This article describes a method for establishing a vaginal infection model using Group B Streptococcus (GBS) in hormone-treated female mice. The protocol includes culturing GBS, preparing the inoculum, and administering it to the mice to study bacterial adherence and infection dynamics.
Take a culture of Group B Streptococcus or GBS, an opportunistic bacterial pathogen.
Dilute the culture in fresh media and incubate to obtain actively dividing bacteria.
Transfer the culture to a conical tube.
Centrifuge to pellet the bacteria, then discard the supernatant.
Wash the bacteria with buffer.
Centrifuge again, remove the supernatant, and resuspend the bacteria in buffer.
Next, use a hormone-treated female mouse with a primed vaginal epithelium, more susceptible to infection.
Restrain the mouse. Using a fine pipette tip, dispense the GBS suspension into the vaginal lumen.
Release the mouse, lift its tail, and let the front paws walk on a firm surface.
This promotes inoculum retention in the vaginal lumen, enabling GBS adherence.
Inspect the vaginal opening for inoculum backflow.
The adhered bacteria colonize the vaginal epithelial surface and establish an infection.
The mouse model with vaginal infection is ready for further studies.
On the same day as the injection, grow a five milliliter, overnight liquid culture of the GBS strain of interest in Todd Hewitt broth, or THB, at 37 degrees Celsius.
The next morning, subculture the GBS at a 1 to 10 volume in fresh THB for a two to three hour incubation at 37 degrees Celsius, until the bacteria reach mid-log phase. Then, transfer the subculture into a sterile, 15 milliliter conical tube, and centrifuge the bacterial cells. Resuspend the pellet in 200 microliters of sterile PBS.
Then, bring the total volume of the suspension up to 1 milliliter of PBS per 10 mice, to reach an OD600 of 0.4 in a new, 5 milliliter culture tube. Transfer the culture to a new, 15 milliliter tube, and re-pellet the bacteria. Then, resuspend the cells in PBS at one tenth the original volume.
Reserve 50 microliters of the bacteria for serial dilution and plating on THB agar, to determine the exact CFU of the inoculum. Then, draw 10 microliters of GBS into a 200 microliter gel-loading pipette tip, and manually secure the loose skin at the scruff of the neck, of the first animal, between the thumb and index finger, followed by immobilization of the tail. Insert the pipette tip 5 to 10 millimeters into the vaginal lumen, and dispense the entire volume of inoculum.
Then, immediately release the scruff and lift the tail to elevate the hind end of the animal, while walking the front paws on a hard surface. After a minute, visually inspect the vaginal opening for any inoculum backflow.
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