JoVE Encyclopedia of Experiments
Microbiology
0 views • 4:22 min • July 1st, 2026
Take a culture of Shigella, a pathogenic bacterium.
Add the bacterial suspension and culture medium to the differentiated human colonic epithelial monolayer in a culture plate.
Centrifuge to promote direct contact between bacteria and epithelial cells.
Incubate to allow bacterial internalization.
Wash with buffer to remove the extracellular bacteria.
Add antibiotic-supplemented culture medium. During incubation, antibiotics selectively eliminate the extracellular bacteria without affecting intracellular ones.
Remove the media. Wash the cells with buffer to remove the dead bacteria.
Add fresh antibiotic-supplemented culture medium to prevent cross-contamination.
Incubate to allow internalized bacteria to multiply.
Remove media and wash the cells with buffer.
Add detergent-containing buffer to lyse the epithelial cells containing bacteria.
Scrape the lysed cells and collect in a microcentrifuge tube.
Vortex to release the bacteria from the lysed cells.
The recovered bacteria are ready for downstream analysis.
To begin, take the overnight-grown Shigella cultures and vortex them. Add 100 microliters of the culture to 5 milliliters of fresh TSB in a culture tube. Incubate the cultures at 37 degrees C
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