Quantification of Bacterial Load in Mouse Spleen and Liver After Systemic Infection

Begin with the spleen and liver tissues isolated from a mouse infected with an intracellular bacterial pathogen.

These organs, which function as immune filters, accumulate bacteria captured by tissue-resident phagocytic cells during systemic infection.

Place each organ into a tube filled with a lysis buffer containing a non-ionic detergent and glass beads to facilitate tissue disruption.

Mechanically agitate the tubes so that the beads repeatedly collide with the tissue, breaking down its structure.

The detergent permeabilizes host cells without solubilizing bacterial cell walls, thereby releasing intact, viable bacteria into the suspension.

Perform serial dilutions of each homogenate to obtain a range of bacterial concentrations.

Spread aliquots from each dilution onto nutrient-rich agar plates.

Incubate the plates to allow individual bacteria to grow into distinct colonies.

Count the colonies to determine the bacterial load in each tissue sample.

To measure the peak infection bacterial load, on day three post infection, restrain the limbs of an experimental animal in the supine position on a dissecting board. And disinfect the skin with 70 percent ethanol.

Using sterile tough cut scissors,