This article details a colony-forming unit (CFU) assay to quantify viable bacteria, specifically Listeria monocytogenes, within macrophages. The methodology includes infection of macrophages, treatment with gentamicin, and subsequent lysis to release intracellular bacteria for colony counting.
To begin the colony-forming unit, or CFU, assay to quantify viable bacteria, take a multi-well plate containing cultured macrophages in growth media. Infect the macrophages with Listeria monocytogenes, a pathogenic bacteria capable of intracellular growth.
During infection, the macrophages engulf the bacteria, internalizing the bacteria within a membrane-bound compartment — a phagosome.
Bacteria secrete a pore-forming toxin and disrupt the phagosomal membrane. Thereby, the bacteria are allowed to enter the cytosol and replicate within the macrophages.
Briefly treat the infected macrophages with gentamicin, an antibiotic that selectively eliminates non-internalized bacteria while preserving intracellular bacteria.
Aspirate the overlying media containing antibiotics. Next, add sterile distilled water over the infected macrophages, creating a hypotonic environment. This leads to osmosis, where water molecules enter the cells and cause macrophage lysis, releasing the intracellular components, including the bacteria.
Spread the cell lysate containing intracellular bacteria onto the lysogeny agar plate, promoting the separation of individual bacterial cells and enabling their independent growth. During incubation, viable bacteria grow on the agar surface and form distinct colonies.
Count the colonies on the plate and obtain the CFU, representing intracellular bacteria that have evaded the host immune response and successfully replicated within the infected macrophages.
To harvest, slightly tilt the dish and carefully use a pipette to remove the entire media from the well. Add 0.5 milliliters of distilled sterile water to the well. After 30 seconds, transfer the resulting water lysate to a 1.5-milliliter microcentrifuge tube and vortex vigorously for 10 seconds.
Add 4.5 microliters and 50 microliters of the water lysate to 450 microliters of distilled sterile water to prepare the 10-fold and 100-fold dilutions. Briefly, vortex to ensure thorough mixing. Add 50 microliters of the original 10-fold diluted and 100-fold diluted samples onto lysogeny broth agar plates and spread using a plate spreader.
To assay for emergent Lm, extract 10 microliters of the overlaying media from the dish, and divide it into two 5-microliter aliquots in microcentrifuge tubes. Add 5 microliters of DMEM/FCS to one aliquot and add 5 microliters of DMEM/FCS containing 5 microliters per milliliter gentamicin to the second aliquot as control.
Wait 5 minutes at room temperature. Add 90 microliters of distilled sterile water to each aliquot and vortex the tube vigorously for 10 seconds. Then, add 50 microliters of the sample on LB agar plates, and spread using a plate spreader. Store the plates in 37-degrees-Celsius incubator for two days before enumerating Lm colonies.
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