Trichomycetes (gut fungi) are obligate symbionts of various arthropods and have been found in marine, freshwater, and terrestrial habitats on every continent except Antarctica. Gut fungi associate commensally with their immature aquatic hosts (including black flies, mayflies, stoneflies, isopods, and others) attaching to the chitinous lining of the mid- or hindgut. Gut fungi are routinely isolated from the host’s digestive tract into axenic cultures, which provide a rich resource of genetic material for phylogenic analysis. Given the obligate endosymbiotic nature of the fungi, unique challenges are encountered upon culturing, thus only 30% of all trichomycetes have been successfully cultured. Our laboratory houses the world’s largest culture collection of trichomycetes. We have been endeavoring to expand the collection by formulating suitable media in which field collected trichomycete specimens can grow. This requires attention to variables such as competing bacterial growth, pH changes required for sporulation, agar density and proper nutrient balance within the media. The continued development and enhancement of culture media is intended to increase the number of isolates that previously were considered "unculturable" and offer more robust samples of genetic material for future analyses.