Intrabronchial Instillation of Streptococcus pneumoniae in Immunocompetent Rodent Models

Obtain a sterile intubation device consisting of a metal cannula with polyethylene tubing connected to a metal needle.

Attach a syringe containing Streptococcus pneumoniae to the needle, and fill the tubing.

Next, position an anesthetized immunocompetent rat in a supine position.

Insert the free end of the metal cannula into the rat's mouth. Advance it into the trachea toward the left bronchus and down the left lung to an appropriate depth.

Advance the tubing to the end of the cannula and instill the bacterial suspension into the left lung lobe.

The bacterial capsule and surface-associated enzymes prevent bacterial entrapment in the mucus. Further, Streptococcus pneumoniae attach to the surface receptors on bronchial epithelial cells, facilitating endocytosis and release to the basolateral surface.

Bacteria secrete toxins that damage the epithelial cells and barriers, triggering an immune response.

However, Streptococcus pneumoniae has developed strategies to evade the host immune response and establish lung infection.

Place a clean disposable mat onto the work surface, close to the water bath.

To prepare the work surface and intubation device, first, sterilize the metal cannula and polyethylene tubing according to the text protocol, and then, transfer it in its glass storage tube to the work surface. Then, remove the cap from the storage tube and slide the handle end of the cannula to the open end of the tube. Using sterile forceps, remove one length of polyethylene tubing from the beaker, and insert it through the inside of the sterile metal cannula, making sure it moves freely. Place a guide mark at a predetermined location with indelible pen.

Next, fit a sterile disposable 25-gauge needle onto the free end of the polyethylene tubing by sliding the needle several millimeters into the tubing. Then, use up sterile saline to fill a fresh disposable 1-milliliter syringe. Attach the syringe to the needle fitted onto the polyethylene tubing, and flush the entire volume of saline through the tubing.

Discard the used syringe in the sharps container. Then, fill a new sterile disposable 1-milliliter syringe with the agar-based inoculum from the water bath. Reattach the 25-gauge needle and tubing and flush agar through the tubing by depressing the plunger just until the tubing is completely filled with agar.

After anesthetizing the rat according to the text protocol, place the animal in a supine position on the disposable mat, with the head facing right and the tail facing left. Next, insert the free end of the metal cannula into the animal's mouth. Turn the cannula so that the free end is angled upward and gently advance it into the trachea, carefully bypassing the laryngeal structures with a slight twisting motion.

Confirm insertion into the trachea as opposed to the esophagus by gently sliding the cannula slightly forward and back several times, while using the left forefinger to palpate the tracheal rings.

To ensure intubation into the trachea and not the esophagus, move the cannula back and forth slightly and feel for the ridges. If the cannula slides smoothly down the throat and the ridges cannot be felt, remove the device and try again.

When the cannula reaches the bifurcation where the trachea splits into the left and right bronchi, make a slight twisting motion toward the animal's left side to ensure that the cannula is inserted into the left bronchus.

A common error is not placing the inocula deep enough into the lung, which can block the airways and inhibit breathing. When a cannula is turned to the proper angle, it should slide easily into the left bronchus, and advance smoothly to the appropriate depth.

Advance the metal cannula until the end is halfway to 3/4 down the left lung, using the previously placed guide mark to confirm that the appropriate depth has been reached. With the metal cannula in place, advance the polyethylene tubing several millimeters using the previously placed guide mark on the tubing to ensure it is advanced only far enough to exit the end of the metal cannula and not puncture the lung.

With both cannulae in place, use the attached syringe to instill 100 microliters of agar suspension deep into the large lobe of the left lung. Withdraw several millimeters of the tubing, and gently remove the intact intubation device. Set it back into the glass storage tube and move the animal into a fresh cage to recover.