JoVE Encyclopedia of Experiments
Microbiology
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Take a culture of mouse peritoneal macrophages and introduce a bacterial pathogen.
Incubate to allow bacterial uptake through phagocytosis into the macrophages, where they alter intracellular degradation pathways and survive.
Remove the supernatant and wash with a buffer to eliminate extracellular bacteria.
Add a medium containing gentamicin antibiotic and incubate to remove any remaining extracellular bacteria.
Wash the infected macrophages and mechanically detach them. Collect the cells, centrifuge, and remove the supernatant.
Add a bacteriolytic enzyme and incubate to lyse any additional extracellular bacteria.
Centrifuge and discard the supernatant. Wash the cells, centrifuge, and remove the supernatant.
Resuspend and aliquot the cells. Add a detergent to lyse them, releasing intracellular bacteria. Serially dilute the lysate in buffer and drop the dilutions onto agar.
Incubate to allow bacterial colony formation. Count the colonies to assess bacterial survival within the macrophages.
Incubate the peritoneal macrophages with Staphylococcus aureus MRSA-252 for two hours. Remove the supernatant and wash the cells twice with PBS. Add 1 milliliter per well of complete DMEM medium containing 100 micrograms per milliliter gentamicin and incubate for two hours at 37 degrees Celsius in 5% carbon dioxide.
After incubation, wash the cells thrice with PBS. Using a cell scraper, collect the peritoneal macrophages into 50 milliliters centrifuge tubes. Centrifuge the samples at 1000 g for 5 minutes.
Add 10 micrograms per milliliter of lysozyme to the macrophages infected with intracellular MRSA-252, and incubate at 37 degrees Celsius for 10 minutes. After washing the cells twice with PBS, resuspend them in 1 milliliter of PBS. Then, take out about 20 microliters of aliquot and add 0.1% Triton X-100 for five minutes to lyse the cells.
Perform serial dilutions of the lysed cell suspension with PBS and drop them onto a Tryptic Soy Agar plate. Incubate the plate overnight at 37 degrees Celsius. The next day, count the bacterial colonies to calculate the intracellular MRSA-252 bacteria in peritoneal macrophages.