minimum 1 week before challenge. All animal studies should
be performed in accordance with relevant guidelines and
regulations.2.4 Analgesia,
Anesthesia and
Euthanasia
- Anesthetic: mix one part Hypnorm®(0.315 mg/ml fentanyl
citrate and 10 mg/ml fluanisone) with two parts of
sterile water. Pour them together gently. Then add one part
5 mg/ml midazolam. Pour together gently! A fresh solution
should be made every time. Store at room temperature
until use. - Analgesia: 5 mg/ml bupivacaine. Store at room temperature.
- Euthanasia: 200 mg/ml pentobarbital with 20 mg/ml lidocain
hydrochloride. Store at room temperature.
2.5 Infection
Procedure
- A bead tipped needle is used when installing the alginate beads
in the mouse lung. - Sutur: ethicon Vicryl, 3-0, FS-2.
- Surgical equipment can be chosen as desired. We use Nasal
scissors, Durogrip needleholder, and tweezers. - Heating plates: LP reptiles vivarium heating mat
1200 280 mm.
2.6 Freeze
Microtomy
- Tissue-Tek Cryomold standard (25 mm20 mm5 mm).
Disposable vinyl specimen molds for placement of lungs. - Tissue-tek O.C.T.™ gel for embedment of lungs before
freezing. - Tissue-Tek Cryo 2000 for sectioning of the lungs.
- Hexane.
- Dry ice.
2.7 Epifluorescence
Microscopy
- Use an epifluorescence microscope with a camera to visualize
GFP of the AHL monitor strains. Images can be obtained using
objectives in the range 40–100.
2.8 Confocal
Scanning Laser
Microscopy (CSLM)
- Image acquisitions of bacteria in the lungs can be performed
with confocal scanning laser microscopy (CSLM) equipped
with a detector and a filter set for monitoring GFP. In addition,
a reflection detector for acquiring bright-field images is
installed. Images can be obtained using objectives in the
range 40–100, we prefer oil-based objectives. Image scan-
ning should be carried out with the 488 nm laser line from an
Ar/Kr laser. Imaris software package can be used to generate
images of the bacteria.
206 Louise Dahl Hultqvist et al.