This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.
I. Preparation of Urethral Catheters
II. Animal Inoculation
III. Measuring CFU in Infected Tissue
IV. Representative Results
Figure 1. Urethral meatus (dark pink tissue) exposed by lifting clitoral hood cephalad with forceps (beyond top of photo)
Figure 2. Catheterized urethra. The bifid clitoris can be seen just above the catheter.
Figure 3. Bladder cfu counts 2 days after transurethral infection of 8 week old female CBA/J mice with uropathogenic E. coli. Bladders were homogenized and cfu/ml determined by serial dilution plating on MacConkey agar. Diamonds indicate cfu/ml for individual mice, horizontal bar indicates median cfu/ml.
Induction of mouse urinary tract infection by transurethral inoculation of bacteria is a long-established but technically demanding procedure11. Hung and Hultgren recently published an excellent review of transurethral techniques of mouse urinary tract infection13. In this paper, we attempt to further illustrate the finer points of this experimental approach.
Novices may practice their technique by performing a midline laparotomy to expose the bladder, injecting a dilute solution of India ink (1% in PBS), and observing for blue coloration of the bladder. We have found that a full bladder is one of the most difficult problems to deal with during mouse urethral injection, and is best prevented by the water deprivation and pre-anesthesia voiding strategies outlined above. In our hands, <10% of mice exhibit overflow of urine during urethral injection. Another potential stumbling block is traumatic catheterization, which can lead to urethral perforation, improper doses of bladder inocula, and even animal death. Gentle technique and patience are key during catheterization; successful catheterization should never require forcing the catheter into the bladder.
Finally, some laboratories may not wish to use a Bullet Blender homogenizer or other “medium throughput” homogenizer, either due to low sample numbers or cost considerations. An intermediate solution is to use a standard homogenizer and clean the device between each sample. The most economical solution is use of glass grinders, which we successfully employed prior to switching to the Bullet Blender homogenizer. Regardless of the tissue homogenization method used, meticulous, aseptic, and consistent technique are crucial to obtaining reproducible results.
The authors have nothing to disclose.
The authors would like to acknowledge Shelly Xie and Jennifer Payne for their expert technical assistance. This work was made possible through grants from the Society of Pediatric Urology and the Stanford Pediatric Research Fund.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Cryovial tubes, 2.0 ml polypropylene (no caps) | E&K Scientific | 640201 | ||
Caps for cryovial tubes, with ‘O’ ring | E&K Scientific | 440011-N | ||
Bullet Blender | Next Advance | BBX24 | ||
Zirconium Oxide Beads (0.5mm) | Next Advance | ZROB05 | ||
Stainless Steel Beads (1.6mm) | Next Advance | SSB16 | ||
Stainless Steel Bead Blend (0.9-2.0mm) | Next Advance | SSB14B | ||
CBA/J Mice, 8-12 week old female | Jackson Laboratories | 000656 | ||
Phosphate Buffered Saline (NaCl 137mM, KCl 2.7mM, Na2HPO4 4.3mM, KH2PO4 1.4mM) | EMD | EM-SX0420-13 (NaCl) EM-PX1405-1 (KCl) EM1.06585.0 (Na2HPO4) EM-5108-1 (KH2PO4) | ||
LB agar | ||||
LB broth | EMD | 1.10285.0500 | ||
Isoflurane (Aerrane) | Baxter | NDC 10019-773-60 | ||
Inoculating loops | Fisher Scientific | 22-363-601 | ||
Eosin-methylene blue (EMB) agar powder | Himedia Laboratories | MM022-500G | ||
MacConkey agar powder | Difco | 212123 | ||
LB agar powder | Difco | 244520 | ||
Intramedic non-radiopaque polyethylene tubing, PE10 [inner dimension 0.28 mm (0.011 inch); outer dimension 0.61 mm (0.024 inch)] | BD Biosciences | 427401 | ||
30 gauge needles, 1/2 inch | BD Biosciences | 305106 | ||
1 ml tuberculin Slip-Tip syringe | BD Biosciences | 309602 | ||
3 ml Luer-Lock syringe | BD Biosciences | 309585 |