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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
- Inoculation of Animals with Bacterial Strains by Injection
- On the morning of injections, remove cryovials of the bacterial strains being tested and thaw at 4 °C for 3-4 h. Thaw 0.5 mL for each mouse that will be injected. Keep vials on ice after thawing and inject mice within 2 h.
- After thawing, transfer each cryovial to a new 2 mL tube and centrifuge at 4,500 x g for 10 min, discard the supernatant, and resuspend the cell pellet in 1 mL of 1x PBS (Phosphate-buffered saline).
- Centrifuge again at 4,500 x g for 10 min. Discard supernatant and resuspend pellet in 1x PBS to a final concentration of 2.5 x 109 CFU/mL (CFU: colony-forming units). The exact amount of 1x PBS used will vary slightly between strains.
- Take 3 samples from final suspension of each strain to validate CFU/mL, genotype, and phenotype as described above.
- For each strain, aliquot 1.5 mL of PBS/cell suspension into one 2 mL tube per 5 mice to limit the number of times the tube is entered. Also, prepare tubes of 1x PBS for control injections.
- Gather mice (10 male and 10 female C57BL/6 per group for this experiment) and materials needed for injections (syringes, needles, sharps containers, markers, pen and paper, etc.). Move to the sterile animal surgical room. Wipe all surfaces with sanitizing wipes.
- To eliminate distress and risk of injury to experimenter, only bring one sex and experimental group of mice to the surgical room at a time (e.g., a group of 10 male mice to be infected with a particular strain). Wear two pairs of latex gloves to eliminate puncture of gloves if bitten. Wear lab coat, safety glasses, and face mask to avoid contamination.
- Begin injections of the control group with 1x PBS. This will ascertain whether any adverse effects result from the injection alone.
- Remove a mouse from the cage. Only remove one mouse at a time.
- Weigh the mouse and mark its tail with a permanent marker to track for weight loss post-injection.
- Open a new 1 mL syringe and 27 G needle (use a new syringe and needle for each mouse to eliminate contamination) and inject 200 µL of sterile 1x PBS.
- Grab the mouse behind its ears using the thumb and forefinger. Pinch to create skin fold at nape of neck to hold onto - a tighter fold reduces neck movement and risk of being bitten during injection. Secure tail into the palm using the pinky to hold mouse flat with little movement.
- Turn the mouse over and insert needle at 30° angle into the peritoneal cavity to the left or right side of midline. Lift the needle slightly once inserted to ensure it was inserted into the intraperitoneal area and not into organs. Slowly inject the PBS and then withdraw the needle.
- Place the used needle in a designated biohazard sharps container. Do not reuse the syringe or needle. A bolus at the site of injection is typical.
- After injection, move the mouse to a separate cage.
- Repeat the procedure with the next mouse. After all the mice of one cage are injected, return them to their original cage immediately.
- After injecting the control group, begin injecting suspensions of strains to be tested following the same procedure.
- Inject 200 µL of the cell suspension. When beginning with cell suspensions of 2.5 x 109 CFU/mL, each mouse receives 5 x 108 CFU.
NOTE: These concentrations were optimized with preliminary animal research to determine lethal doses of each strain. The dosing may need to be adjusted for other strains/species.
- Once all injections are complete, return the mice to the housing room to alleviate distress. Clean work area with sanitizing wipes.
- Monitor the animals for mortality following injection by checking cages for dead mice every 3 h for 72 h and every 12 h for 10 days. Record the weight of mice every 12 h to determine weight loss due to illness.