The Cotton Rat as a Model for Staphylococcus aureus Nasal Colonization in Humans - Bacterial Pathogenesis: Methods and Protocols - page 231

20 μL) by applying continuous gentle pressure on the syringe plunger
and slowly withdrawing the catheter tip from the nostril ( see Note 16 ).
5. Gently message the nose of the cotton rat to disperse the semi-solid and open
the nares to allow normal breathing.
6. Lay the cotton rat down on its back and do not proceed to the second nostril
until normal breathing is resumed.
7. Once the animal is breathing normally, repeat steps 3 and 4 to instill the semi-
solid in the second nostril ( see Note 17 ).
8. Return animals breathing normally to their cages laying them on their backs.
9. Repeat the treatment with all animals and groups to be treated.
10. Monitor the animals until they are demonstrating purposeful movements.
11. Return food and water ad libitum and cover the cage with a barrier cover.

(usually

∼

3.2.3. Recovery
1. The noses of colonized cotton rats can be harvested 24 h after S. aureus instil-
lation ( see Note 18 ).
2. Prepare one 15-mL snap cap test tube per animal containing 500 μL ( see Note
19 ) PBS + Tween 20 + any appropriate neutralizer needed ( see Note 20 ).
3. Sacrifice the animals as appropriate to the guidelines of the presiding IACUC.
We use 100% CO 2 delivered at 10 psi.
4. Cleanse the outside of the nose and the area around the nose well ensuring that
the bridge of the nose is also cleansed with a sterile 70% alcohol prep pad ( see
Note 21 ). This removes any S. aureus colonizing the skin around the nose.
5. Sanitize fine-tipped forceps and fine-bladed dissecting scissors by flaming with
100% ethanol ( see Note 22 ) and then let them cool prior to reuse.
6. Lay the cotton rat on its back on a sanitized dissection board.
7. Hold the chin of the cotton rat between the thumb and forefinger of one hand
and place the blade of a scalpel with a #21 blade just under the upper lip and
against the teeth of the cotton rat ( see Fig. 1A ). Slide the blade down the teeth
and back along the bridge of the nose removing the anterior nares and soft tissue
and bony cartilage along the bridge of the nose ( see Fig. 1B ).
8. Grasp the cartilage with the flamed forceps, and using the scissors, bisect the
nostrils laterally ( see Figs. 1C and 1D ) exposing the nostril lumen.
9. Place the bisected nose into an appropriately labeled recovery tube and cap the
tube.
10. Vortex the tube on maximum for 10 s to release adherent bacteria ( see Note
23 ).
11. To prepare for the processing of the next animal, rinse the forceps, scissors, and
dissecting board with 70% ethanol and wipe with a clean paper towel. Dispose
of scalpel blade and flame forceps and scissors as above.