3 Methods
3.1 Preparation of a
Single-Cell
Suspension
1. Inoculate 6 mL of complete 7H9 medium in a 30-mL PETG
bottle with a frozen stock of the mycobacterial reporter strain,
diluted 50 to 100 times (see Note 11).
2. Grow the culture in a 37 ˚C incubator with shaking at 100 rpm
for about 18 h (in case of fast-growing species), or with shaking
at 50 rpm for about 7 days (in case of slow-growing species),
until
the
culture
reaches
mid-exponential
phase
(OD 600 ¼ 0.5–0.8) (see Note 12).
3. Equilibrate a 5-μm filter with 1 mL of fresh 7H9 medium,
using a 1-mL syringe firmly threaded into the filter, let the
liquid drip by gravity, and discard it.
4. Load 1 mL of exponential-phase culture into the syringe barrel,
gently insert the syringe plunger into the barrel, and push
slowly until the culture has been filtered, to remove large
bacterial clumps.
5. The clear filtrate contains predominantly single-cell bacteria
and can be used for imaging.
3.2 Agarose Pad
Microscopy
1. Prepare 1.5% (w/v) low-melting point agarose in 10 mL of
sterile deionized water in a 50-mL tube and dissolve in the
microwave (see Note 13).
2. After the agarose has cooled down to around 40–50
C, add
10 mL of prewarmed (37
C) 2Â complete 7H9 medium (see
Note 2) and mix by vortexing.
3. Gently dispense 1 mL onto a glass cover slide placed inside a
100-mm petri dish on a flat surface.
4. Place a glass cover slip (24 Â 50 mm) on the top to create an
agarose sandwich (see Note 14).
5. Close the petri dish and wait for 30–60 min till the agarose
solidifies (see Note 15).
6. Remove the upper cover slip gently using a scalpel blade and
cut out the agarose pad to a size smaller than the coverslip used
for imaging (24 Â 60 mm; 21 Â 26 mm; 25 mm round), using
a scalpel blade or a biopsy punch and place it on a glass slide.
7. Seed 2 μL of the single-cell suspension on top of the agarose
pad and wait for 5 min so that the excess of medium dries out
and the cells settle on the agarose surface.
8. Flip the agarose pad gently onto a coverslip and carry out
snapshot imaging of the bacteria from the bottom through
the coverslip (see Note 16).
9. Alternatively, for short-term time-lapse microscopy experiments, the agarose pad (or several agarose pads, each with a
different strain of bacteria) can be flipped onto the bottom of a
212
Giulia Manina and Neeraj Dhar
3.1 Preparation of a
Single-Cell
Suspension
1. Inoculate 6 mL of complete 7H9 medium in a 30-mL PETG
bottle with a frozen stock of the mycobacterial reporter strain,
diluted 50 to 100 times (see Note 11).
2. Grow the culture in a 37 ˚C incubator with shaking at 100 rpm
for about 18 h (in case of fast-growing species), or with shaking
at 50 rpm for about 7 days (in case of slow-growing species),
until
the
culture
reaches
mid-exponential
phase
(OD 600 ¼ 0.5–0.8) (see Note 12).
3. Equilibrate a 5-μm filter with 1 mL of fresh 7H9 medium,
using a 1-mL syringe firmly threaded into the filter, let the
liquid drip by gravity, and discard it.
4. Load 1 mL of exponential-phase culture into the syringe barrel,
gently insert the syringe plunger into the barrel, and push
slowly until the culture has been filtered, to remove large
bacterial clumps.
5. The clear filtrate contains predominantly single-cell bacteria
and can be used for imaging.
3.2 Agarose Pad
Microscopy
1. Prepare 1.5% (w/v) low-melting point agarose in 10 mL of
sterile deionized water in a 50-mL tube and dissolve in the
microwave (see Note 13).
2. After the agarose has cooled down to around 40–50
C, add
10 mL of prewarmed (37
C) 2Â complete 7H9 medium (see
Note 2) and mix by vortexing.
3. Gently dispense 1 mL onto a glass cover slide placed inside a
100-mm petri dish on a flat surface.
4. Place a glass cover slip (24 Â 50 mm) on the top to create an
agarose sandwich (see Note 14).
5. Close the petri dish and wait for 30–60 min till the agarose
solidifies (see Note 15).
6. Remove the upper cover slip gently using a scalpel blade and
cut out the agarose pad to a size smaller than the coverslip used
for imaging (24 Â 60 mm; 21 Â 26 mm; 25 mm round), using
a scalpel blade or a biopsy punch and place it on a glass slide.
7. Seed 2 μL of the single-cell suspension on top of the agarose
pad and wait for 5 min so that the excess of medium dries out
and the cells settle on the agarose surface.
8. Flip the agarose pad gently onto a coverslip and carry out
snapshot imaging of the bacteria from the bottom through
the coverslip (see Note 16).
9. Alternatively, for short-term time-lapse microscopy experiments, the agarose pad (or several agarose pads, each with a
different strain of bacteria) can be flipped onto the bottom of a
212
Giulia Manina and Neeraj Dhar