Modeling Murine Vaginal Colonization by Anaerobic Bacteria

Abstract

Source: Morrill, S. R., et al. Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria. J. Vis. Exp. (2022)

This video demonstrates a protocol for modeling vaginal colonization in mice using antibiotic-resistant anaerobic bacteria. It begins with hormonal treatment to increase epithelial susceptibility, followed by vaginal inoculation and co-inoculation under anaerobic conditions. The procedure concludes with post-inoculation sampling and culture to evaluate bacterial colonization and biofilm formation.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of inoculum

NOTE: This section contains the general steps for the preparation of inocula from anaerobically grown streptomycin-resistant Gardnerella vaginalis JCP8151B-SmR. All steps in this section should be done in an anaerobic chamber.

1. Cycle any reagents, including prepared New York City broth III (NYCIII), agar, and Phosphate-Buffered Saline (PBS), into an anaerobic chamber to equilibrate for at least 24 h prior to use.
   NOTE: Here, a v....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
β-estradiol 17-valerate	Sigma	E1631	estrogenization of mice
1x PBS	Fisher	MT21040CM	vaginal lavage, G. vaginlalis inoculum prep, tissue collection and homogenization
Anaerobic chamber	Coy	7200000	Growth of anaerobic bacteria
Bacto agar	Fisher	DF0140074	G. vaginalis, F. nucleatum, and P. bivia agar plates
Glucose	Sigma	G7528	NYCIII media for G. vaginalis growth
HEPES	Cellgro	25-060-Cl	NYCIII media for G. vaginalis growth
Horse serum	Hemostat	SHS500	NYCIII media for G. vaginalis growth
Hydrogen gas mix (5% hydrogen, 10% CO₂, 85% nitrogen)	Matheson		Gas for anaerobic chamber
Milli-Q Water Purifier	Millipore	IQ-7000	for making media and homogenization
NaCl	Sigma	S3014	NYCIII media for G. vaginalis growth
Nitrogen gas	Airgas		Gas for anaerobic chamber
p-200 filter tips	ISC Bio	P-1237-200	vaginal lavages and bacterial inoculations
Sesame oil	Fisher	ICN15662191	estrogenization of mice
Spectrophotometer	BioChrom	80-3000-45	measuring bacterial OD₆₀₀
Streptomycin	Gibco	11860038	add to agar media to make selective plates
Yeast extract	Fisher	DF0127-17-9	NYCIII media for G. vaginalis growth