27. Spin at 3000 Â g for 15 min, and discard the supernatant (see
Note 71).
28. Sanitize the tubes containing the cell pellet. Bring the tubes to
a top loading balance and write down the wet weight on the
tube label. Place tubes into three biohazard bags and seal
tightly with tape. Store at À80
C. The cell pellet is ready for
γ-irradiation or 2:1 chloroform:methanol added for lipid
extraction (see Note 72).
29. Place a sterile 4 L bottle and VacuCap into the BSC. Using
autoclave tape, fasten the tubing from the vacuum pump to the
BSC to prevent the tubing from causing accidental spillage.
30. Open the 4 L bottle and VacuCap package. Insert an aspiration
pipette into the CFP collection flask and insert the other end
into the VacuCap hose. Place the VacuCap on the mouth of the
bottle and attach the tubing from the vacuum pump to the
VacuCap and turn on vacuum. Proceed per CFP harvest (see
Subheading 3.8, step 31).
31. Unhook the empty vessel from the control unit and place in the
BSC. Remove the pH probe from the vessel. Wipe/clean the
probe with ethanol, cap, and put in a biosafety bag to be
Fig. 12 Bioreactor at harvest. Lower image shows a close-up of the cell paste at
the bottom of the container
Culturing Mycobacteria
49
Note 71).
28. Sanitize the tubes containing the cell pellet. Bring the tubes to
a top loading balance and write down the wet weight on the
tube label. Place tubes into three biohazard bags and seal
tightly with tape. Store at À80
C. The cell pellet is ready for
γ-irradiation or 2:1 chloroform:methanol added for lipid
extraction (see Note 72).
29. Place a sterile 4 L bottle and VacuCap into the BSC. Using
autoclave tape, fasten the tubing from the vacuum pump to the
BSC to prevent the tubing from causing accidental spillage.
30. Open the 4 L bottle and VacuCap package. Insert an aspiration
pipette into the CFP collection flask and insert the other end
into the VacuCap hose. Place the VacuCap on the mouth of the
bottle and attach the tubing from the vacuum pump to the
VacuCap and turn on vacuum. Proceed per CFP harvest (see
Subheading 3.8, step 31).
31. Unhook the empty vessel from the control unit and place in the
BSC. Remove the pH probe from the vessel. Wipe/clean the
probe with ethanol, cap, and put in a biosafety bag to be
Fig. 12 Bioreactor at harvest. Lower image shows a close-up of the cell paste at
the bottom of the container
Culturing Mycobacteria
49