3.10 Quantification
of M. tuberculosis
Survival in
Macrophages
1. To lyse macrophages, remove medium and add 0.5 mL of lysis
buffer to each well.
2. Incubate at 37
C for 10 min.
3. Observe cells under an inverted light microscope. Macrophages should appear swollen (Fig. 1a) compared to normal,
healthy cells (Fig. 1b).
4. Using a P1000 (1 mL) pipettor and pipette tip, systematically
scrape the well with the pipette tip. Following scraping, pipette
the bacterial suspension up and down three times. Utilize a
consistent technique for all of the wells.
5. In a 96-well assay plate, serially dilute the lysed macrophage
suspension by 10
À1 , 10
À2 , and 10
À3 .
6. Plate 50 μL of the undiluted sample and each dilution onto a
quadrant of the appropriate 7H10 agar plate. Spread the lysed
macrophage solution onto the agar with either a sterile plate
spreader or a disposable inoculation loop.
7. Place plates in a Ziploc
® bag and incubate in a 37
C incubator.
Colonies will begin to appear approximately 2 or 3 weeks after
plating, although when they appear can vary considerably.
Count colonies at 2–4 weeks postinfection to account for all
colonies.
8. For quantifying survival of fluorescent M. tuberculosis reporters
in 96- or 384-well plates, use clear bottom, black-sided plates,
and for luciferase reporter assays use solid, white plates.
3.11 Imaging M.
tuberculosis-Infected
Macrophages
1. Autoclave the round glass coverslips in a small beaker lined on
the bottom with Kimwipes.
2. In a biosafety cabinet, add a single coverslip to each well of a
24-well plate (see Note 17).
3. Seed macrophages in the same manner as described above (see
Subheading 3.2).
4. Allow macrophages to adhere overnight in macrophage infection medium and infect macrophages as described above (see
Subheading 3.8).
5. To prepare the coverslips for mounting on microscope slides,
remove the medium from each well and wash the wells twice
with 1 mL of tissue culture-grade PBS.
6. To fix cells, remove PBS and add 1 mL of 4% paraformaldehyde
(PFA) in PBS to each well. Seal the plate in a plastic Ziploc
®
bag and incubate at 4
C overnight.
7. Remove the PFA and discard, wash twice with 1 mL of PBS,
and then add 0.5 mL of PBS to each well.
8. Add 10 μL of ProLong
® Gold antifade reagent to the center of
a glass microscope slide.
178
Benjamin K. Johnson et al.
of M. tuberculosis
Survival in
Macrophages
1. To lyse macrophages, remove medium and add 0.5 mL of lysis
buffer to each well.
2. Incubate at 37
C for 10 min.
3. Observe cells under an inverted light microscope. Macrophages should appear swollen (Fig. 1a) compared to normal,
healthy cells (Fig. 1b).
4. Using a P1000 (1 mL) pipettor and pipette tip, systematically
scrape the well with the pipette tip. Following scraping, pipette
the bacterial suspension up and down three times. Utilize a
consistent technique for all of the wells.
5. In a 96-well assay plate, serially dilute the lysed macrophage
suspension by 10
À1 , 10
À2 , and 10
À3 .
6. Plate 50 μL of the undiluted sample and each dilution onto a
quadrant of the appropriate 7H10 agar plate. Spread the lysed
macrophage solution onto the agar with either a sterile plate
spreader or a disposable inoculation loop.
7. Place plates in a Ziploc
® bag and incubate in a 37
C incubator.
Colonies will begin to appear approximately 2 or 3 weeks after
plating, although when they appear can vary considerably.
Count colonies at 2–4 weeks postinfection to account for all
colonies.
8. For quantifying survival of fluorescent M. tuberculosis reporters
in 96- or 384-well plates, use clear bottom, black-sided plates,
and for luciferase reporter assays use solid, white plates.
3.11 Imaging M.
tuberculosis-Infected
Macrophages
1. Autoclave the round glass coverslips in a small beaker lined on
the bottom with Kimwipes.
2. In a biosafety cabinet, add a single coverslip to each well of a
24-well plate (see Note 17).
3. Seed macrophages in the same manner as described above (see
Subheading 3.2).
4. Allow macrophages to adhere overnight in macrophage infection medium and infect macrophages as described above (see
Subheading 3.8).
5. To prepare the coverslips for mounting on microscope slides,
remove the medium from each well and wash the wells twice
with 1 mL of tissue culture-grade PBS.
6. To fix cells, remove PBS and add 1 mL of 4% paraformaldehyde
(PFA) in PBS to each well. Seal the plate in a plastic Ziploc
®
bag and incubate at 4
C overnight.
7. Remove the PFA and discard, wash twice with 1 mL of PBS,
and then add 0.5 mL of PBS to each well.
8. Add 10 μL of ProLong
® Gold antifade reagent to the center of
a glass microscope slide.
178
Benjamin K. Johnson et al.