Preparation of Bioluminescent Enterohemorrhagic Escherichia coli Inoculum

Begin with a frozen stock of enterohemorrhagic Escherichia coli or EHEC, a pathogenic intestinal bacterium.

These bacteria carry a plasmid that expresses luciferase for real-time bioluminescent imaging and an antibiotic resistance gene for selection.

Streak the bacteria onto an agar plate containing the appropriate antibiotic and incubate to isolate individual plasmid-bearing colonies.

Pick a single colony and culture it in antibiotic-supplemented broth. Incubate to propagate a genetically uniform, plasmid-stable population.

Expand the culture under aerated conditions to synchronize cells in logarithmic growth.

Monitor the bacterial growth until the desired optical density is reached.

Next, centrifuge the culture to pellet the bacteria.

Remove the supernatant, and wash the pellet with sterile saline.

Centrifuge again. Discard the supernatant and resuspend the pellet in a smaller volume of saline.

This concentrated EHEC suspension is now ready for bioluminescence-based tracking.

To begin, streak a negative 80 degree Celsius stock of E.coli O157 H7 EDL933 bacteria harboring a luciferase plasmid onto an LB agar plate with 50 micrograms per milliliter of kanamycin. Grow the bacteria for 16 to 18