Quantification of Bacterial Infection in Host Cells by the Colony-Forming Unit Assay

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.