Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

• Authors: Adrianne Edwards, Emory University; Jose M. Suarez, Emory University; Shonna McBride, Emory University
• Journal Title: Journal of Visualized Experiments
• Volume: Volume 2013, Number 79
• Publisher: Journal of Visualized Experiments (JoVE) | 2013-09-14, Pages e50787-e50787
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.3791/50787
• Subjects: Biology, Microbiology; Health Sciences, Immunology
• Keywords: Immunology; Issue 79; Genetics; Bacteria; Anaerobic; Gram-Positive Endospore-Forming Rods; Spores; Bacterial; Gram-Positive Bacterial Infections; Clostridium Infections; Bacteriology; Clostridium difficile; Gram-positive; anaerobic chamber; spore; culturing; maintenance; cell culture

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.