39. Do not try to inject more than 30 μL of bacterial suspension.
40. It is good practice to chill the homogenizer on ice without
opening the autoclave bag before putting in the tissue.
41. After about a minute or two of homogenization, place the
homogenizer on ice for couple of minutes.
42. This will be a hard and somewhat sticky pellet. Use 5 mL glass
pipette to dislodge pellet from the tube wall and then resuspend by gentle pipetting. Avoid frothing and use chilled media.
Adjust resuspending media quantity according to the size of
the bacterial pellet such that while adjusting AFB numbers (see
step 30) another centrifugation and resuspension step will not
be needed.
43. Best to use a 5 mL syringe and do it as slow as possible to avoid
frothing.
44. Make at least three serial dilutions 1:10, 1:100, 1:1000 of this
suspension for AFB count. Follow Fite-Faraco staining protocol (see Fig. 8) and Shepard’s method for counting M. leprae
(see Subheading 3.6).
45. DO NOT incubate for more than 3 h at 37
C as M. leprae
rapidly loses viability at this temperature. This step is done to
kill other contaminating bacteria. Use these 3 h to prepare AFB
slides and to determine bacterial count.
46. Live M. leprae should be used in experiments within 24–48 h of
harvest from the footpads, provided it is not contaminated and
has satisfactory viability (see steps 34 and 35).
47. It is usual to see bacterial growth in the BEFORE antibiotic
treatment plates but there should not be any growth in the
AFTER antibiotic treatment plates. Growth indicates that the
suspension is contaminated with other bacteria.
48. There should not be any growth on the L-J slant. Growth
indicates that the suspension is contaminated most likely with
other mycobacteria.
49. For highly viable M. leprae,
14
C CPM should be >6000 at
24 h.
50. This mixture is light sensitive, so keep lights off.
51. The mixture may turn yellow at this point. Continue stirring
until the mixture changes from a yellow paste to a yellow clear
solution. This can take up to 30 min. Remember to keep
lights off.
52. The solution dries quickly, so you must work fast.
53. These strips can be used for up to 6 months when stored
properly.
54. Each M. leprae suspension should at least be tested in triplicate.
55. DO NOT tighten, air should be able to escape the glass vial.
54
Elizabeth Wallace et al.
40. It is good practice to chill the homogenizer on ice without
opening the autoclave bag before putting in the tissue.
41. After about a minute or two of homogenization, place the
homogenizer on ice for couple of minutes.
42. This will be a hard and somewhat sticky pellet. Use 5 mL glass
pipette to dislodge pellet from the tube wall and then resuspend by gentle pipetting. Avoid frothing and use chilled media.
Adjust resuspending media quantity according to the size of
the bacterial pellet such that while adjusting AFB numbers (see
step 30) another centrifugation and resuspension step will not
be needed.
43. Best to use a 5 mL syringe and do it as slow as possible to avoid
frothing.
44. Make at least three serial dilutions 1:10, 1:100, 1:1000 of this
suspension for AFB count. Follow Fite-Faraco staining protocol (see Fig. 8) and Shepard’s method for counting M. leprae
(see Subheading 3.6).
45. DO NOT incubate for more than 3 h at 37
C as M. leprae
rapidly loses viability at this temperature. This step is done to
kill other contaminating bacteria. Use these 3 h to prepare AFB
slides and to determine bacterial count.
46. Live M. leprae should be used in experiments within 24–48 h of
harvest from the footpads, provided it is not contaminated and
has satisfactory viability (see steps 34 and 35).
47. It is usual to see bacterial growth in the BEFORE antibiotic
treatment plates but there should not be any growth in the
AFTER antibiotic treatment plates. Growth indicates that the
suspension is contaminated with other bacteria.
48. There should not be any growth on the L-J slant. Growth
indicates that the suspension is contaminated most likely with
other mycobacteria.
49. For highly viable M. leprae,
14
C CPM should be >6000 at
24 h.
50. This mixture is light sensitive, so keep lights off.
51. The mixture may turn yellow at this point. Continue stirring
until the mixture changes from a yellow paste to a yellow clear
solution. This can take up to 30 min. Remember to keep
lights off.
52. The solution dries quickly, so you must work fast.
53. These strips can be used for up to 6 months when stored
properly.
54. Each M. leprae suspension should at least be tested in triplicate.
55. DO NOT tighten, air should be able to escape the glass vial.
54
Elizabeth Wallace et al.