5. Once filled, tuck all tubing back under the lid of vessel. Remove
the vessel from the BSC and place at 37
C overnight to check
for contamination.
6. Prepare initial inoculum (1 L of M. tuberculosis grown for
2 weeks in a Fernbach (see Subheading 3.8 and Note 66).
Place the initial culture inoculum and bioreactor vessels into
the BSC. Gently swirl the flask to create a whirlpool and set
down to allow the cells to settle. This will help settle the cells
into a pellet in the center of the flask.
7. Pipette half of the cell pellet from the flask into each of the
bioreactor vessels through the pH probe port.
8. Sanitize pH probe with 2.5% Vesphene and then 70% ethanol
(see Note 67). Aseptically install probe, and seal with parafilm.
9. Sanitize the exterior of the fermentor vessels before removing
the vessels from the BSC. Bring the vessels to the bioreactor
(for example, a BioFlo unit) and attach the pH cables to the pH
probes. Gently insert DO probes and temperature probes into
the vessels. Attach the exhaust heater to the filtered exhaust
tube and wrap the incubator jacket around the vessel. Attach
the gas lines to the sparge filters and place the rotor motors on
top of the vessels. Set up the bioreactor to the desired parameters for normoxic growth (37
C, DO set to 35–40, pH set
to 6.6, agitation low limit of 45 and a high limit of 105, and gas
flow set to a low limit of 0 and a high limit of 1 VVM (gas
volume flow per unit of liquid volume per minute).
10. Sample removal may not always be necessary, but it is useful for
timepoint-specific analysis.
11. Check cultures 2 days postinoculation for contamination.
Check all equipment and tanks for proper functioning. Continue monitoring cultures remotely (see Note 68).
12. Remove two 40 mL samples from each vessel using the sample
port and syringe. Place sample syringes onto Vesphene-soaked
Wypalls (see Note 69).
13. Inside a BSC, transfer syringe contents into 50 mL conical vial.
Centrifuge at 3000 Â g for 10 min. Transfer supernatant to
new 50 mL conical.
14. If CFP is needed, attach the 50 mL conical tube containing the
decanted supernatant to a Steriflip conical. Use the vacuum
pump to filter CFP to the new container. Cap and wrap with
parafilm. Freeze at À80
C or store at 4
C (see Note 35). If
Steriflips are unavailable, transfer the supernatant into a reservoir boat. Pull up the liquid with a syringe and attach a 0.8/0.2
syringe filter. Slowly press the liquid through the filter into a
new 50 mL conical tube.
Culturing Mycobacteria
47
the vessel from the BSC and place at 37
C overnight to check
for contamination.
6. Prepare initial inoculum (1 L of M. tuberculosis grown for
2 weeks in a Fernbach (see Subheading 3.8 and Note 66).
Place the initial culture inoculum and bioreactor vessels into
the BSC. Gently swirl the flask to create a whirlpool and set
down to allow the cells to settle. This will help settle the cells
into a pellet in the center of the flask.
7. Pipette half of the cell pellet from the flask into each of the
bioreactor vessels through the pH probe port.
8. Sanitize pH probe with 2.5% Vesphene and then 70% ethanol
(see Note 67). Aseptically install probe, and seal with parafilm.
9. Sanitize the exterior of the fermentor vessels before removing
the vessels from the BSC. Bring the vessels to the bioreactor
(for example, a BioFlo unit) and attach the pH cables to the pH
probes. Gently insert DO probes and temperature probes into
the vessels. Attach the exhaust heater to the filtered exhaust
tube and wrap the incubator jacket around the vessel. Attach
the gas lines to the sparge filters and place the rotor motors on
top of the vessels. Set up the bioreactor to the desired parameters for normoxic growth (37
C, DO set to 35–40, pH set
to 6.6, agitation low limit of 45 and a high limit of 105, and gas
flow set to a low limit of 0 and a high limit of 1 VVM (gas
volume flow per unit of liquid volume per minute).
10. Sample removal may not always be necessary, but it is useful for
timepoint-specific analysis.
11. Check cultures 2 days postinoculation for contamination.
Check all equipment and tanks for proper functioning. Continue monitoring cultures remotely (see Note 68).
12. Remove two 40 mL samples from each vessel using the sample
port and syringe. Place sample syringes onto Vesphene-soaked
Wypalls (see Note 69).
13. Inside a BSC, transfer syringe contents into 50 mL conical vial.
Centrifuge at 3000 Â g for 10 min. Transfer supernatant to
new 50 mL conical.
14. If CFP is needed, attach the 50 mL conical tube containing the
decanted supernatant to a Steriflip conical. Use the vacuum
pump to filter CFP to the new container. Cap and wrap with
parafilm. Freeze at À80
C or store at 4
C (see Note 35). If
Steriflips are unavailable, transfer the supernatant into a reservoir boat. Pull up the liquid with a syringe and attach a 0.8/0.2
syringe filter. Slowly press the liquid through the filter into a
new 50 mL conical tube.
Culturing Mycobacteria
47