3. Place Beakers 1 and 2 on each of two hot plates and bring to a
rolling boil with constant stirring.
4. Wearing sterile gloves, cut strips of dialysis membrane tubing,
7–9 in. (~18–23 cm) in length, using sterile scissors. Gently tie
off one end of the tubing using a simple overhand knot by
forming a loop and passing the free end of the tubing through
the loop.
5. Place the tied tubing into Beaker 1 and replace the aluminum
foil cover.
6. Repeat steps 4 and 5 for each animal/DMC, plus 1–2 extra.
7. Boil tubing for 20 min with constant stirring. Be sure to keep
the foil cover loose enough to allow steam to escape. As
needed, use a sterile 10-ml serological pipette or extra-long
forceps to push the tubing back into the boiling liquid.
8. After 20-min boiling, transfer tubing to Beaker 2 using sterile
10 ml pipette or extra-long forceps. Replace aluminum foil
cover and boil tubing for 20 min. With constant stirring.
9. While strips are boiling in Beaker 2, place Beaker 3 on the hot
plate and bring to a boil.
10. After 20 min boiling, transfer tubing to Beaker 3 as described
in step 8.
11. After 20 min boiling, turn off heating/stirring for Beaker
3. Once cool to the touch, transfer Beaker 3 to a biological
safety cabinet (BSC). To ensure sterility, the beaker containing
the tubing should remain in the BSC until the tubing is transferred to a sterile container for storage.
12. Transfer dialysis membrane tubing to the bottom portion of a
0.22-μm filter unit using sterile blunt tip forceps. Replace the
filter unit top and filter in ~200 ml of Ultrapure-Q water from
Beaker 3. Seal the bottom portion of the filter unit using the
sterile cap provided by the manufacturer (see Note 7). Going
forward, the container should be opened only within the BSC.
3.2 In Vitro
Cultivation
of Leptospira
and Culture Dilution
1. Transfer 10 ml of EMJH to a sterile 15-ml conical tube.
2. Inoculate with leptospires to give a final density of 10
3 to
10
5 leptospires/ml.
3. Incubate at 30
C until the culture reaches mid- to latelogarithmic phase (usually 5–10 days).
4. On the day of DMC implantation, enumerate leptospires using
Petroff-Hausser chamber or other suitable method.
5. Dilute the culture in 3–4 ml EMJH to 10
6 leptospires/ml.
6. Distribute 10 ml of high-BSA EMJH into sterile 50-ml conical
tubes, one per DMC. Prewarm media to 37
C in a bacteriological incubator.
234
Andre Alex Grassmann and Melissa J. Caimano
rolling boil with constant stirring.
4. Wearing sterile gloves, cut strips of dialysis membrane tubing,
7–9 in. (~18–23 cm) in length, using sterile scissors. Gently tie
off one end of the tubing using a simple overhand knot by
forming a loop and passing the free end of the tubing through
the loop.
5. Place the tied tubing into Beaker 1 and replace the aluminum
foil cover.
6. Repeat steps 4 and 5 for each animal/DMC, plus 1–2 extra.
7. Boil tubing for 20 min with constant stirring. Be sure to keep
the foil cover loose enough to allow steam to escape. As
needed, use a sterile 10-ml serological pipette or extra-long
forceps to push the tubing back into the boiling liquid.
8. After 20-min boiling, transfer tubing to Beaker 2 using sterile
10 ml pipette or extra-long forceps. Replace aluminum foil
cover and boil tubing for 20 min. With constant stirring.
9. While strips are boiling in Beaker 2, place Beaker 3 on the hot
plate and bring to a boil.
10. After 20 min boiling, transfer tubing to Beaker 3 as described
in step 8.
11. After 20 min boiling, turn off heating/stirring for Beaker
3. Once cool to the touch, transfer Beaker 3 to a biological
safety cabinet (BSC). To ensure sterility, the beaker containing
the tubing should remain in the BSC until the tubing is transferred to a sterile container for storage.
12. Transfer dialysis membrane tubing to the bottom portion of a
0.22-μm filter unit using sterile blunt tip forceps. Replace the
filter unit top and filter in ~200 ml of Ultrapure-Q water from
Beaker 3. Seal the bottom portion of the filter unit using the
sterile cap provided by the manufacturer (see Note 7). Going
forward, the container should be opened only within the BSC.
3.2 In Vitro
Cultivation
of Leptospira
and Culture Dilution
1. Transfer 10 ml of EMJH to a sterile 15-ml conical tube.
2. Inoculate with leptospires to give a final density of 10
3 to
10
5 leptospires/ml.
3. Incubate at 30
C until the culture reaches mid- to latelogarithmic phase (usually 5–10 days).
4. On the day of DMC implantation, enumerate leptospires using
Petroff-Hausser chamber or other suitable method.
5. Dilute the culture in 3–4 ml EMJH to 10
6 leptospires/ml.
6. Distribute 10 ml of high-BSA EMJH into sterile 50-ml conical
tubes, one per DMC. Prewarm media to 37
C in a bacteriological incubator.
234
Andre Alex Grassmann and Melissa J. Caimano