14. Mince the footpads with scissors and then transfer aseptically
into the handheld glass homogenizer by two 4 mL washes
using 10 mL sterile glass pipette and RPMI-10 medium.
15. Add up to 8 mL of RPMI-10 medium and homogenize gently,
taking care not to generate too much heat while doing
so. Homogenize until suspension looks milky, around 4 min
(see Note 41).
16. Place homogenizer tube (without the plunger) on ice, at an
angle to help larger particles collect to one corner of the tube.
Let it stand for 2–3 min.
17. With a 10 mL sterile glass pipette, transfer the suspension,
without the larger fragments settled at bottom, into a sterile
50 mL Oakridge tube.
18. Centrifuge at 10,000 Â g for 15 min at 4
C.
19. Discard supernatant.
20. Thoroughly resuspend pellet in 2–6 mL RPMI-10 medium
using a 5 mL sterile glass pipette (see Note 42).
21. Once fully resuspended, gently pass the suspension 2–3 times
through 27G needle (see Note 43) to get an even single-cell
distribution of M. leprae.
22. Streak 100 μL of the suspension each on a blood agar and a
chocolate agar plate. Incubate at 37
C. These are the
BEFORE antibiotic treatment plates.
23. Remove 100 μL of suspension for AFB counts (see Notes 44
and 45).
24. Add gentamycin and ampicillin to the suspension at a final
concentration of 50 μg/mL each.
25. Incubate M. leprae suspension for exactly 3 h at 37
C (see Note
46).
26. Following incubation, streak 100 μL of the suspension each on
a blood agar and a chocolate agar plate. Incubate at 37
C.
These are the AFTER antibiotic treatment plates.
27. Inoculate thioglycolate medium with 50 μL and L-J slant with
100 μL of the suspension and incubate at 37
C.
28. Adjust suspension to 1 Â 10
9 M. leprae/mL using RPMI-10.
29. Perform radiorespirometry to ascertain M. leprae viability (see
Subheading 3.11).
30. Aliquot M. leprae suspension as needed for end users and store
at 4
C (see Note 46).
31. Observe BEFORE and AFTER blood and chocolate agar
plates as well as thioglycolate medium at 24 h and day 4. Report
any growth (see Note 47).
40
Elizabeth Wallace et al.
into the handheld glass homogenizer by two 4 mL washes
using 10 mL sterile glass pipette and RPMI-10 medium.
15. Add up to 8 mL of RPMI-10 medium and homogenize gently,
taking care not to generate too much heat while doing
so. Homogenize until suspension looks milky, around 4 min
(see Note 41).
16. Place homogenizer tube (without the plunger) on ice, at an
angle to help larger particles collect to one corner of the tube.
Let it stand for 2–3 min.
17. With a 10 mL sterile glass pipette, transfer the suspension,
without the larger fragments settled at bottom, into a sterile
50 mL Oakridge tube.
18. Centrifuge at 10,000 Â g for 15 min at 4
C.
19. Discard supernatant.
20. Thoroughly resuspend pellet in 2–6 mL RPMI-10 medium
using a 5 mL sterile glass pipette (see Note 42).
21. Once fully resuspended, gently pass the suspension 2–3 times
through 27G needle (see Note 43) to get an even single-cell
distribution of M. leprae.
22. Streak 100 μL of the suspension each on a blood agar and a
chocolate agar plate. Incubate at 37
C. These are the
BEFORE antibiotic treatment plates.
23. Remove 100 μL of suspension for AFB counts (see Notes 44
and 45).
24. Add gentamycin and ampicillin to the suspension at a final
concentration of 50 μg/mL each.
25. Incubate M. leprae suspension for exactly 3 h at 37
C (see Note
46).
26. Following incubation, streak 100 μL of the suspension each on
a blood agar and a chocolate agar plate. Incubate at 37
C.
These are the AFTER antibiotic treatment plates.
27. Inoculate thioglycolate medium with 50 μL and L-J slant with
100 μL of the suspension and incubate at 37
C.
28. Adjust suspension to 1 Â 10
9 M. leprae/mL using RPMI-10.
29. Perform radiorespirometry to ascertain M. leprae viability (see
Subheading 3.11).
30. Aliquot M. leprae suspension as needed for end users and store
at 4
C (see Note 46).
31. Observe BEFORE and AFTER blood and chocolate agar
plates as well as thioglycolate medium at 24 h and day 4. Report
any growth (see Note 47).
40
Elizabeth Wallace et al.