12. 4 L bottles (see Note 5) sterile.
13. 0.2 μm filtration units for bottles (VacuCap with tubing).
14. 230 mL polycarbonate centrifuge conical that matches centrifuge rotor/insert.
15. Balances (equal arm/trip beam and top loading).
16. Sterile pyrogen-free, 0.2 μm filtered water.
17. 37
C warm room (see Note 7).
18. Sodium azide (NaN 3 ).
19. Roller bottle apparatus.
2.9 Inoculation
of Athymic Nude
Mouse Footpad
with M. leprae
1. Athymic nude mice 4–6 weeks old (see Note 8).
2. PPE: laboratory coats or surgical gowns, shoe covers, hair
bonnets, gloves, mask, and safety glasses.
3. Freshly harvested viable M. leprae suspension adjusted to
1 Â 10
9
/mL (see Note 9).
4. 0.5 mL graduated sterile syringes.
5. 27 or 28 G needles.
6. Alcohol swabs.
7. Absorbent bench liner.
2.10 Harvesting Live
M. leprae from
Athymic Nude Mouse
Footpads
1. RPMI-10: To sterile RPMI 1640 (Thermo Fisher Scientific,
USA), add FBS aseptically to a final concentration of 10% v/v.
2. Heat-inactivated FBS.
3. Ampicillin.
4. Gentamicin.
5. 70% ethanol.
6. Betadine (povidone-iodine 10%) solution.
7. Blood agar plates.
8. Chocolate agar plates.
9. Thioglycolate broth.
10. L-J agar slants (see Note 1): To 367.5 mL of DI water, add
30.0 g potato flour, 3.6 g asparagine, 2.4 g monopotassium
phosphate, 0.6 g magnesium citrate, 0.4 g malachite green,
0.24 g magnesium sulfate, and 7.5 mL glycerol. Heat and stir
to dissolve. Autoclave at 121
C for 15 min. Cool to 50–60
C
before aseptically adding 625 mL of egg base. The egg base is a
uniform suspension of fresh eggs prepared under aseptic conditions. Avoid whipping air into suspension during the collection and mixing. Once the media and the egg base are
combined, pour into screw top slants. Place into slant position.
Heat to 85
C for 45 min (see Note 4).
Culturing Mycobacteria
27
13. 0.2 μm filtration units for bottles (VacuCap with tubing).
14. 230 mL polycarbonate centrifuge conical that matches centrifuge rotor/insert.
15. Balances (equal arm/trip beam and top loading).
16. Sterile pyrogen-free, 0.2 μm filtered water.
17. 37
C warm room (see Note 7).
18. Sodium azide (NaN 3 ).
19. Roller bottle apparatus.
2.9 Inoculation
of Athymic Nude
Mouse Footpad
with M. leprae
1. Athymic nude mice 4–6 weeks old (see Note 8).
2. PPE: laboratory coats or surgical gowns, shoe covers, hair
bonnets, gloves, mask, and safety glasses.
3. Freshly harvested viable M. leprae suspension adjusted to
1 Â 10
9
/mL (see Note 9).
4. 0.5 mL graduated sterile syringes.
5. 27 or 28 G needles.
6. Alcohol swabs.
7. Absorbent bench liner.
2.10 Harvesting Live
M. leprae from
Athymic Nude Mouse
Footpads
1. RPMI-10: To sterile RPMI 1640 (Thermo Fisher Scientific,
USA), add FBS aseptically to a final concentration of 10% v/v.
2. Heat-inactivated FBS.
3. Ampicillin.
4. Gentamicin.
5. 70% ethanol.
6. Betadine (povidone-iodine 10%) solution.
7. Blood agar plates.
8. Chocolate agar plates.
9. Thioglycolate broth.
10. L-J agar slants (see Note 1): To 367.5 mL of DI water, add
30.0 g potato flour, 3.6 g asparagine, 2.4 g monopotassium
phosphate, 0.6 g magnesium citrate, 0.4 g malachite green,
0.24 g magnesium sulfate, and 7.5 mL glycerol. Heat and stir
to dissolve. Autoclave at 121
C for 15 min. Cool to 50–60
C
before aseptically adding 625 mL of egg base. The egg base is a
uniform suspension of fresh eggs prepared under aseptic conditions. Avoid whipping air into suspension during the collection and mixing. Once the media and the egg base are
combined, pour into screw top slants. Place into slant position.
Heat to 85
C for 45 min (see Note 4).
Culturing Mycobacteria
27