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C. difficile spreads from host to host as an infectious spore. The only natural environment known to support C. difficile spore formation is the gastrointestinal tract, yet we know very little about how spore formation occurs. Once ingested, C. difficile spores germinate when exposed to bile salts in the intestine, yielding the vegetative form of the bacterium. At some point prior to exiting the colon, these vegetative cells initiate the sporulation pathway and again become spores. Because C. difficile is a strict anaerobe, spore formation is absolutely critical to the survival of the bacterium outside of the host. Our data suggest that sporulation of C. difficile is initiated in response to signals found within the host intestine, through unknown and unique genetic mechanisms. The goals of this project are to identify the genetic mechanisms required for spore initiation and determine how sporulation is initiated in the host. Understanding how C. difficile forms a spore is the first step in the development of rational strategies to prevent spore formation, which would greatly decrease transmission of disease.

Abstract

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

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Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

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