Summary

אינדוקציה Transurethral של דלקת בדרכי השתן עכבר

Published: August 05, 2010
doi:

Summary

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

Abstract

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37°C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

Protocol

I. הכנה של צנתרים השופכה במשך כל שלפוחית ​​השתן העכבר כליה להיות שנדגמו, מקום 5-6 חרוזים נירוסטה (שלפוחית ​​השתן, בגודל 1.6 מ"מ או תערובת 0.9-2.0 מ"מ) או חרוזים זירקוניום אוקסיד (כליות בגודל 0.5 מ"מ) הגליל האחד cryovial, בהת…

Discussion

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 dilu…

Declarações

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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  

Referências

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Citar este artigo
Thai, K. H., Thathireddy, A., Hsieh, M. H. Transurethral Induction of Mouse Urinary Tract Infection. J. Vis. Exp. (42), e2070, doi:10.3791/2070 (2010).

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