4. Peristaltic pump.
5. Dissolved oxygen (DO) probe with proper calibration (see
Note 17).
6. pH probe with proper calibration (see Note 18).
7. M. tuberculosis large-scale inoculum (see Subheading 3.8).
8. 2.5% Vesphene solution.
9. 70% ethanol.
10. NEB Bioreactor incubator jacket.
11. NEB Bioreactor control unit.
12. 0.8/0.2 μm syringe filter or Steriflip 50 mL conical filter.
13. 60 mL syringe.
14. Aerosol-resistant centrifuge with rotors and buckets for 15 and
50 mL conicals.
15. 50 or 230 mL centrifuge tube.
16. Sterile 4 L glass bottle.
17. VacuCap.
3 Methods
3.1 Preparation
of Bacterial Smears
1. To stain growth from liquid culture, place a drop on the
microscope glass slide.
2. For cultures grown on solid media, place a drop of water,
saline, or broth onto the slide. Select a single colony with a
loop and suspend it in the drop on the slide and let air-dry.
3. Fix the smear to the slide, either by means of heat or methanol
(see Note 19).
(a) To heat-fix the smear, place the slide over an open flame
for a few seconds, or on a hot plate (100
C) for up to a
minute.
(b) To fix the smear using methanol, flood the slide with cold
methanol for 5 min.
(c) Air-dry in the BSC. Slides can then be removed from the
BSC for staining.
3.2 Gram Staining
1. Place fixed slide over basin or sink.
2. Add Gram crystal violet to cover slide.
3. Wait 60 s and rinse gently.
4. Add Gram iodine to cover slide.
5. Wait 60 s and rinse gently.
6. Hold the slide at an angle near running water.
Culturing Mycobacteria
31
5. Dissolved oxygen (DO) probe with proper calibration (see
Note 17).
6. pH probe with proper calibration (see Note 18).
7. M. tuberculosis large-scale inoculum (see Subheading 3.8).
8. 2.5% Vesphene solution.
9. 70% ethanol.
10. NEB Bioreactor incubator jacket.
11. NEB Bioreactor control unit.
12. 0.8/0.2 μm syringe filter or Steriflip 50 mL conical filter.
13. 60 mL syringe.
14. Aerosol-resistant centrifuge with rotors and buckets for 15 and
50 mL conicals.
15. 50 or 230 mL centrifuge tube.
16. Sterile 4 L glass bottle.
17. VacuCap.
3 Methods
3.1 Preparation
of Bacterial Smears
1. To stain growth from liquid culture, place a drop on the
microscope glass slide.
2. For cultures grown on solid media, place a drop of water,
saline, or broth onto the slide. Select a single colony with a
loop and suspend it in the drop on the slide and let air-dry.
3. Fix the smear to the slide, either by means of heat or methanol
(see Note 19).
(a) To heat-fix the smear, place the slide over an open flame
for a few seconds, or on a hot plate (100
C) for up to a
minute.
(b) To fix the smear using methanol, flood the slide with cold
methanol for 5 min.
(c) Air-dry in the BSC. Slides can then be removed from the
BSC for staining.
3.2 Gram Staining
1. Place fixed slide over basin or sink.
2. Add Gram crystal violet to cover slide.
3. Wait 60 s and rinse gently.
4. Add Gram iodine to cover slide.
5. Wait 60 s and rinse gently.
6. Hold the slide at an angle near running water.
Culturing Mycobacteria
31