The Cotton Rat as a Model for Staphylococcus aureus Nasal Colonization in Humans - Bacterial Pathogenesis: Methods and Protocols - page 234

Nephelo culture flasks (125 mL) work well for larger groups of animals (10 or
greater).
4. A Spectronic 20 (Theromo Scientific, Waltham, MA) with 1-cm diameter sterile
glass test tubes works well for this, allowing the %T for the whole suspension
to be determined. Alternatively, the %T of an aliquot of suspended bacteria can
be determined using a cuvette and a conventional visible light spectrometer set
to 650 nm.
5. A T (transmittance) of 5×10 8 S. aureus per milliliter

of buffer at a 1-cm path length, but it is best to determine this empirically for
each strain of S. aureus . A dose >10 8 S. aureus instilled in the nares will ensure
consistent nasal colonization even if much of the initial dose is expelled during
instillation ( see Note 7 ). Consistent colonization can be achieved with initial
inoculums of as low

10% is equivalent to

∼ ∼

10 5 per animal, but care must be taken that the animal
does not expel any of the lower inoculum volume. Instillation of <10 5 S. aureus
have not resulted in consistent nasal colonization in our hands.
6. The injection volumes given in Table 1 will usually render a cotton rat
unconscious in

∼

5 min, and they will remain that way for about 20-30 min.
Occasionally, an individual animal will not be rendered unconscious by the
initial dose of anesthesia; in this case, additional doses of 50-100 μL depending
on the weight of the animal can be administered until the animal in unconscious.
It is important that the cotton rats be fully unconscious prior to instillation of
the bacteria ( see Note 7 ).
7. Even when unconscious, cotton rats retain a strong expiratory response, that is,
they will often “snort-out” much of the S. aureus inoculum. For safety reasons,
a full face mask and hair cover should be worn when instilling S. aureus in
cotton rat nares. Expiration can be controlled to some extent by gently rolling
the loose skin on the bridge of the nose over the nostrils with your forefinger
after instillation. This is particularly necessary when instilling a lower inoculum
of bacteria.
8. Occasionally, instilled cotton rats will appear to have difficulty in breathing
demonstrating a gasping appearance because of nasal blockage by the bacterial
bolus. The animal will usually clear its own airway within about a minute, but
occasionally artificial respiration in the form of gentle compressions on the rib
cage are needed to aid the animal's recovery from the instillation.
9. Female rats can be housed five per 18.5” × 10” cage, but after manipulation of
any kind, male rats must be housed individually or they will fight. This fighting
often involves biting on the nose, which will interfere with this model and can
lead to active infection. The cotton rats should begin to demonstrate purposeful
movement about 20-30 min after bacterial instillation.
10. All cage bedding should be considered as biohazardous and treated appropriately
as S. aureus continues to be shed by the animals for a couple of days after
instillation.

∼