Processing of Infected Tissues for Bacterial Enumeration in a Mouse Co-Infection Model

Begin with a mouse co-infected with a pathogenic bacterium and a virus.

The virus damages the epithelial lining of the mouse’s upper respiratory tract, facilitating the spread of bacteria from the nasopharynx to the lungs.

Place the harvested mouse lungs into a dish containing phosphate-buffered saline, or PBS.

Mince the lungs into small pieces and mix well.

Transfer half of the lung tissue into a tube containing PBS.

Place a sterilized tissue homogenizer probe inside the tube containing the lung tissue.

Homogenize the tissue to release the bacteria into the PBS.

Dilute the homogenate and plate the dilutions on a nutrient-rich agar plate.

Incubate the plate to allow bacterial colonies to form.

Count these colonies to quantify the lung bacterial load, which reflects bacterial burden in the lungs.

For lung collection, using dissection scissors, cut the sides of the exposed rib cage and gently pull the ribs up toward the head of the mouse to expose the heart. Insert a 25-gauge needle attached to a 10 milliliter syringe prefilled with PBS into the right ventricle and begin slowly perfusing. Look for bleaching of the lungs as an indicator of successful perfusion.

Flush slowly to avoid breaking the