6. WBC and hematocrit are run in an Element HT5 veterinary
hematology analyzer (HESKA).
7. Liver enzymes (LDH, ALT, and AST) are run in an Element
DC veterinary chemistry analyzer (HESKA) using the manufacturer’s recommended reagents.
2.13 Harvesting Liver
and Spleen from
M. leprae-Infected
Armadillos,
and Purification
of M. leprae from
Harvested Tissues
1. Gentamicin: Vetone gentamicin (100 mg/mL), 1 mL.
2. Penicillin antibiotic solution: Aspen Bactracillin G benzathine
(Penicillin G Benzathine and Penicillin G Procain, 300,000 U/
mL), 2 mL (see Subheading 2.12, items 2 and 3).
3. Infected liver or spleen tissue (see Note 13).
4. L-J agar slants (see Subheading 2.10, item 10).
5. 7H11 agar slants: Prepare Middlebrook 7H11 agar as in Subheading 2.8, item 2, under and prepare in slant pour into screw
top slants. Place in slant position.
6. 1 L sterile beaker (see Note 14).
7. 400 mL sterile beaker.
8. 250 mL sterile beakers (see Note 14).
9. 14 50 mL Teflon Oakridge (Nalgene) tubes (see Note 14).
10. Homogenization chamber (see Note 14).
11. Homogenizer (see Note 14).
12. Sterile stir bars (see Note 14).
13. Sterile scissors (see Note 14).
14. Brain Heart Infusion (BHI) broth.
15. Milli-Q water.
16. Tryptic Soy Agar (TSA).
17. 100 mm Petri plates.
18. Inoculation loop.
19. 10 mM Na 2 EDTA: Dissolve 3.72 g of Na 2 EDTA in 900 mL of
DI water. Adjust pH to 7.1–7.2 and then complete to 1 L with
DI water. Autoclave, using a liquid cycle for 20 min. Store at
4
C, use cold.
20. 10 mM Na 2 EDTA/0.1 M NaOH buffer: Dissolve 3.72 g of
Na 2 EDTA in 900 mL of DI water. Adjust pH to 7.1–7.2.
Complete to 1 L with DI water. Add 4.0 g NaOH. Autoclave
using a liquid cycle for 20 min. Store at 4
C, use cold.
21. 0.1 M Tris–HCl (ph 7.2). Dissolve 15.76 g Tris–HCl in
800 mL DI water. Adjust pH to 7.2 and complete volume to
1 L. Autoclave using a liquid cycle for 20 min. Store at room
temperature.
Culturing Mycobacteria
29
hematology analyzer (HESKA).
7. Liver enzymes (LDH, ALT, and AST) are run in an Element
DC veterinary chemistry analyzer (HESKA) using the manufacturer’s recommended reagents.
2.13 Harvesting Liver
and Spleen from
M. leprae-Infected
Armadillos,
and Purification
of M. leprae from
Harvested Tissues
1. Gentamicin: Vetone gentamicin (100 mg/mL), 1 mL.
2. Penicillin antibiotic solution: Aspen Bactracillin G benzathine
(Penicillin G Benzathine and Penicillin G Procain, 300,000 U/
mL), 2 mL (see Subheading 2.12, items 2 and 3).
3. Infected liver or spleen tissue (see Note 13).
4. L-J agar slants (see Subheading 2.10, item 10).
5. 7H11 agar slants: Prepare Middlebrook 7H11 agar as in Subheading 2.8, item 2, under and prepare in slant pour into screw
top slants. Place in slant position.
6. 1 L sterile beaker (see Note 14).
7. 400 mL sterile beaker.
8. 250 mL sterile beakers (see Note 14).
9. 14 50 mL Teflon Oakridge (Nalgene) tubes (see Note 14).
10. Homogenization chamber (see Note 14).
11. Homogenizer (see Note 14).
12. Sterile stir bars (see Note 14).
13. Sterile scissors (see Note 14).
14. Brain Heart Infusion (BHI) broth.
15. Milli-Q water.
16. Tryptic Soy Agar (TSA).
17. 100 mm Petri plates.
18. Inoculation loop.
19. 10 mM Na 2 EDTA: Dissolve 3.72 g of Na 2 EDTA in 900 mL of
DI water. Adjust pH to 7.1–7.2 and then complete to 1 L with
DI water. Autoclave, using a liquid cycle for 20 min. Store at
4
C, use cold.
20. 10 mM Na 2 EDTA/0.1 M NaOH buffer: Dissolve 3.72 g of
Na 2 EDTA in 900 mL of DI water. Adjust pH to 7.1–7.2.
Complete to 1 L with DI water. Add 4.0 g NaOH. Autoclave
using a liquid cycle for 20 min. Store at 4
C, use cold.
21. 0.1 M Tris–HCl (ph 7.2). Dissolve 15.76 g Tris–HCl in
800 mL DI water. Adjust pH to 7.2 and complete volume to
1 L. Autoclave using a liquid cycle for 20 min. Store at room
temperature.
Culturing Mycobacteria
29