This article describes a method for quantifying bacterial burden in mouse spleen tissue following infection. The process involves homogenizing the spleen, performing serial dilutions, and culturing the bacteria on agar plates to determine colony-forming units (CFUs).
Begin with a mouse spleen pre-infected with pathogenic bacteria.
Place the spleen in cold phosphate-buffered saline to preserve bacterial viability.
Using a homogenizer, homogenize the spleen to mechanically disrupt it and release the intracellular bacteria into the buffer.
This generates a bacterial suspension that reflects the total bacterial burden within the spleen.
Perform serial dilutions of the homogenate to reduce bacterial density in the solution for accurate bacterial count determination.
Next, take a nutrient agar plate divided into two sections.
Plate the same bacterial dilution onto each section of the agar plate to generate replicates.
Allow the plate to dry, then invert it and incubate. During incubation, viable bacteria multiply and form distinct colonies.
Count the colonies from each section of the plate to calculate the average colony-forming units, or CFUs, which represent the total bacterial burden in the spleen during infection.
The spleen in a 15 milliliter tube containing two milliliters of PBS. Next, use individual, disposable homogenizers per sample to macerate the tissues on ice, followed by serial dilution of the tissue slurries.
Plate the dilutions, in duplicate, on separate sides of a single TSA plate, and allow the samples to briefly dry. Then, invert the plates and culture the samples in a static 37 degrees Celsius incubator overnight, averaging the bacterial colonies from both sides of the plate and from each dilution the next morning.
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