Preparation of Gardnerella vaginalis Inoculum to Model Recurrent Urinary Tract Infections in Mice

Abstract

Source: O'Brien, V.P. et al. Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice. J. Vis. Exp. (2020)

This video demonstrates the preparation of a Gardnerella vaginalis inoculum for establishing a mouse model of recurrent urinary tract infection.

Protocol

1. Prepare Gardnerella vaginalis inoculum
   NOTE: All steps are performed in an anaerobic chamber. Ideal culture incubation times vary among different strains of G. vaginalis, with some strains entering the stationary phase and even beginning to die more quickly than others. Thus, incubation times should be determined empirically for a given strain prior to performing experiments in mice. It is unknown whether other/all strains of G. vaginalis will trigger the same effects in this model.
2. Streak G. vaginalis strain from -80 °C freezer stock onto an NYCIII plate (without antibiotics). Incubate the plate at 37 °C anaerobically for 24 h.
3. In the anaerobic chamber, inoculate 5 mL of anaerobic NYCIII media with a 1 μL loopful of cells (a single colony is insufficient) from the NYCIII plate and incubate culture statically at 37 °C under anaerobic conditions for 18 h. Do not include antibiotics in the growth medium.
4. Determine the OD⁶⁰⁰ of the culture using a spectrophotometer
   1. Centrifuge a defined volume (X) of culture at 9600 × g for 1 min and aspirate the media. Calculate the volume (Y) of Phosphate-buffered saline (PBS) to re-suspend the pellet to achieve the desired inoculum optical density (OD) to achieve 10⁸ colony forming units (CFU) in 50 μL using the following equation:X mL × ODᶜᵘˡᵗᵘʳᵉ = Y mL × OD ^inoculum , solve for YY = (X ml × ODᶜᵘˡᵗᵘʳᵉ) / OD ^inoculum
      NOTE: The OD ^inoculum for JCP8151B^SmR is 5, but this must be determined empirically for other G. vaginalis strains. For example, if spinning 3 mL of a JCP8151B^SmR overnight liquid culture with ODᶜᵘˡᵗᵘʳᵉ = 2.0: Y= (3 mL × 2.0) / 5.0; therefore, resuspend pellet in 1.2 mL PBS.
   2. Resuspend the bacterial pellet in PBS to the desired concentration. Serially dilute and plate the inoculum to determine the actual inoculum dose that has been used in each experiment. Do not simply rely on the OD values.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glucose	Sigma	G7528	For NYCIII G. vaginalis growth media
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	Cellgro	25-060-Cl	For NYCIII G. vaginalis growth media
Sodium chloride	Sigma	S3014	For NYCIII G. vaginalis growth media
Proteose peptone #3	Fisher	DF-122-17-4	For NYCIII G. vaginalis growth media
Yeast extract	Fisher	DF0127-17-9	For NYCIII G. vaginalis growth media
Spectrophotometer	BioChrom	80-3000-45	Measuring bacterial OD600
Streptomycin	Gibco	11860038	Add to G. vaginalis NYCIII selectiveplates (1 mg/mL)