Convenient Protocol for Production and Purification of Clostridioides difficile Spores for Germination Studies

Clostridioides difficile, an obligate anaerobic bacterium, causes infections leading to prolonged diarrhea. The bacterium produces dormant spores that can withstand an aerobic environment, resulting in easy environmental transfer. Here, we present a convenient sporulation and purification protocol that can be practiced in any lab setting using a portable anaerobic glove bag. This protocol also optimizes existing cell growth methods and presents a detailed trouble shooting guide. This protocol is a modification of those previously reported by Edwards and McBride (2016) and Shen et al. (2016).