The Cotton Rat as a Model for Staphylococcus aureus Nasal Colonization in Humans - Bacterial Pathogenesis: Methods and Protocols - page 229

Table 1B
Administration Volume of Standard
Anesthesia Preparation by Animal Weight
(i.m. with 1-mL syringe, 27-guage needle)

Cotton rat weight (g) Dose (μL)

50 150

52 160

54 170

56 180

58 190

60 200

62 210

64 220

66 230

68 240

70 250

4. Chromagar Staph aureus (Chromoagar Microbiology, Paris, France): As per the
manufacturer's instruction, 82.5 g/L (agar is light sensitive and can be stored for
1 month at 4 °C after preparation).
5. Sterile alcohol prep pads: 70% isopropyl alcohol.
6. Sterile scalpel blades, #21.
7. Sterile disposable snap cap test tubes, 15 mL.

3. Methods
3.1. Instillation
1. Inoculate a CSA plate from a thawed TSB stock of S. aureus using a sterile
swab. Cover the agar surface completely and allow any liquid to absorb into the
agar.
2. Incubate the inoculated CSA plate at 35-37 °C for 16-24 h.
3. Harvest the S. aureus from the inoculated CSA plate into PBS.
4. Aliquot 1 mL of PBS per animal to be instilled into a sterile glass test tube,
using a separate test tube for each different S. aureus strain ( see Note 3 ).
5. Determine the percent transmittance (% T ) of the blank PBS at 650 nm on a
spectrometer and set to T = 100% ( see Note 4 ).
6. Harvest the S. aureus from the CSA plate using a sterile swab resuspending the
bacteria in the PBS and vortex briefly to disperse the bacteria. Check the %T at
650 nm.
7. Add bacteria to the PBS until the %T is 10% for a 1-cm path length ( see Note 5 ).

8. Pellet the S. aureus by centrifugation at 7750 × g for 7.5 min.

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