Quantification of Clostridium difficile in Fecal Samples from an Infected Mouse Model

Begin with a mouse whose gastrointestinal tract is colonized by Clostridium difficile, an anaerobic, spore-forming bacterial pathogen.

Restrain the mouse and allow it to defecate naturally into a sterile, pre-weighed tube.

Determine the net weight of the fecal sample by subtracting the empty tube weight from the total weight to prepare appropriate dilutions.

Transfer the sample into an anaerobic environment to preserve the viability of the oxygen-sensitive bacterium.

Add a buffer to the sample and mechanically disrupt it to release its contents into suspension.

Incubate to allow larger debris to settle, retaining bacterial cells and spores in the supernatant.

Perform serial dilutions of the supernatant in the buffer.

Plate the dilutions onto selective media and incubate under anaerobic conditions.

The media promote spore germination and selective growth of Clostridium difficile while inhibiting competing bacteria, resulting in distinct colonies.

Count the colonies to quantify the Clostridium difficile load in the feces and evaluate gastrointestinal colonization.

To collect fecal samples from mice for assessment, first lightly but firmly restrain the subject mouse by the loose skin on the neck and back, ensuring the animal is not in distress.

Using the pinky finger, gently lift the animal's tail, exposing the anus. Hold a sterile microcentrifuge tube of known weight directly under the animal's anus, ensuring the tube does not directly touch the mouse. When the animal defecates, collect the fecal pellet in the tube.

Store the tube at room temperature until all necessary samples are collected. Weigh the tubes containing feces on an analytical scale and record the mass to four decimal places. Calculate the weight of the fecal pellet by subtracting the weight of the tube.

Next, calculate a one to ten dilution of the contents and resuspend into 1x PBS. Use the tip of the pipette to gently disrupt the fecal matter into solution. Incubate the samples at room temperature for 30 minutes, allowing the contents to settle.

Maintaining the solutions anaerobically, make serial dilutions for each sample in 1x PBS. Transfer 100 microliters of each serial dilution onto TCCFA plates. Use a sterile L-shaped spreader and evenly apply the media to the plates.

Incubate the plates in anaerobic conditions at 37 degrees Celsius for 24 hours. Finally, enumerate the C.difficile colonies and calculate the colony forming unit per gram of fecal content.