Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

This method will help investigators study the biology and pathophysiology of anaerobic bacteria that live in and cause infections in the female reproductive tract. Previous models of murine vaginal inoculation have focused on bacteria grown and recovered under aerobic conditions. Our method instructs on the inoculation and recovery of obligately anaerobic bacteria, which require additional strategies to minimize oxygen exposure.

Depending on how fastidious they are, new bacterial strains used in this model may require changes to certain steps. We have provided suggestions for potential modifications in table one of the manuscript. To begin with, add 200 microliters of a 16-hour-old bacterial culture to a 1.5-milliliter cuvette with 800 microliters of fresh media in an anaerobic chamber.

Then measured the optical density, or OD, of the diluted culture at 600 nanometers in a spectrophotometer, using a separate cuvette with only media as a blank. To get the actual OD value of the culture, multiply the spectrophotometer reading by five, then determine the volume of inoculum required for the experiment and calculate the volume of the bacterial culture needed to achieve the required inoculum density. Next, pipette the required volume of bacterial culture to a sterile microcentrifuge tube and centrifuge at 16, 000 times G for one to three minutes.

After removing the supernatant, resuspend the pellet in anaerobic PBS to achieve the required volume of inoculum calculated before. To determine the number of bacterial colony-forming units in the prepared inoculum, first, use 10 microliters of the bacterial suspension to make several serial dilutions in PBS and use a multi-channel pipette to spot plate inoculum dilutions on agar plates. At the same time, 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.

After inoculating all the mice, get the post-inoculum colony-forming units by plating serial dilutions of the inoculum in the anaerobic chamber. To collect mouse organs, prepare five-milliliter organ collection tubes in the anaerobic chamber by adding one milliliter of sterile anaerobic PBS to each tube for vaginas and cervices. Add 750 milliliters of PBS to the tubes marked for uterine horns.

For collecting the final lavage at sacrifice, prepare another set of vaginal lavage tubes as demonstrated previously. Also, for rinsing and sterilizing dissection instruments, prepare 50-milliliter beaker of deionized water and a 50-milliliter beaker of 70 to 90%ethanol. Immediately before or after sacrificing the mice, perform the final lavage as demonstrated earlier.

After sacrifice, spray the abdomen and back of a mouse with 70%ethanol. Then, use ethanol-sterilized and dried scissors to open the abdominal pelvic cavity of the mouse with vertical cuts. Next, use the forceps to pull back the skin.

To expose the reproductive tract, gently push the intestines out of the body cavity to the side of the open field and remove the bladder from the mouse to access the lower portion of the reproductive tract. After that, clip the center of the pubic bone with scissors without cutting the vagina. Then, use two sets of forceps to grab each side of the pelvis and pull the pelvis open laterally to expose the lower portion of the vagina underneath.

Gently grab the cervix with forceps and pull it up to expose the colon. After that, use small scissors to release the vagina from the external connective tissues. Carefully release the vagina from the colon without puncturing the intestinal tract.

When the vagina is fully released from the colon, use scissors to flank the vagina at the introitus and then snip to remove it from the mouse body. Maintaining a gentle grip on the cervix, pull it up to make the uterine horns more visible and cut directly below the ovaries on the right and left sides to release the uterine horns. Next, remove the reproductive tract from the mouse body and place it in a sterile Petri dish.

Then, using a razor blade, separate the uterine horns, cervix, and vagina. Place each tissue in its respective labeled collection tube. For homogenizing the organs, make a handheld homogenizer ready in a biosafety cabinet.

First, prepare a set of three 50-milliliter tubes with deionized water, 70%ethanol, and PBS, respectively, for washing the homogenizer blades after homogenizing each organ. To clean the homogenizer, dip the blade in the water tube and turn on the machine at full speed for about three seconds. Then, shake the blade on a paper towel to remove excess water.

Rinse the blade successively in 70%ethanol and PBS. After the initial rinsing, lower the homogenizer probe to the bottom of a tube with organs to trap the tissue underneath. Then, turn on the homogenizer to grind the tissue and gently move the probe up and down about five times, keeping it submerged.

Turn off and remove the homogenizer and visually ensure complete tissue disruption. After homogenization, cycle the sample back into the anaerobic chamber and perform serial dilution for CFU recovery. Following this protocol, recovery of viable bacteria and enumeration of colony-forming units from the vaginal washes could be successfully done for the bacterial strains, Gardnerella vaginalis, Prevotella bivia, and Fusobacterium nucleatum.

Moreover, the number of Gardnerella vaginalis colony-forming units obtained from vaginal washes correlated with those recovered from the vaginal tissue homogenates. This method also revealed that the Prevotella bivia titers and mouse uterine tissues were significantly higher during co-infection with Gardnerella vaginalis versus Prevotella mono-infection. Furthermore, this method could be successfully used to compare the vaginal persistence of a mutant Fusobacterium nucleatum strain with that of the wild type at 24 and 72 hours post-inoculation.

To investigate the immune response to vaginal bacterial inoculation, cytokine assays can be run on the tissue homogenates. Alternatively, tissues could be saved for histological examination to assess for signs of inflammation.