15. Freeze the cell pellet for analysis. For lipid analysis or chemical
inactivation, cells can instead be transferred to a glass tube and
inactivated with 2:1 chloroform:methanol. If RNA extraction
is desired, this must be done immediately when sample is taken.
Label the 2 Â 50 mL centrifuge tubes or glass tubes with the
appropriate information (strain, lot number, date, medium,
and technician name).
16. Initiate hypoxic conditions in one vessel 4 days after inoculation by changing the set point of the DO to 1. If the DO fails to
fall naturally within 1 day, change the cascade gas flow lower
limit to 0.2. Remotely monitor transition to hypoxic
conditions.
17. Harvest hypoxic culture 10 days post hypoxic condition initiation (14 days postinoculation).
18. Harvest normoxic culture 10 days postinoculation (see Note
70).
19. Place two empty 2.8 L Fernbach collection flasks into the BSC.
20. Turn off the gas flow from the bioreactor unit to the vessel and
remove the heating jacket. Attach the vacuum compressor
pump exhaust port to the filter of the gas overlay port of the
reactor vessel. Clamp shut all other exhaust and gas ports so the
vessel is completely sealed.
21. Turn off the DO, pH, and temperature probes, leaving the
agitation at 105 rpm.
22. Attach the custom harvesting line to the harvest port of the
vessel using the appropriate adaptor piece. Wrap parafilm
around the connection. Run the other end of the harvest line
into the BSC and tape the end to the empty collection flask, so
the vessel culture in pumped into the flask. Unscrew the
exhaust regulator of the pump all the way and then turn on
the pump. VERY slowly increase the pressure until the culture
begins to flow in the BSC collection flask (see Fig. 12).
23. When the collection flask is full, turn off the pump and allow
the cells to settle. Carefully transfer the CFP into the second
empty flask (CFP collection flask). Then continue pumping the
vessel culture in the cell collection flask until the vessel is empty.
24. Unhook the empty vessel from the bioreactor and place in the
BSC. Remove the pH probe from the vessel.
25. Pipette out the remaining media along with the cell pellet into
a 50 or 230 mL conical tube (record tare weight prior to
adding cell mixture).
26. When all the cells have been transferred, remove as much media
supernatant as possible.
48
Elizabeth Wallace et al.
inactivation, cells can instead be transferred to a glass tube and
inactivated with 2:1 chloroform:methanol. If RNA extraction
is desired, this must be done immediately when sample is taken.
Label the 2 Â 50 mL centrifuge tubes or glass tubes with the
appropriate information (strain, lot number, date, medium,
and technician name).
16. Initiate hypoxic conditions in one vessel 4 days after inoculation by changing the set point of the DO to 1. If the DO fails to
fall naturally within 1 day, change the cascade gas flow lower
limit to 0.2. Remotely monitor transition to hypoxic
conditions.
17. Harvest hypoxic culture 10 days post hypoxic condition initiation (14 days postinoculation).
18. Harvest normoxic culture 10 days postinoculation (see Note
70).
19. Place two empty 2.8 L Fernbach collection flasks into the BSC.
20. Turn off the gas flow from the bioreactor unit to the vessel and
remove the heating jacket. Attach the vacuum compressor
pump exhaust port to the filter of the gas overlay port of the
reactor vessel. Clamp shut all other exhaust and gas ports so the
vessel is completely sealed.
21. Turn off the DO, pH, and temperature probes, leaving the
agitation at 105 rpm.
22. Attach the custom harvesting line to the harvest port of the
vessel using the appropriate adaptor piece. Wrap parafilm
around the connection. Run the other end of the harvest line
into the BSC and tape the end to the empty collection flask, so
the vessel culture in pumped into the flask. Unscrew the
exhaust regulator of the pump all the way and then turn on
the pump. VERY slowly increase the pressure until the culture
begins to flow in the BSC collection flask (see Fig. 12).
23. When the collection flask is full, turn off the pump and allow
the cells to settle. Carefully transfer the CFP into the second
empty flask (CFP collection flask). Then continue pumping the
vessel culture in the cell collection flask until the vessel is empty.
24. Unhook the empty vessel from the bioreactor and place in the
BSC. Remove the pH probe from the vessel.
25. Pipette out the remaining media along with the cell pellet into
a 50 or 230 mL conical tube (record tare weight prior to
adding cell mixture).
26. When all the cells have been transferred, remove as much media
supernatant as possible.
48
Elizabeth Wallace et al.