3 Methods
3.1 General
Procedure for Bacterial
Labeling
Metabolic labeling of mycobacteria can be done in a variety of
experimental contexts, each of which may require some optimization of key variables (e.g., one-step vs. two-step labeling, probe and
secondary reagent concentrations, incubation times). Here, representative labeling experiments done using M. smegmatis in the
commonly used 96-well plate format are described.
1. Transfer 3 mL of Middlebrook 7H9 medium into a sterile
culture tube.
2. Inoculate medium with a single isolated colony of
M. smegmatis.
3. Incubate at 37
C with shaking at 220 rpm until
mid-logarithmic phase (typically overnight).
4. Read OD 600 . The target OD 600 is ~0.5–1.0.
5. Dilute culture with Middlebrook 7H9 medium to an OD 600
of 0.2.
6. Transfer 198μL of diluted culture into a sterile 96-well flatbottom plate.
7. Add 2μL of appropriate stock solution of probe prepared in
Subheadings 2.1 or 2.2 (or vehicle control) to achieve a desired
final concentration. For instance, to achieve a final concentration of 50μM, add 2μL of 5 mM stock solution (see Note 5).
Mix the well contents by pipetting up and down gently.
8. Incubate the 96-well plate with shaking at 37
C and 220 rpm
for the desired time period, typically 0.5–16 h (see Note 6),
optionally acquiring a growth curve by reading OD 600 during
the incubation (see Note 7).
9. Transfer cells into a 96-well V-bottom plate.
10. Centrifuge for 5 min at 3200 Â g at room temperature.
11. Gently pipette and discard the supernatant without disturbing
the pellet.
12. Wash the cells with 150μL of PBSB by gently pipetting up and
down to resuspend the pellet, centrifuge as above, and discard
the supernatant.
13. Repeat the wash step for a total of three washes.
14. Depending on the probe being used, follow the procedures in
Subheadings 3.2 or 3.3 for one-step or two-step labeling
approaches, respectively.
3.2 One-Step
Labeling
with Fluorescent
Probes
This procedure is applicable to metabolic labeling experiments with
FITC-Tre, DMN-Tre, O-FITC-TMM, and other fluorophoreconjugated trehalose analogs. This procedure uses a final probe
concentration of 50μM, although this can be adjusted as needed.
Trehalose Probe Labeling
391
3.1 General
Procedure for Bacterial
Labeling
Metabolic labeling of mycobacteria can be done in a variety of
experimental contexts, each of which may require some optimization of key variables (e.g., one-step vs. two-step labeling, probe and
secondary reagent concentrations, incubation times). Here, representative labeling experiments done using M. smegmatis in the
commonly used 96-well plate format are described.
1. Transfer 3 mL of Middlebrook 7H9 medium into a sterile
culture tube.
2. Inoculate medium with a single isolated colony of
M. smegmatis.
3. Incubate at 37
C with shaking at 220 rpm until
mid-logarithmic phase (typically overnight).
4. Read OD 600 . The target OD 600 is ~0.5–1.0.
5. Dilute culture with Middlebrook 7H9 medium to an OD 600
of 0.2.
6. Transfer 198μL of diluted culture into a sterile 96-well flatbottom plate.
7. Add 2μL of appropriate stock solution of probe prepared in
Subheadings 2.1 or 2.2 (or vehicle control) to achieve a desired
final concentration. For instance, to achieve a final concentration of 50μM, add 2μL of 5 mM stock solution (see Note 5).
Mix the well contents by pipetting up and down gently.
8. Incubate the 96-well plate with shaking at 37
C and 220 rpm
for the desired time period, typically 0.5–16 h (see Note 6),
optionally acquiring a growth curve by reading OD 600 during
the incubation (see Note 7).
9. Transfer cells into a 96-well V-bottom plate.
10. Centrifuge for 5 min at 3200 Â g at room temperature.
11. Gently pipette and discard the supernatant without disturbing
the pellet.
12. Wash the cells with 150μL of PBSB by gently pipetting up and
down to resuspend the pellet, centrifuge as above, and discard
the supernatant.
13. Repeat the wash step for a total of three washes.
14. Depending on the probe being used, follow the procedures in
Subheadings 3.2 or 3.3 for one-step or two-step labeling
approaches, respectively.
3.2 One-Step
Labeling
with Fluorescent
Probes
This procedure is applicable to metabolic labeling experiments with
FITC-Tre, DMN-Tre, O-FITC-TMM, and other fluorophoreconjugated trehalose analogs. This procedure uses a final probe
concentration of 50μM, although this can be adjusted as needed.
Trehalose Probe Labeling
391