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

Take a stock of an antibiotic-resistant strain of G. vaginalis, an anaerobic bacterium associated with urinary tract infections in mice.

During an initial bladder infection, uropathogenic E. coli establishes quiescent reservoirs within bladder epithelial cells.

Subsequent exposure to G. vaginalis triggers toxin-mediated shedding of superficial cells, allowing dormant UPEC to re-emerge and cause recurrent UTIs.

To prepare the G. vaginalis inoculum, streak the stock onto a nutrient agar plate without antibiotics to enable bacterial recovery.

Incubate anaerobically to obtain actively growing colonies.

Transfer a loopful of dense colonies into antibiotic-free nutrient broth.

Incubate statically under anaerobic conditions until the desired bacterial concentration is reached.

Measure optical density to estimate the concentration.

Transfer the suspension to a tube, centrifuge to pellet the bacteria, discard the supernatant, and resuspend in a buffer.

Prepare serial dilutions and plate them on antibiotic-containing media to allow selective growth of G. vaginalis and prevent contamination. Count colonies to determine the inoculum concentration.

Begin by cycling the G. vaginalis strain from a minus 80 degree Celsius freezer stock into an anaerobic chamber. Streak the bacteria onto an NYCIII plate without antibiotics. Incubate the plate at 37 degrees Celsius anaerobically for 24 hours.

In the anaerobic chamber, inoculate five milliliters of anaerobic NYCIII media with a one-microliter loop full of cells from the NYCIII plate, and incubate the culture statically at 37 Celsius under anaerobic conditions for 18 hours. Do not include antibiotics in the growth medium. Determine the OD₆₀₀ of the culture using a spectrophotometer.

Then, centrifuge it at 9,600 times g for one minute and aspirate the media. Resuspend the bacterial pellet in PBS to the desired concentration and serially dilute and plate the inoculum.