A Technique to Establish a Bacterial Infection Model in an Ex Vivo Organ Culture

Before beginning the infection, inoculate a single L. monocytogenes colony from a streaked brain-heart infusion agar plate in 3 milliliters of brain-heart infusion broth. Incubate the culture overnight in a slanted position at 30 degrees celsius to allow the bacterial growth to reach a stationary phase.

The next morning, use sterile tweezers to remove any floating organ culture pieces that did not invade into the extracellular matrix, and carefully aspirate the medium from the bottom of each transwell. Next, gently pipette 1 milliliter of warm PBS into the upper and lower transwells two times, removing each wash with careful aspiration.

After the second wash, gently pipette 1 milliliter of the appropriate medium to the upper and lower transwells taking care not to disturb the organ cultures and return the cultures to the incubator for one hour.

At the end of the incubation, replace the medium in the top of the transwells with 1 milliliter of inoculum, and place the plates back in the cell culture incubator to facilitate the bacterial invasion, replacing all of the medium in each well daily.