Visualizing Intracellular Bacterial Communities in an Infected Mouse Bladder

Abstract

Source: Conover, M. S. et. al., Establishment and Characterization of UTI and CAUTI in a Mouse Model. J. Vis. Exp. (2015)

This video demonstrates the detection of intracellular bacterial communities (IBCs) in mouse bladder tissue infected with β-galactosidase-expressing Escherichia coli. Following tissue fixation, permeabilization, and staining with X-gal, IBCs appear as blue puncta under a dissecting microscope, confirming bacterial invasion during urinary tract infection.

Protocol

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

1. Prepare urinary tract infections89 (UTI89) inoculum (start 72 hr prior to the inoculation day)
   1. 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.
   2. Inoculate 10 µl of overnight culture i....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material for infections:
Isoflourane – Isothesia	Butler Schein	29405	250 ml
Clear glass straight-sided jar	Kimble Chase	5413289V 21
Stainless steel tea infuser	Schefs-Amazon		Premium loose leaf tea infuser by Schefs - stainless steel - large capacity -
Non-sterile cotton balls	Fisherbrand	22-456-880
50 ml Falcon tubes	VWR	89039-660
Isotec 3 -vaporizer	Ohmeda	1224478
Ear punch	Fisher Scientific	13-812-201	(when necessary)
Betadine solution	Betadine solution		10% Povidie-iodine topical solution
Q-tips	Fisher Scientific	22-037-924	6 inches
Diapers for bench	Fisherbrand	14206 63	Absorbent underpads (20”X36”mats)
Surgical lubricant	Surgilube	0281-0205-36
Dissecting scissor	Fine Science tools, INC	14084-08
Micro-Adson forceps	Fine Science tools, INC	11018-12
1 ml syringe	BD	309659	Tuberculin slip tip
Parafilm	Bemis	PM996	4 inches x 125 FT
Eppendorf rack	Fisherbrand	05-541-1
Eppendorf tubes	MIDSCI	AVX-T-17-C
IBC materials:
6-well tissue culture test plate	Techno Plastic Products	92006
Pins	Fine Science Tools	26002-20
Sylgard 184	Dow Corning	3097358-1004	Silicone elastomer kit
X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactoside)	Invitrogen	15520-034	Ultrapure
N, N-dimethylformamide	Sigma Aldrich	D4551
Magnesium chloride	Sigma Aldrich	M8266
Sodium deoxycholate	Sigma Aldrich	D6750
Nonidet-P40	Roche	11754599001	Octylphenolpoly(ethyleneglycolether)n
Potassium hexacyanoferrate(II) trihydrate (K-ferrocyanide)	Sigma Aldrich	P3289
Potassium hexacyanoferrate(III) (K-ferricyanide)	Sigma Aldrich	60299