Take a restrained mouse and clean its tail with alcohol to reduce contamination at the injection site and dilate the vein.
Inject Listeria monocytogenes, a pathogenic bacterium, into the tail vein to deliver it directly into the bloodstream.
Apply gentle pressure at the injection site to stop bleeding, then allow the mouse to recover.
The injected bacteria circulate systemically and reach the heart. They then colonize the heart, establishing an infection.
Later, harvest the pathogen-infected heart from the euthanized mouse.
Homogenize the heart using a homogenizer to release bacteria from the tissue.
Perform serial dilutions of the homogenate in a multi-well plate.
Spot small volumes from each dilution onto an agar plate and allow them to dry.
Incubate the plate to allow the bacteria to proliferate and form visible colonies.
Count the colonies to determine the bacterial load in the infected mouse heart.
To inoculate L. monocytogenes into mice, place one animal into a harness in order to restrain the animal during injection. Use an alcohol pad to clean the injection site and dilate the tail vein.
Then, with a 1 milliliter syringe and a 27.5 gauge needle, gently inject the tail vein of the mouse with 200 microliters of the desired culture. Use a paper towel to stop any bleeding that may occur from the injection before placing the animal into a fresh cage. After sacrificing the animals up to 72 hours later, according to the text protocol under the biosafety cabinet, secure the animal onto a dissection block and use 70% ethanol to saturate it.
Make a Y-shaped incision on the abdomen and thorax that extends from the vaginal opening up to the xiphoid process, progressing to the axilla of each arm. Pull back the skin, remove the needles from the legs, and use them to hold open the dissection and cut gently through the peritoneum, exposing the intestines, stomach, liver, and spleen.
Now, locate the diaphragm and gently cut along the rib cage in order to visualize the thorax. Cut through the xiphoid process and sternum up to the neck to open the thorax, exposing the heart and lungs. Using forceps, gently grab the heart by its apex and lift it to create tension in the aorta and pulmonary vessels. Then cut the vessels to free the heart and place it into a separate tube of water.
After preparing a homogenizer according to the text protocol, homogenize one liver for at least two minutes or until no visible portions of the organ remain. Use tweezers to remove any large debris from the probe.
Once all organ samples have been homogenized, prepare serial dilutions of 1 to 10 to 1 to 10,000 for the liver and spleen samples, and 1 to 10 to 1 to 1000 for the heart. Spot plate the dilutions onto pre-warmed LB agar plates. Undiluted samples can be plated on separate plates to ensure that organs were sufficiently infected.
After the plates dry, place the plates in the incubator at 37 degrees Celsius. Use parafilm to wrap the 96-well plates of diluted samples and place in a minus 80 degrees Celsius freezer.
The following day, count the number of colonies and use the dilutions to calculate the total number of bacteria per organ.
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