autoclaved. Add a copious amount of Vesphene into the vessel
through the open pH port and place in a large autoclave bag
for disposal.
4 Notes
1. This may be purchased ready-to-use from qualified scientific
vendors and manufacturers. Manufacturer should provide documentation to assure quality control and will provide recommendations for use and storage.
2. Boil the water and gelatin with stirring until the gelatin is
thoroughly dissolved. Allow to cool before adding the phenol
solution.
3. The 100Â field constant for each microscope with the eyepiece
and 100Â objective pair that will be used for actual counting
must be determined beforehand with the help of a stage
micrometer.
4. Media is light and temperature sensitive; protect from light,
excessive heat, moisture, and freezing. Store between 2 and
8
C.
5. If this procedure is for mass spectometry purposes, the containers used here should be made of glass to minimize background
noise originated from plastic containers. Use of glass is not
recommended or allowed in most BSL3 laboratory facilities,
so this requirement would require special attention and review
by the institutional biosafety review board.
6. Roller bottles are used only if harvesting CFP is needed.
7. If a warm room maintained at 37
C is unavailable, use an
incubator, environmental shaker tables, and/or environmental
roller bottle apparatus as needed.
8. Female mice are preferred as they are easier to manage.
9. The cell suspension can be in PBS, Hanks buffer, or RPMI
1640. All reagents used should be endotoxin free. M. leprae
harvested within the last 24 h and checked for other bacterial
contamination, and viability should be used for footpad
inoculation.
10. PPO: 2,5-Diphenyoxazole. POPOP: 1,4,-bis-2-(4-methyl-5phenyloxazolyl) benzene.
11. This is 2 N NaOH in methanol and can be stored at room
temperature.
12. Sodium azide can be added (to a final concentration of 0.04%)
if storing for longer than a week. This media can be used for up
to 4 weeks when stored properly.
50
Elizabeth Wallace et al.
through the open pH port and place in a large autoclave bag
for disposal.
4 Notes
1. This may be purchased ready-to-use from qualified scientific
vendors and manufacturers. Manufacturer should provide documentation to assure quality control and will provide recommendations for use and storage.
2. Boil the water and gelatin with stirring until the gelatin is
thoroughly dissolved. Allow to cool before adding the phenol
solution.
3. The 100Â field constant for each microscope with the eyepiece
and 100Â objective pair that will be used for actual counting
must be determined beforehand with the help of a stage
micrometer.
4. Media is light and temperature sensitive; protect from light,
excessive heat, moisture, and freezing. Store between 2 and
8
C.
5. If this procedure is for mass spectometry purposes, the containers used here should be made of glass to minimize background
noise originated from plastic containers. Use of glass is not
recommended or allowed in most BSL3 laboratory facilities,
so this requirement would require special attention and review
by the institutional biosafety review board.
6. Roller bottles are used only if harvesting CFP is needed.
7. If a warm room maintained at 37
C is unavailable, use an
incubator, environmental shaker tables, and/or environmental
roller bottle apparatus as needed.
8. Female mice are preferred as they are easier to manage.
9. The cell suspension can be in PBS, Hanks buffer, or RPMI
1640. All reagents used should be endotoxin free. M. leprae
harvested within the last 24 h and checked for other bacterial
contamination, and viability should be used for footpad
inoculation.
10. PPO: 2,5-Diphenyoxazole. POPOP: 1,4,-bis-2-(4-methyl-5phenyloxazolyl) benzene.
11. This is 2 N NaOH in methanol and can be stored at room
temperature.
12. Sodium azide can be added (to a final concentration of 0.04%)
if storing for longer than a week. This media can be used for up
to 4 weeks when stored properly.
50
Elizabeth Wallace et al.