have been conceived [46–51]. In the end, we also provide some
practical examples of how to analyze the single-cell dynamics of
mycobacteria, from videos produced using time-lapse microfluidic
microscopy.
2 Materials
Prepare all growth media and reagents using Milli-Q ultrapure
water. Filter-sterilize each solution, and store all reagents at 4
C
unless differently specified.
2.1 Preparation of a
Single-Cell
Suspension
1. Glycerol: 50% (v/v).
2. Tyloxapol: 10% (w/v). Protect from light (see Note 1).
3. Albumin-Dextrose-Saline supplement (ADS): 5% Bovine
Serum Albumin (Fraction V), 2% D-glucose, 0.81% sodium
chloride in water.
4. Complete 7H9 medium: Dissolve 4.7 g Middlebrook 7H9
broth base (Difco) in 900 mL deionized water. Add 10 mL of
50% glycerol, 2 mL of 10% Tyloxapol and 100 mL of ADS
supplement (see Note 2).
5. Vacuum filtration units (0.22 μm).
6. Vacuum pump.
7. 30-mL PETG bottles (Nalgene).
8. 5-μm filter unit, PVDF membrane.
9. 1-mL syringes.
10. Tuberculocidal disinfectant, such as 0.5% sodium hypochlorite
or 0.8% Vesphene (STERIS Life Sciences).
2.2 Agarose Pad
Microscopy
1. Low-melting point agarose.
2. 2Â Complete 7H9 medium: Dissolve 4.7 g Middlebrook 7H9
broth base (Difco) in 400 mL deionized water. Add 10 mL of
50% glycerol, 2 mL of 10% Tyloxapol and 100 mL of ADS
supplement (see item 2 under Subheading 2.1) (see Note 2).
3. Sterile 50-mL and 15-mL polypropylene conical centrifuge
tubes.
4. #1 Glass cover slips: 24 Â 60 mm; 21 Â 26 mm; 25 mm round.
5. 100-mm Petri dishes.
6. Scalpel blade.
7. Biopsy punch (Harris Uni-core™, different sizes).
8. μ-Dish (Ibidi).
9. Fast-drying glue.
10. Immersion oil.
Microfluidic Microscopy of Mycobacteria
207
practical examples of how to analyze the single-cell dynamics of
mycobacteria, from videos produced using time-lapse microfluidic
microscopy.
2 Materials
Prepare all growth media and reagents using Milli-Q ultrapure
water. Filter-sterilize each solution, and store all reagents at 4
C
unless differently specified.
2.1 Preparation of a
Single-Cell
Suspension
1. Glycerol: 50% (v/v).
2. Tyloxapol: 10% (w/v). Protect from light (see Note 1).
3. Albumin-Dextrose-Saline supplement (ADS): 5% Bovine
Serum Albumin (Fraction V), 2% D-glucose, 0.81% sodium
chloride in water.
4. Complete 7H9 medium: Dissolve 4.7 g Middlebrook 7H9
broth base (Difco) in 900 mL deionized water. Add 10 mL of
50% glycerol, 2 mL of 10% Tyloxapol and 100 mL of ADS
supplement (see Note 2).
5. Vacuum filtration units (0.22 μm).
6. Vacuum pump.
7. 30-mL PETG bottles (Nalgene).
8. 5-μm filter unit, PVDF membrane.
9. 1-mL syringes.
10. Tuberculocidal disinfectant, such as 0.5% sodium hypochlorite
or 0.8% Vesphene (STERIS Life Sciences).
2.2 Agarose Pad
Microscopy
1. Low-melting point agarose.
2. 2Â Complete 7H9 medium: Dissolve 4.7 g Middlebrook 7H9
broth base (Difco) in 400 mL deionized water. Add 10 mL of
50% glycerol, 2 mL of 10% Tyloxapol and 100 mL of ADS
supplement (see item 2 under Subheading 2.1) (see Note 2).
3. Sterile 50-mL and 15-mL polypropylene conical centrifuge
tubes.
4. #1 Glass cover slips: 24 Â 60 mm; 21 Â 26 mm; 25 mm round.
5. 100-mm Petri dishes.
6. Scalpel blade.
7. Biopsy punch (Harris Uni-core™, different sizes).
8. μ-Dish (Ibidi).
9. Fast-drying glue.
10. Immersion oil.
Microfluidic Microscopy of Mycobacteria
207