The Cotton Rat as a Model for Staphylococcus aureus Nasal Colonization in Humans - Bacterial Pathogenesis: Methods and Protocols - page 230

9. Decant or remove the supernatant by pipette and discard as biohazardous waste.
10. Resuspend the bacterial pellet in 10 μL of PBS per animal to be instilled (e.g., if
original volume was 6 mL, use 60 μL of PBS to resuspend bacteria) and vortex
the resuspension on maximum speed for 3 s.
11. Anesthetize the cotton rats by intramuscular injection in either hind leg muscle
with a 27-gauge needle using volumes appropriate for various body weights as
indicated in Table 1B ( see Notes 1 and 6 ).
12. Briefly revortex the S. aureus suspension on maximum speed prior to instillation.
13. Hold the unconscious cotton rat on its back in the palm of your hand and restrain
the chin with your thumb.
14. Using a small (20-μL size) disposable pipette tip, evenly distribute a 10-μL
volume of S. aureus suspension between each nostril drop-wise (usually 2-3
drops per nostril) without touching the nose of the animal. Allow the rat to draw
the drops into its nose by natural respiration ( see Note 7 ).
15. Hold the rat on its back until it is breathing normally and has cleared its nose
( see Note 8 ).
16. Return the rat to the cage, laying the rat on its back ( see Note 9 ).
17. Repeat the instillation with each cotton rat in the group and each group of cotton
rats until all animals have been instilled.
18. Monitor the animals until they are demonstrating purposeful movements.
19. Return food and water ad libitum and cover the cage with a barrier cover ( see
Note 10 ).
20. Perform serial dilutions in PBS of the remaining S. aureus inoculum and plate
the serial dilutions on blood agar or TSA to determine the actual number of
S. aureus instilled per animal ( see Note 2 ).
3.2. Treatment
If desired, the nasally colonized cotton rats can be treated intranasally to
affect nasal colonization ( see Note 11 ).
3.2.1. Nasal Instillation of Liquid Formulations
1. Liquid formulations of various compounds can be instilled into the cotton rat
nares in the same manner as the initial bacterial instillation ( see Note 12 ).
3.2.2. Nasal Instillation of Semi-Solid Formulations
1. Remove the metal needle from a flexible catheter and affix the flexible catheter
to a 1-mL syringe.
2. Load the semi-solid formulation into a 1-mL syringe fitted with the 22-gauge
flexible catheter for nasal delivery ( see Note 13 ).
3. Anesthetize the cotton rats as described above ( see Note 14 ).
4. Once the cotton rats have succumbed to the anesthesia, gently insert the catheter
tip 2-3 mm into one nostril ( see Note 15 ) and dispense the desired volume