The Cotton Rat as a Model for Staphylococcus aureus Nasal Colonization in Humans - Bacterial Pathogenesis: Methods and Protocols - page 236

19. A recovery buffer (PBS + Tween 20) volume of 500 μL will allow sufficient
buffer to plate 100 μL on one recovery media or 2 × 75 μL on two recovery
media. Greater volumes of buffer can be used if further recovery media are
required, but these reduce the sensitivity of the recovery correspondingly.
20. Topical applications of compounds like antibiotics can be retained in the nose
above MIC/MBC levels for >24 h in certain formulations and can lead to ex vivo
killing of the bacteria and false positive results, thus an appropriate neutralizer
should be used to ensure that any apparent antibacterial activity by the administered
compound actually occurs in the nares and not in the recovery tube (18) .
21. Hold the head of the animal down so that any excess alcohol does drip into the
nostrils.
22. We use an IBS Integra Biosciences Fireboy Plus with an ignition sensor instead
of a constant open flame like a bunsen burner for safety around open containers
of ethanol.
23. Vigorous vortexing of the nose recovers the vast majority of the colonizing
S. aureus . We have followed the vigorous vortexing of samples with sonication
to try to release more S. aureus and found no greater recovery of S. aureus
following sonication.
24. The food and bedding from the instilled cotton rat cages should be considered
contaminated and disinfected by autoclaving or other appropriate methods.
25. Unlike the mouse (16) , the cotton rat has a variety of bacteria as normal nasal
flora that are recovered by this procedure ( Table 3 ), so the nasal supernatant must
be plated on a selective media to ensure accurate enumeration of the instilled
colonizing S. aureus . Depending on the characteristics of the S. aureus strain
instilled, a variety of bacterial media can be used for recovery from the cotton
rat nares. Antibiotic-resistant strains, for example, streptomycin-resistant strains,
MRSA, or gene knock-outs with antibiotic resistance markers, are the easiest to
recover because the appropriate antibiotic can be added to the TSA + 7.5% NaCl
agar to inhibit the growth of most other native flora. The cotton rat native nasal
flora includes a prevalent Gram-negative organism that is naturally erythromycin
resistant but which will not grow in the presence of 7.5% NaCl. The cotton
rat nose also has a variety of coagulase negative staphylococci that will also
grow on 7.5% NaCl. If the instilled strain of S. aureus is not antibiotic resistant,
Chromagar Staph aureus works well for recovery of S. aureus with the instilled
S. aureus growing as large purple colonies. It should be noted that a naturally
occurring Staphylococcus muscae found in the cotton rat nose will grow as a
small purple colony on Chromagar Staph aureus, so care should be taken to
differentiate these bacteria from the instilled S. aureus that will grow as a larger
purple colony.
26. The S. aureus colonies grow more slowly on the high salt agar than on other
agars thus 48 h incubation at 37 °C is required for accurate enumeration.
27. S. aureus colonization varies by strain ( see Table 2 ), but generally, the number
of colonies recovered per plate from the primary sample tube are within the
countable range (<2000 CFUs) for a colony counter that can count up to 2000
colonies per plate. If hand counting is done, then it may be beneficial to dilute