Isolating and Purifying Clostridium difficile Spores

Abstract

The ability for the obligate anaerobe, Clostridium difficile to form a metabolically dormant spore is critical for the survival of this organism outside of the host. This spore form is resistant to a myriad of environmental stresses, including heat, desiccation, and exposure to disinfectants and antimicrobials. These intrinsic properties of spores allow C. difficile to survive long-term in an oxygenated environment, to be easily transmitted from host-to-host, and to persist within the host following antibiotic treatment. Because of the importance of the spore form to the C. difficile life cycle and treatment and prevention of C. difficile infection (CDI), the isolation and purification of spores are necessary to study the mechanisms of sporulation and germination, investigate spore properties and resistances, and for use in animal models of CDI. Here we provide basic protocols, in vitro growth conditions, and additional considerations for purifying C. difficile spores for a variety of downstream applications.

Keywords: Anaerobe; Anaerobic chamber; Antibiotic-associated diarrhea; Clostridium difficile; Endospore; Sporulation.

Figure 1

Phase contrast micrographs of C. difficile strains 630 [32, 33], R20291 [34], 5325 (ATCC BAA-1875) and VPI 10463 (ATCC 43255) grown on 70:30 sporulation medium agar plates or BHIS agar plates for the indicated amount of time (h). All strains were obtained from the ATCC except R20291, which was a gift from Linc Sonenshein. Images were corrected for exposure differences, which was applied uniformly across each image.