23. Pipette cell pellets from each flask into this collection flask.
24. Combine the spent media, devoid of cell biomass, into as few
flasks as possible. Spent GAS media from these flasks is generally discarded and not used for CFP. However, if roller bottles
are not used and CFP from flasks needs to be collected, proceed with step 31. Otherwise, discard according to institutional regulations for bulk liquids.
25. Cells from the roller bottles will be pelleted as in step 21 and
added to the step 22 collection flask.
26. After the cells have been harvested, the spent media from the
roller bottles will be collected and filtered to produce CFP (see
Note 32).
27. Place a sterile 4 L filtration bottle, a 0.2 μm filtration unit, and
6–10 roller bottle cultures inside the BSC (see Note 33).
28. Lean the roller bottles against the back wall and each other to
allow the cells to settle in a pile.
29. Uncap a roller bottle and keep it tilted slightly to allow cell
pellet to stay settled (see Note 34).
30. Pipette out the cell pellet from each bottle and add it to the
pellet collection flask from step 22.
31. Insert an aspiration pipette (or a serological pipette with the
cotton removed) into the vacuum tubing hose and aspirate the
media into the bottle, through the filter disc.
32. Avoid cells as much as possible as these can clog the filter (see
Figs. 9 and 10).
33. Repeat with remaining roller bottles.
34. When the 4 L bottle contains 4 L of CFP, place the 0.2 μm
filtration unit in the biohazard trash.
35. Store the five 4 L bottles containing sterile-filtered culture
supernatant at 4
C (see Note 35).
36. Discard remaining media according to institutional guidelines
for bulk liquid waste.
37. Add 1 L of sterile 0.2 μm filtered water to the cell collection
flask.
38. Swirl flask to wash cells.
39. Pipet used water from spent flask and discard appropriately.
40. Add another 1 L of sterile 0.2 μm filtered water to cells, wash
cells, and discard ~three-fourths of this water.
41. Weigh an empty 230 mL conical tube and record weight.
42. Aseptically transfer the washed bacterial cells into a 230 mL
conical (see Note 36) and cap tightly.
43. Centrifuge at 3000 Â g, at 4
C for 10–15 min.
Culturing Mycobacteria
37
24. Combine the spent media, devoid of cell biomass, into as few
flasks as possible. Spent GAS media from these flasks is generally discarded and not used for CFP. However, if roller bottles
are not used and CFP from flasks needs to be collected, proceed with step 31. Otherwise, discard according to institutional regulations for bulk liquids.
25. Cells from the roller bottles will be pelleted as in step 21 and
added to the step 22 collection flask.
26. After the cells have been harvested, the spent media from the
roller bottles will be collected and filtered to produce CFP (see
Note 32).
27. Place a sterile 4 L filtration bottle, a 0.2 μm filtration unit, and
6–10 roller bottle cultures inside the BSC (see Note 33).
28. Lean the roller bottles against the back wall and each other to
allow the cells to settle in a pile.
29. Uncap a roller bottle and keep it tilted slightly to allow cell
pellet to stay settled (see Note 34).
30. Pipette out the cell pellet from each bottle and add it to the
pellet collection flask from step 22.
31. Insert an aspiration pipette (or a serological pipette with the
cotton removed) into the vacuum tubing hose and aspirate the
media into the bottle, through the filter disc.
32. Avoid cells as much as possible as these can clog the filter (see
Figs. 9 and 10).
33. Repeat with remaining roller bottles.
34. When the 4 L bottle contains 4 L of CFP, place the 0.2 μm
filtration unit in the biohazard trash.
35. Store the five 4 L bottles containing sterile-filtered culture
supernatant at 4
C (see Note 35).
36. Discard remaining media according to institutional guidelines
for bulk liquid waste.
37. Add 1 L of sterile 0.2 μm filtered water to the cell collection
flask.
38. Swirl flask to wash cells.
39. Pipet used water from spent flask and discard appropriately.
40. Add another 1 L of sterile 0.2 μm filtered water to cells, wash
cells, and discard ~three-fourths of this water.
41. Weigh an empty 230 mL conical tube and record weight.
42. Aseptically transfer the washed bacterial cells into a 230 mL
conical (see Note 36) and cap tightly.
43. Centrifuge at 3000 Â g, at 4
C for 10–15 min.
Culturing Mycobacteria
37