- Fixate each mouse on its back. Use a rubber band fastened
between two needles to hold the head of the mouse fixated. We
use a tray off styrofoam (29 cm39 cm) as the operation area. - Sterilize the throat of the mouse with 70% (vol/vol) ethanol.
- Make an incision in the skin above the trachea with a pair of
scissors and expand the incision using the pair of scissors by
making it larger between the cutting area. - Move the muscles to the sides to locate the trachea using the
pair of scissors. Again do this by making the cutting area larger.
Do not cut the muscles. - Use the scissors to make an incision in the membrane sur-
rounding the trachea. - Make a way into the trachea using an 18-G needle.
- Use a bead tipped needle to install 0.04 ml of the bead suspen-
sion in the left lung, 11 mm from the tracheal penetration site. - Suture the incision with silk.
- Sterilize the surgical equipment between each mouse using
70% (vol/vol) ethanol. - Euthanize four mice right after challenge and isolate the lungs
(seeSubheading3.3). The lungs are used to estimate the infec-
tion inoculum. - Alternative: make two tenfold serial dilutions of the bead sus-
pension and plate the appropriate dilutions on blue plates. - Use an 18-G needle to apply one drop of bupivacaine on the
incision site for postoperative pain. - Inject the mice s.c. with 0.75 ml of 0.9% (wt/vol) NaCl in the
neck-skin area to avoid dehydration. - Place the mice on a row on top of bedding in the cage and
cover them with a paper napkin for recovering after surgery.
Place the part of the cage where the mice are placed on top of
the heating pad. There should also be an area of the cage,
which is free from heating. Heating should be no more than
28 C. - Check the mice after 4–6 h and thereafter minimum once a day
(seeNote 4).
3.3 Quantitative
Bacteriology
- Injecting pentobarbital (10.0 ml/kg body weight) intraperito-
neal (i.p.) to euthanize the mice. Ensure that local animal care
regulations are followed. - Fixate each dead mouse on its back and apply 70% (vol/vol)
ethanol on the chest and stomach to avoid contamination. - To isolate the lungs, first remove the skin with a pair of scissors.
Then open the thoracic cavity without damaging the lungs.
ImagingN-Acyl Homoserine Lactone Quorum Sensing In Vivo 209