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 nec