Preparation of Spores with the 70:30 Method.

Clostridioides difficile spores are highly resistant, dormant structures that play a critical role in the transmission and pathogenesis of C. difficile infections. The purification of C. difficile spores is essential for understanding the mechanisms of spore formation, germination, and the disease. In this chapter, a method for the purification of C. difficile spores using a 70:30 medium is described, consisting of 70% SMC (sporulation medium for Clostridia) and 30% BHIS (brain-heart infusion supplement). The 70:30 medium is used to induce sporulation and produce high yields of pure C. difficile spores. A protocol for the preparation of the 70:30 medium, spore purification, and characterization of the purified spores is described. This method is a valuable tool for researchers studying the biology and pathogenesis of C. difficile and will facilitate the development of effective strategies for the prevention and treatment of C. difficile infections.