48. Package the 230 mL conical with pellet in three layers of
biohazard bags with disinfectant spray between layers one
and two.
49. Seal each layer with tape (see Note 38).
3.9 Inoculating
Athymic Nude Mouse
Footpads
with M. leprae
1. Cover the work area with absorbent bench liner.
2. Draw the M. leprae suspension into the syringe, avoiding any
air bubbles.
3. Assistant firmly holds the cervical area of the mouse in one
hand and the tail with the other and puts the mouse ventral side
up with its footpads showing.
4. Clean both hind footpads with alcohol swab.
5. Extend the footpad by grasping on the toes.
6. Inject 30 μL of M. leprae suspension subdermally into each
hind footpad (see Note 39).
7. Release the mouse in its cage.
8. Footpads will be ready for harvest between 5 and 7 months (see
Fig. 4).
3.10 Harvesting Live
M. leprae from
Infected Athymic Nude
Mouse Footpads
1. Prepare 100 mL of sterile RPMI1640 with FBS (10% v/v)
(RPMI-10). Chill on ice.
2. Chill the glass homogenizer on ice (see Note 40).
3. Dispense 20 mL of Betadine solution in a sterile cup. Set aside.
4. Dispense 40 mL of 70% ethanol in each of four sterile cups. Set
aside.
5. Euthanize nude mouse by CO 2 asphyxiation. Place mouse on a
sterile gauze.
6. Ensure euthanization by cervical dislocation.
7. Clamp each thigh with hemostat and cut it off above the clamp.
8. Immerse legs in 20 mL Betadine solution (see step 5) for
20 min.
9. Remove legs from Betadine and immerse in 70% ethanol for
30 s. Repeat three more 70% ethanol rinses.
10. Clamp the three middle toes with another hemostat.
11. Over a fresh sterile gauze, position foot with heel pointing
away or up while the toes are pointing down or towards the
harvester.
12. Slice tissue at the heel working your way down to the toes. Use
forceps to grab the skin and tease footpad tissue away from skin
using a scalpel.
13. Discard skin and place the tissue on a sterile Petri plate.
Culturing Mycobacteria
39
biohazard bags with disinfectant spray between layers one
and two.
49. Seal each layer with tape (see Note 38).
3.9 Inoculating
Athymic Nude Mouse
Footpads
with M. leprae
1. Cover the work area with absorbent bench liner.
2. Draw the M. leprae suspension into the syringe, avoiding any
air bubbles.
3. Assistant firmly holds the cervical area of the mouse in one
hand and the tail with the other and puts the mouse ventral side
up with its footpads showing.
4. Clean both hind footpads with alcohol swab.
5. Extend the footpad by grasping on the toes.
6. Inject 30 μL of M. leprae suspension subdermally into each
hind footpad (see Note 39).
7. Release the mouse in its cage.
8. Footpads will be ready for harvest between 5 and 7 months (see
Fig. 4).
3.10 Harvesting Live
M. leprae from
Infected Athymic Nude
Mouse Footpads
1. Prepare 100 mL of sterile RPMI1640 with FBS (10% v/v)
(RPMI-10). Chill on ice.
2. Chill the glass homogenizer on ice (see Note 40).
3. Dispense 20 mL of Betadine solution in a sterile cup. Set aside.
4. Dispense 40 mL of 70% ethanol in each of four sterile cups. Set
aside.
5. Euthanize nude mouse by CO 2 asphyxiation. Place mouse on a
sterile gauze.
6. Ensure euthanization by cervical dislocation.
7. Clamp each thigh with hemostat and cut it off above the clamp.
8. Immerse legs in 20 mL Betadine solution (see step 5) for
20 min.
9. Remove legs from Betadine and immerse in 70% ethanol for
30 s. Repeat three more 70% ethanol rinses.
10. Clamp the three middle toes with another hemostat.
11. Over a fresh sterile gauze, position foot with heel pointing
away or up while the toes are pointing down or towards the
harvester.
12. Slice tissue at the heel working your way down to the toes. Use
forceps to grab the skin and tease footpad tissue away from skin
using a scalpel.
13. Discard skin and place the tissue on a sterile Petri plate.
Culturing Mycobacteria
39