This study investigates the susceptibility of female mice to bacterial infections in the vaginal environment following hormonal treatment. The methodology includes inoculating the vaginal lumen with anaerobic bacterial pathogens and assessing the adherence and growth of these bacteria.
Take a female mouse treated with a hormone that conditions the vaginal environment, increasing its susceptibility to infection.
Lift the mouse by its tail while it grips the cage with its forepaws to elevate the hind quarters and expose the vaginal opening.
Rinse the vaginal lumen with an anaerobic buffer to flush out mucus, a protective barrier against pathogens, along with dead epithelial cells, and endogenous vaginal microbes.
Immediately inoculate the vagina with a suspension of an anaerobic bacterial pathogen, while keeping the mouse restrained to minimize fluid loss.
For co-inoculation, administer a suspension of another anaerobic bacterial pathogen using the same volume as before.
House the inoculated mouse separately from uninoculated mice to prevent cross-contamination.
Following inoculation, the bacteria adhere to the vaginal epithelium, grow into colonies, and develop a protective biofilm layer that acts as a barrier against host immune responses.
Prepare the vaginal lavage tubes in the anaerobic chamber by pipetting 70 microliters of sterile anaerobic PBS into 1.5-milliliter microcentrifuge tubes labeled with mouse numbers.
Close the tubes tightly and take them out of the chamber. For performing vaginal lavage, first, place a mouse on top of a cage that it can grab with its forepaws. Then, gently grip the middle part of the tail and lift it so that the hind quarters are slightly elevated and the vaginal opening is exposed.
After drawing up 50 microliters of PBS from the labeled wash tube, gently insert the pipette tip two to three millimeters into the vaginal lumen to wash it with PBS by pipetting in and out three to four times. Then, transfer the wash material back to the original wash tube with the remaining 20 microliters of unused PBS. Immediately after this vaginal lavage, inoculate each mouse by vaginally transferring 20 microliters of the bacterial suspension or vehicle control.
For co-inoculation experiments with two different bacterial strains, separately inoculate with 10 microliters of each bacterial suspension, rather than mixing the strains beforehand. To prevent the administered volume from immediately accumulating at the vaginal introitus, continue holding up the mouse's hind part for 10 to 20 seconds. Transfer the inoculated mouse to a new cage or with already inoculated cage mates, and repeat the procedure with the other mice.
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