Establishing a Mouse Model of Vaginal Group B Streptococcus Infection

Abstract

Source: Patras, K. A., Doran, K. S. A Murine Model of Group B Streptococcus Vaginal Colonization. J. Vis. Exp. (2016)

This video demonstrates the preparation and vaginal inoculation of a hormone-treated female mouse with Group B Streptococcus to model infection. It explains how to culture and prepare the bacteria, administer the inoculum into the vaginal lumen, and ensure successful infection.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Vaginal Inoculation with Group B Streptococcus (GBS)

1. One day prior to inoculation, grow a 5-ml overnight liquid culture of a GBS strain of interest, such as A909 (serotype Ia), in Todd Hewitt broth (THB) at 37 °C.
2. Subculture the GBS overnight culture at a 1:10 volume into fresh THB and incubate at 37 °C. Grow the bacteria to mid-log phase (OD₆₀₀ = 0.4-0.5).

NOTE: This will typically take 2 - 3 hr, depending on the strain of GBS.

3. Transfer the subculture to a sterile 15-ml conical tube and pellet bacteria at 3,000 × g for 5 min. Aspirate the supernatant. Resuspend the bacterial pellet in 200 µl of sterile phosphate-buffered saline (PBS).
4. Using the resuspended pellet, bring 3 - 5 ml of PBS (1 ml per 10 mice) to exactly OD₆₀₀ = 0.4 in a new 5-ml culture tube. This will be a concentration of ~ 1 × 10⁸ colony-forming units (CFU)/ml. Transfer to a new 15-ml conical tube and re-pellet the bacteria at 3,000 × g for 5 min. Aspirate the supernatant.
5. Resuspend the pellet in PBS at 1/10 the original volume. For example, if 3 ml of OD₆₀₀ = 0.4 was pelleted, then resuspend it in 300 µl of PBS.

NOTE: This is the final bacterial suspension (~ 1 × 10⁹ CFU/ml) used for animal inoculation.

6. Reserve 50 µl of this suspension for serial dilution and plating on THB agar to determine the exact inoculum.
7. Inoculate each mouse with 10 µl of the final bacterial suspension so that 1 × 10⁷ CFU is administered to each mouse.
   1. To inoculate, restrain the mouse manually by securing the loose skin at the scruff of the neck between the handler's thumb and index finger and then immobilizing the tail.
   2. Draw up 10 µl of the GBS into a 200-µl gel loading pipette tip. Insert the tip 5 to 10 mm into the vaginal lumen and dispense the 10 µl of inoculum.
      NOTE: Gel loading tips are preferred over standard 200-µl tips to minimize the risk of organ trauma or injury, particularly in younger or smaller mice.
   3. Immediately following inoculation, release the scruff of the neck and elevate the hind end of the mouse, lifting the mouse by the tail and walking the front paws on a hard surface for ~ 1 min.
   4. Visually inspect the vaginal opening for any backflow of inoculum. If backflow is observed, a fresh pipette tip may be used to manipulate or enlarge the vaginal opening, facilitating uptake of the backflow into the lumen. Additionally, backflow may be aspirated via a pipette and re-inoculated.
      NOTE: If administering topical agents, probiotic organisms, or proteins of interest, a volume up to 20 µl in a physiologic buffer may be given in the vaginal tract.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
200 μl gel loading pipette tips	USA Scientific	1252-0610
Todd Hewitt Broth	Hardy Diagnostics	7161C
Dulbecco's Phosphate-Buffered Salt Solution 1x	Corning	21-031-CV