Biology Reference
In-Depth Information
4. To process throat swabs for GAS CFU enumeration, add DTT to 0.7 m
M
(7 μL of
a 0.1
M
stock DTT solution), vortex, and incubate for 15 min at room temperature.
DTT facilitates the release of GAS from the swab into the surrounding PBS.
5. Vortex samples a second time following the 15-min DTT incubation.
6. Make serial dilutions in PBS and plate 100 μL of neat, 10
−
1
,10
−
2
, and 10
−
3
dilutions onto select agar plates.
7. A single plate is used per throat swab per dilution, resulting in 64 plates per
time-point (8 animals × 2 swabs × 4 dilutions).
8. Incubate plates at 37 °C with 5% CO
2
for 24 h.
9. Following incubation, enumerate GAS colonies and determine colonization
levels for each animal. Note that a single GAS strain may produce colonies with
several different morphologies. Coupled with the natural microbial flora of the
macaque posterior pharynx, this complicates the task of GAS CFU enumeration.
10. To confirm that scored bacterial colonies are indeed GAS, amplify the
emm
gene by colony PCR from several colonies per throat swab per time-point.
emm
encodes the M protein, a major GAS-specific virulence factor
(28)
.
3.4.4. Saliva Isolation
Saliva samples from infected macaques enable testing of IgA production
during the course of infection. Approximately 1 mL of saliva is collected by
aspiration from the cheek pouch while the animals are under anesthesia. Saliva
samples from each animal are placed into individual blue-capped 2-mL tubes
and put on ice until all animal manipulations are completed. No processing of
the saliva samples is required, with the tubes simply being stored at -80 °C
until needed.
3.4.5. Nasal Washes
Nasal washes also provide a means to test IgA production during infection.
Washes are collected by instilling 0.7 mL of sterile PBS into each nostril and
immediately aspirating wash fluid and secretions with a sterile syringe. Nasal
washes are placed into white-capped 2-mL tubes on ice until all animal manip-
ulations are completed. Nasal wash samples are processed by centrifugation in
a microfuge at 500 ×
g
for 10 min at 4 °C, removing the supernatant to new
white-capped tubes and storing at -80 °C.
3.5. Post-experiment Antibiotic Treatment of Animals
Following sample isolations on day 28 of the experiment, the macaques
are each given an intramuscular injection of penicillin G (20,000 U/kg). Two
additional doses of penicillin G are given during the following week. Following
antibiotic treatment, throat swabs are taken of the animals and used to confirm
that they are culture-negative for GAS and hence have cleared the infection.
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