Begin with an anesthetized mouse with a Petri dish lid placed under its abdomen to keep the back horizontal.
The mouse exhibits a burn wound on its back that has been infected with Acinetobacter baumannii, a Gram-negative bacterial pathogen.
The bacteria are genetically engineered to carry a bioluminescence operon.
Position the mouse inside a bioluminescence imaging chamber maintained at an optimum temperature and begin imaging.
At the infection site, the luxA and luxB genes of the bioluminescence operon encode the luciferase enzyme. The luxCDE genes encode enzymes that synthesize the luciferase substrate.
Luciferase catalyzes substrate oxidation and produces visible light, making the bacteria luminescent.
The bioluminescence signal indicates the presence of metabolically active bacteria at the wound site.
Using imaging software, quantify the bioluminescence signal intensity to estimate the viable bacterial load.
Immediately following the inoculation, carry out bioluminescence imaging by starting the live imaging software. In the control panel, click Initialize. Then wait until the color of the temperature box turns green, indicating that the temperature of the stage in the specimen chamber has reached to 37 degrees Celsius.
Place the anesthetized mouse on the warm stage in the specimen chamber with the infected burns directly under the camera. Next, in the control panel, put a check mark next to “Luminescence”.
Then select "Auto exposure" so that the exposure time for imaging will be optimized by the live imaging software based on the bioluminescence intensity. Select "C" from the "Field of View" drop-down list. Select the scan mid-range option to let the software determine the focal distance.
Then put a check mark next to overlay. Now, click "Acquire" to capture the image. Then, in the "Edit Image Label" box, click ok. An "Image Window" and "Toll Palette" will appear. Set the Auto-ROI parameters for auto-selection.
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