Optimizing in vitro spherulation cues in the fungal pathogen .

. are part of a group of thermally dimorphic fungal pathogens, which grow as filamentous cells (hyphae) in the soil and transform to a different morphology upon inhalation into the host. The host form, the spherule, is unique and highly under characterized due to both technical and biocontainment challenges. Each spherule arises from an environmental spore (arthroconidium), matures, and develops hundreds of internal endospores, which are released from the spherule upon rupture. Each endospore can then go on to form another spherule in a cycle called spherulation. One of the foremost technical challenges has been reliably growing spherules in culture without the formation of contaminating hyphae, and consistently inducing endospore release from spherules. Here, we present optimization of in vitro spherule growth and endospore release, by closely controlling starting cell density in the culture, using freshly-harvested arthroconidia, and decreasing the concentration of multiple salts in spherulation media. We developed a minimal media to test spherule growth on various carbon and nitrogen sources. We defined a critical role for the dispersant Tamol in both early spherule formation and prevention of the accumulation of a visible film around spherules. Finally, we examined how the conditions under which arthroconidia are generated influence their transcriptome and subsequent development into spherules, demonstrating that this is an important variable to control when designing spherulation experiments. Together, our data reveal multiple strategies to optimize in vitro spherulation growth, enabling characterization of this virulence-relevant morphology.