6. Non-mycobacterial contaminants are tested in blood agar,
thioglycolate media, and TSB that are incubated at 37
C for
7 days and checked daily.
7. The non-contaminated tissues are stored dry in Cole-Parmer
wide-mouth sample containers (30 mL) at À80
C until contamination check is complete.
3.14 Purification
of M. leprae from
Armadillo Tissue
All work with open tissues/homogenate is to be done inside of a
BSC in a BSL2-certified laboratory. Follow institutional regulations
for PPE, material handling, and decontamination procedures. This
process takes 3 days.
1. Day 1. Remove tissue from À80
C at the end of the day, place
in a sterile 250 mL beaker. Cover beaker with foil and place in
4
C overnight to thaw.
2. Day 2. Aseptically chop tissue with scissors into approximately
1 cm pieces and pour into sterile homogenization chamber
(stainless steel).
3. Rinse beaker with cold 10 mM Na 2 EDTA and add to homogenization chamber. Add 10 mM Na 2 EDTA to the chamber to a
final volume of 30 mL per 10 g of tissue.
4. Homogenize tissue on ice using setting 5.5 for 3 min (see Note
57).
5. Pour homogenate into sterile Oakridge tubes, balance and
centrifuge at 500 Â g at 4
C for 5 min.
6. While centrifuging, perform a sterility test with the small
amount of homogenate left in the chamber: take a loop full of
homogenate and streak on an L-J slant. Repeat the process and
inoculate a TSA plate and BHI media (parafilm the TSA plate
to ensure that agar does not dry out). Incubate all three at
37
C (see Note 58).
7. Pour supernatant into sterile 250 mL beaker with a stir bar and
stir on medium speed to maintain homogenization.
8. Resuspend pellet by vortexing in a minimal amount of
Na 2 EDTA buffer (approx. 20 mL) and pour into the previously
used homogenization chamber.
9. Homogenize as in step 4.
10. Combine homogenate with the supernatant. Rinse chamber
with ~5 mL of 10 mM Na 2 EDTA buffer and add to homogenate/supernatant combination.
11. Continue to stir for ~2 min to decrease the volume of foamy
layer on the top.
12. Pour homogenate into the same 50 mL Oakridge centrifuge
tube as before, being sure to avoid pouring the stir bar into
tubes (see Note 59). More tubes may be used if needed.
Culturing Mycobacteria
43
thioglycolate media, and TSB that are incubated at 37
C for
7 days and checked daily.
7. The non-contaminated tissues are stored dry in Cole-Parmer
wide-mouth sample containers (30 mL) at À80
C until contamination check is complete.
3.14 Purification
of M. leprae from
Armadillo Tissue
All work with open tissues/homogenate is to be done inside of a
BSC in a BSL2-certified laboratory. Follow institutional regulations
for PPE, material handling, and decontamination procedures. This
process takes 3 days.
1. Day 1. Remove tissue from À80
C at the end of the day, place
in a sterile 250 mL beaker. Cover beaker with foil and place in
4
C overnight to thaw.
2. Day 2. Aseptically chop tissue with scissors into approximately
1 cm pieces and pour into sterile homogenization chamber
(stainless steel).
3. Rinse beaker with cold 10 mM Na 2 EDTA and add to homogenization chamber. Add 10 mM Na 2 EDTA to the chamber to a
final volume of 30 mL per 10 g of tissue.
4. Homogenize tissue on ice using setting 5.5 for 3 min (see Note
57).
5. Pour homogenate into sterile Oakridge tubes, balance and
centrifuge at 500 Â g at 4
C for 5 min.
6. While centrifuging, perform a sterility test with the small
amount of homogenate left in the chamber: take a loop full of
homogenate and streak on an L-J slant. Repeat the process and
inoculate a TSA plate and BHI media (parafilm the TSA plate
to ensure that agar does not dry out). Incubate all three at
37
C (see Note 58).
7. Pour supernatant into sterile 250 mL beaker with a stir bar and
stir on medium speed to maintain homogenization.
8. Resuspend pellet by vortexing in a minimal amount of
Na 2 EDTA buffer (approx. 20 mL) and pour into the previously
used homogenization chamber.
9. Homogenize as in step 4.
10. Combine homogenate with the supernatant. Rinse chamber
with ~5 mL of 10 mM Na 2 EDTA buffer and add to homogenate/supernatant combination.
11. Continue to stir for ~2 min to decrease the volume of foamy
layer on the top.
12. Pour homogenate into the same 50 mL Oakridge centrifuge
tube as before, being sure to avoid pouring the stir bar into
tubes (see Note 59). More tubes may be used if needed.
Culturing Mycobacteria
43