32. Observe L-J slant at 6–8 weeks. Report any growth (see Note
48).
33. Read radiorespirometry (
14
C CPM) at 24 h and day 7 (see
Note 49).
3.11 Radiorespirometry
of M. leprae
1. To prepare 70–80 strips for radiorespirometry, add 15 g PPO
to 20 mL liquid scintillation and concentrate under a
fume hood.
2. Mix in a beaker (wrapped in aluminum foil) for at least 10 min
using a magnetic stirrer (see Note 50).
3. Once the PPO is completely dissolved, add 5.0 mL of Triton-X
100 and mix.
4. Add 5.0 mL NaOH–MeOH. Mix thoroughly (see Note 51).
5. Dissolve completely with vigorous stirring (approx.
20–30 min).
6. Dip the filter paper strips in the solution and remove immediately making sure that each strip is covered (see Note 52).
7. Spread out the strips individually over aluminum foil and cover
loosely with another sheet of aluminum foil to protect from
light and allow to dry overnight.
8. Store in a tightly covered container at room temperature in
dark (see Note 53).
9. Set up radiorespirometry of M. leprae.
(a) Add 1.0 mL of radiorespirometry media to a sterile
6.0 mL glass vial.
(b) Add 1 Â 10
7 M. leprae (10 μL of 1 Â 10
9 /mL) to the vial
(see Note 54).
(c) Place screwcap on glass vial loosely (see Note 55).
(d) Place the 6.0 mL glass vial inside a plastic 20 mL liquid
scintillation vial along with a radiorespirometry strip.
(e) Place screwcap on plastic vial tightly (see Note 56).
(f) Record
14
C-CPM daily for 7 days.
3.12 Infecting
Armadillos
with M. leprae
1. The armadillos are anesthetized using a combination of Ketamine (0.6 mL) and Dexdomitor (0.4 mL) given IM.
2. Clean the skin at the injection site with 70% ethanol.
3. The inoculum of 1 Â 10
9 M. leprae in a maximum of 1 mL is
injected slowly in the saphenous vein using a tuberculin
syringe.
4. Disease progression is to be monitored during the following
18–24 months. M. leprae is expected to localize in the nerves
and other tissues.
Culturing Mycobacteria
41
48).
33. Read radiorespirometry (
14
C CPM) at 24 h and day 7 (see
Note 49).
3.11 Radiorespirometry
of M. leprae
1. To prepare 70–80 strips for radiorespirometry, add 15 g PPO
to 20 mL liquid scintillation and concentrate under a
fume hood.
2. Mix in a beaker (wrapped in aluminum foil) for at least 10 min
using a magnetic stirrer (see Note 50).
3. Once the PPO is completely dissolved, add 5.0 mL of Triton-X
100 and mix.
4. Add 5.0 mL NaOH–MeOH. Mix thoroughly (see Note 51).
5. Dissolve completely with vigorous stirring (approx.
20–30 min).
6. Dip the filter paper strips in the solution and remove immediately making sure that each strip is covered (see Note 52).
7. Spread out the strips individually over aluminum foil and cover
loosely with another sheet of aluminum foil to protect from
light and allow to dry overnight.
8. Store in a tightly covered container at room temperature in
dark (see Note 53).
9. Set up radiorespirometry of M. leprae.
(a) Add 1.0 mL of radiorespirometry media to a sterile
6.0 mL glass vial.
(b) Add 1 Â 10
7 M. leprae (10 μL of 1 Â 10
9 /mL) to the vial
(see Note 54).
(c) Place screwcap on glass vial loosely (see Note 55).
(d) Place the 6.0 mL glass vial inside a plastic 20 mL liquid
scintillation vial along with a radiorespirometry strip.
(e) Place screwcap on plastic vial tightly (see Note 56).
(f) Record
14
C-CPM daily for 7 days.
3.12 Infecting
Armadillos
with M. leprae
1. The armadillos are anesthetized using a combination of Ketamine (0.6 mL) and Dexdomitor (0.4 mL) given IM.
2. Clean the skin at the injection site with 70% ethanol.
3. The inoculum of 1 Â 10
9 M. leprae in a maximum of 1 mL is
injected slowly in the saphenous vein using a tuberculin
syringe.
4. Disease progression is to be monitored during the following
18–24 months. M. leprae is expected to localize in the nerves
and other tissues.
Culturing Mycobacteria
41