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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.
1. Uropathogenic Escherichia coli (UPEC) Bacterial Inoculum Preparation
- Prepare urinary tract infections89 (UTI89) inoculum (start 72 hr prior to the inoculation day)
- Streak UTI89 onto an LB (Luria-Bertani) agar plate from a frozen stock. Incubate overnight at 37 °C. Pick a colony and inoculate it into 10 ml of LB in a 125 ml flask. Incubate at 37 °C for 24 hr, statically.
- Inoculate 10 µl of overnight culture into 10 ml of LB in a new 125 ml flask for an additional 24 hr at 37 °C, statically.
- Transfer 3 ml (will vary based on strain and desired inoculum size) to sterile 1.5 ml tubes and centrifuge at 7,000 x g for 3 min and resuspend the pellet in 1x phosphate-buffered saline (PBS) and centrifuge a second time. Resuspend the bacterial pellet in 1 mL 1x PBS.
- Measure bacterial optical density and adjust concentration to the desired inoculum density. The standard concentration of the inoculum used is 107 bacteria; however, this may vary due to the design of the study.
2. Bacterial Inoculation
- Clean the workstation with 70% ethanol and cover the area with absorbent paper (or use a sterile flow hood).
- Draw up to 0.9 ml of the prepared bacterial inoculum into a 1 ml Tuberculin (TB) syringe (remove air bubbles). Attach a prepared sterile inoculation needle with polyethylene with a size designation of 10 (PE10) tubing onto the syringe containing the inoculum, then sterilely trim the polyethylene tubing.
NOTE: Leave 1 mm of tubing above the tip of the needle to avoid puncturing the bladder. - Cut a 1-inch square piece of parafilm and put a dab of surgical lubricant (approximately the size of a dime) on top.
- Anesthetize female C3H/HeN mice by putting them in a vaporizer chamber (following manufactures protocol) until unconscious but still breathing normally (1 breath/sec).
CAUTION: Isoflurane is an inhalation anesthetic. Use in a well-ventilated area and minimize inhalation. - Remove the mouse from the vaporizer and place it on its back on a paper towel and spread the legs.
- Cover the nose of the mouse with a nose cone (a tube connected to the vaporizer that provides a controlled isoflurane dose that comes equipped on some vaporizer units) to maintain anesthetization.
- Gently palpitate the bladder to induce urination and ensure a voided bladder. Wipe the periurethral area with a 100% ethanol wipe. Dab the inoculation needle/syringe, point first, into the surgical lubricant.
- Inoculate each mouse with 50 µl of the bacteria solution by inserting the inoculation needle transurethrally, approximately 12 mm, and pressing down on the syringe plunger gently to dispense the inoculum into the bladder (10 µl/sec). Remove the inoculation needle from the mouse.
NOTE: Immediate return of inoculum at the urethral opening when beginning to inoculate indicates improper or incomplete insertion of the needle. - Remove the mouse from the nose cone and return it to its cage. Repeat steps 2.3-2.8 for each mouse. When/if switching inoculum conditions/strains, dispose of the syringe and inoculation needle in an approved sharps container and start again at step 2.2.
NOTE: Inoculation with uropathogenic organisms generally does not cause severe pain symptoms. However, in rare instances, administering large doses of pathogens may cause fever, reduction in food and water intake, and abnormal behavior. Animal health should be monitored throughout the experiment. If overt pain symptoms are noticed, an analgesic, such as Buprenorphine (0.05−0.1 mg/kg given subcutaneously), can be applied.
3. Intracellular bacterial communities (IBC) Enumeration
- Aseptically remove the bladder and place it in 1x PBS in a 6-well plate with a silicone bottom. Plates can be prepared using a silicone elastomer kit and pouring approximately 1 cm of silicone in each well. Cut the bladder in half using scissors.
- Using small metal pins, gently splay the bladder so that the bladder lumen is facing upward. Be sure to place the pins at the outermost edges of the bladder sections to ensure the maximal amount of urothelium is exposed.
- After splaying, gently wash the bladder once with 1x PBS. Remove the 1x PBS by pipette. Fix the bladder by adding enough 3% paraformaldehyde to cover the entire splayed bladder.
CAUTION: Paraformaldehyde is carcinogenic. Proper protective equipment, including gloves, should be worn at all times. Disposal should follow institutional guidelines. - Incubate at room temperature for 1 hr. Remove the paraformaldehyde solution. Wash once with 1x PBS.
- Wash with LacZ wash solution (2 mM MgCl2, 0.01% sodium deoxycholate and 0.02% nonidet-p40 in 1x PBS) three times for 5 min per wash.
- Remove wash solution and add enough LacZ stain (9.5 ml LacZ wash solution, 0.4 ml 25 mg/ml X-gal, and 0.1 ml of 100 mM K-ferrocyanide/K-ferricyanide) to cover the bladders. Incubate at 30 °C overnight protected from light.
- Remove splayed bladders from the incubator and observe IBCs, which appear as blue puncta as visualized with a dissecting scope, 40-60X magnification.
NOTE: Sometimes a mouse may urinate prior to placement of the tube under the urethra. In this case, wait 15-20 minutes before attempting to collect urine again.