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 pulmonary tissue. Lift the heart with forceps and make a cut to separate the lungs and heart. Once separated, pick up all lobes of the lung with forceps and rinse in a dish with sterile PBS to remove any residual blood.

In a Petri dish, mince the lung into small pieces and mix well. Remove half of the lung mix to determine the bacterial CFU or viral PFU and place it in a round-bottom 15 milliliter tube prefilled with 0.5 milliliters of PBS for homogenization.

To homogenize the collected tissue, clean the homogenizer probe by putting it in 70% ethanol and turning on the homogenizer at 60% power for 30 seconds. Repeat this step in sterile water for 10 seconds.

Homogenize each tissue for 1 minute. For enumeration of bacterial numbers, plate serial dilutions on blood agar plates. To calculate the total CFU use 10 microliters to plate and note the final volume in milliliters for each sample. Incubate overnight at 37 degrees Celsius and 5% carbon dioxide.