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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
- Obtaining Samples for Clostridium difficile (C. difficile) Enumeration
NOTE: Prior to obtaining feces or cecal content, weigh sterile microcentrifuge tubes on an analytical scale. Record the tube weight on the side of the tube, rounding to four decimal places (e.g., 0.9864 g). Denote this weight as the "tube weight." Each sample collected should be placed in a sterile, weighed microcentrifuge tube.
1. Sterile collection of mouse feces- Gently restrain each mouse by grasping the animal by the loose skin of the neck and back in order to immobilize the head. Use the pinky finger to gently restrain the animal's tail, thus exposing the anus. Ensure that the animal does not vocalize or show other signs of distress during restraint.
- Hold a sterile, weighed microcentrifuge tube directly under the animal's anus. It is imperative that the tube does not come into direct contact with the mouse.
- As the animal defecates, collect the fecal pellet into the tube. Keep the tube at room temperature until all fecal samples are collected. It can take several minutes for a mouse to defecate, thus necessitating repeated attempts to collect feces.
- Weigh the tubes containing feces on the analytical scale. Record the tube weight, rounding to four decimal places (e.g., 1.0021 g). Denote the obtained weight as the "final tube weight."
- Pass the fecal samples into the anaerobic chamber for bacterial enumeration directly after stool collection.
- Bacterial Enumeration of C. difficile
- Calculate the final weight of the contents (feces) that will be enumerated for C. difficile. Performed this by subtracting the "final tube weight" from the "tube weight."
- Calculate a 1:10 dilution of the contents into 1X phosphate-buffered saline (PBS) and resuspend accordingly. For fecal pellets, use the tip of the pipette to gently disrupt the pellet into solution.
- Anaerobically incubate these resuspended samples at room temperature for 30 min, allowing the contents to settle.
- Make serial dilutions for each sample in 1x PBS for enumeration of the colony-forming units (CFU) per g of content (feces). Perform this anaerobically.
- Using aseptic technique, transfer 100 µl of each serial dilution to taurocholate cycloserine cefoxitin fructose agar (TCCFA) plates. Using a sterile L-shaped spreader, spread the dilution applied to the media to evenly spread it across the plate. Even spreading of the dilution is essential for accurate enumeration of colonies.
- Incubate the plates anaerobically for 24 hr at 37 °C. At this time, enumerate C. difficile colonies to obtain the CFU per g of content (feces). C. difficile colonies are flat and pale yellow and have irregular edges and a ground-glass appearance.
NOTE: This protocol can be used to enumerate C. difficile from other murine gastrointestinal (GI) content, including ileal and colonic content. Polymerase chain reaction (PCR) plates can be used for making serial dilutions.