyde or acetone. After fixation, FISH probes are incubated with samples to allow for the hybridization of probes to the desired target. To remove excess background, the animals are washed again, and then examined on microscope slides or using automated approaches. Overall, this protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available. SUMMARYGut microbiome bacteria and intestinal intracellular pathogens, like the Orsay virus and microsporidia, are often found associated with wild Caenorhabditis nematodes. This protocol presents RNA FISH as a method for the detection, quantification, and identification of colonizing or infectious microbes within the context of intact C. elegans nematodes.