The overall goal of this procedure is to build a model to study the interactions between an individual bacterium and a single fungus. This message can help to answer key questions in the field of pathogen interactions, such as what factors are involved in cross-talk and interspecies dialect. The main advantage of this technique is the data generated from this in vivo model have clinical relevance.
To intranasally install the fungus, first dilute two times ten to the sixth CFU of C.albicans per milliliter in sterile PBS. Next, after confirming the loss of the righting reflex in an adult hypotonic mouse, aspirate at least 50 microliters of the fungal suspension into a 200 microliter pipette. Now, grasp the mouse with one hand and turn the animal so that it is nestled on its back with its abdomen facing upright.
When the mouse is in position, use the index finger of the hand holding the mouse to support the head, and the thumb to keep the jaw closed, and move the pipette tip close to the animal's nose. Then, carefully dispense a 50 microliter drop of fungus onto the nostrils, allowing the drop to be inhaled by the spontaneous breathing of the mouse. And place the mouse alone in a cage until it is fully recovered.
Four days after C.albicans installation, shave the abdomen of the euthanized animal, followed by ethanol sterilization of the exposed skin. Next, use scissors to open the skin from the sternum to the mid-abdomen. Then, extend the incision along the rib cage on either side of the midline and fold back the skin on either side of the thorax.
When the rib cage is visible, make a vertical incision on either side of the chest toward the clavicles, and use the sternum to recline the entire anterior chest wall to visualize the heart and lungs. Then, using a pre-heparinized syringe, puncture the heart next to the intraventricular artery, and withdraw a minimum of 500 microliters of blood. Place the blood sample on ice.
Then, make a midline cervical incision to reveal the trachea. Carefully dissect the fascia around the trachea, followed by the placement of a suture under the trachea. Then, use a 20-gauge modified gavage needle to catheterize the trachea, securing the cannula with the previously placed suture.
To perform a bronchoalveolar lavage, gently dispense 500 microliters of ice-cold PBS into the lung. Then, aspirate the lavage fluid and transfer the fluid into a two milliliter centrifuge tube on ice. After collecting two more 500 microliter samples, remove the lungs from the chest, and place a piece of lung into a 1.5 milliliter centrifuge tube for immediate storage at minus 80 degrees Celsius.
Then, place another piece of lung tissue into a pre-weighed hemolysis tube containing PBS on ice. Finally, make an incision in the left side of the abdomen. Harvest the spleen into a second hemolysis tube containing one milliliter of PBS and place the tube on ice.
A four days persistence model is obtained by intranasal installation of five times ten to the fifth CFU of C.albicans per mouse. During these four days, mice gain weight, but do not exhibit lung injury. As the CFU of the inoculum increases, however, so does the lung injury.
With the maximum lung injury observed between 24 to 36 hours after infection. The bacterial burden, by contrast, demonstrates a one log CFU per milliliter decrease every 24 hours, with a cumulative bacterial dissemination increase observed over each day. In the bronchoalveolar lavage harvested from infected mice, neutrophils are widely recruited, with the differential cell count demonstrating a 90 percent neutrophil, 10 percent macrophage lymphocyte infiltrating immune cell population.
Following this procedure, other methods like analysis of the bronchoalveolar lavage can be performed to answer additional questions about the characterization of the inflammatory response. After watching this video, you should have a good understanding of how to build a colonization Pseudomonas. Don't forget that working with luminescent Candida can be extremely hazardous, and the precautions, such as wearing protective gear to avoid droplet contamination, should always be taken while performing this procedure.
