Examining the Vector–Host–Pathogen Interface With Quantitative Molecular Tools - Bacterial Pathogenesis: Methods and Protocols - page 121

3.1.3. Preparation of Standard Curve Samples to Quantitate Y. pestis
in Fleabite-Site Skin Biopsies
1. Collect skin biopsies from a euthanized, uninfected mouse of the same size as
the skin biopsies to be collected from flea-bitten mice. Several skin biopsies
can be collected from a single animal. Freeze individual skin biopsies in sterile
microfuge tubes for future use.
2. Thaw a stock bacterial standard aliquot, vortex, and make 1:10 serial dilutions in
sterile PBS. Six standards containing 1 × 10 2 to 1×10 7 bacteria in 100 μL PBS
in a sterile 1.5-mL microfuge tube are needed.
3. Add a thawed, uninfected skin biopsy to each of the six standard sample tubes
and to a tube containing 100 μL sterile PBS for use as a negative control. Proceed
to DNA extraction.

3.1.4. Preparation of Standard Curve Samples to Quantitate Y. pestis in
Rat Lymph Nodes
1. Aseptically dissect lymph nodes from euthanized, uninfected rats. Several lymph
nodes can be collected from an individual animal. For example, bilateral inguinal,
axillary, and maxillary lymph nodes are easy to locate and can be dissected
rapidly.
2. Immediately after dissection, disrupt each lymph node by pressing it through a
70-μm cell strainer into 10 mL of RNA protect. Transfer the suspension to a
15-mL conical tube, mix, and dispense 1-mL aliquots to 1.5-mL microfuge tubes.
3. Centrifuge tubes at 13.5 × g for 5 min. Remove the RNA protect supernatant and
store the pellets at -80 °C.
4. Thaw a stock bacterial standard aliquot ( see Subheading 3.1.1. ), vortex, and
make 1:10 serial dilutions in sterile PBS. Seven standards containing 1 × 10 3 to

1×10 9 bacteria in 500 μL PBS are needed.
5. Thaw seven uninfected lymph node pellets from step 3 . Add one of the 500
μL PBS bacterial suspensions from step 5 to the seven lymph node pellets and
resuspend. Proceed to DNA extraction.

3.2. Preparation of Unknown Samples
After they are prepared, samples can be stored at -80 °C prior to DNA
extraction.

3.2.1. Fleas
1. Surface-sterilize fleas as described in step 1, Subheading 3.1.2 .
2. Add individual fleas to 1.5-mL microfuge tubes containing 100 μL BHI and 20 μL
glass bead slurry. Triturate fleas and collect supernatant as described in step 4,
Subheading 3.1.2 .