This article describes a protocol for preparing a concentrated suspension of enterohemorrhagic Escherichia coli (EHEC) for bioluminescence-based tracking. The method involves culturing EHEC with a luciferase plasmid and antibiotic selection to ensure a genetically uniform population.
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 hours at 37 degree Celsius. Pick a single colony from the overnight plate and inoculate three milliliters of LB medium with 50 micrograms per milliliter of kanamycin.
Then, incubate the culture at 37 degree Celsius and 220 rpm for 16 to 18 hours. The following day, prepare a 2 milliliters of a 1 to 100 dilution of the overnight culture and inoculate it into 200 milliliters of LB with kanamycin. Incubate the culture at 37 degree Celsius and 200 rpm for 2.5 to 3 hours or until the optical density at 600 nanometers reaches between 0.9 and 1.
Next, centrifuge the re-cultured bacteria at 8000 times gravity and four degree Celsius for 30 minutes. Discard the supernatant without disturbing the pellet and wash the bacteria with 100 milliliters of 0.9% sterile normal saline by gentle agitation. Following the wash, centrifuge the bacterial culture again and gently discard the supernatant.
Then, use 0.9% sterile normal saline to concentrate the pellet 100-fold.
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