3. Streak three to five large plates to later upscale to three to five
flasks.
4. Incubate at 37
C, check plates periodically until a thick lawn
has formed. Complete growth will require 3–6 weeks depending on the strain (see Note 28).
5. Once a lawn has formed, harvest cells.
6. Scrape cells and use the scraper to transfer cells to one flask (see
Note 29).
7. Repeat with remaining plates and flasks. Incubate the flasks on
an orbital platform shaker for 2 weeks (see Note 30) at 37
C at
65 rpm.
8. Two weeks later (see Note 28), place one flask with good cell
growth into the BSC, along with eight flasks containing fresh
GAS media.
9. Visually inspect flasks for signs of contamination, such as significant turbidity or color change.
10. Gently swirl flask then allow cells to settle to the bottom into a
pile in the center.
11. Using a 50 mL pipette, remove approximately one-eighth of
the bacterial pellet and transfer it to one of the new flasks.
12. Repeat the previous three steps for the seven remaining flasks.
13. Place the eight newly inoculated flasks on an orbital platform
shaker at 37
C for 2 weeks (see Note 28).
14. Place roller bottles with 400 mL of GAS medium inside the
BSC (see Note 31).
15. Place one flask with 2 weeks of bacterial growth inside the BSC,
visually inspecting as before.
16. Swirl the flask gently to dislodge cells from the sides, and then
set the flask down to allow the cells to settle in the center.
17. Once the cells have settled, remove approximately 1/20 of
bacterial pellet and aseptically transfer it to one of the roller
bottles and tighten cap.
18. Repeat for remaining 19 bottles.
19. Place the inoculated roller bottles on roller bottle apparatus at
37
C for 2 weeks (see Note 28).
20. Once placed on the apparatus, the roller bottles should be
rotated at a slow speed to ensure proper aeration of the cells
while avoiding over-agitation.
21. To harvest cells, start by placing the flask cultures into the BSC
and allow cell pellets to settle.
22. Gently swirl the flasks to create a cell pellet in each. One flask
will be used to pool cell biomass (“collection flask”).
36
Elizabeth Wallace et al.
flasks.
4. Incubate at 37
C, check plates periodically until a thick lawn
has formed. Complete growth will require 3–6 weeks depending on the strain (see Note 28).
5. Once a lawn has formed, harvest cells.
6. Scrape cells and use the scraper to transfer cells to one flask (see
Note 29).
7. Repeat with remaining plates and flasks. Incubate the flasks on
an orbital platform shaker for 2 weeks (see Note 30) at 37
C at
65 rpm.
8. Two weeks later (see Note 28), place one flask with good cell
growth into the BSC, along with eight flasks containing fresh
GAS media.
9. Visually inspect flasks for signs of contamination, such as significant turbidity or color change.
10. Gently swirl flask then allow cells to settle to the bottom into a
pile in the center.
11. Using a 50 mL pipette, remove approximately one-eighth of
the bacterial pellet and transfer it to one of the new flasks.
12. Repeat the previous three steps for the seven remaining flasks.
13. Place the eight newly inoculated flasks on an orbital platform
shaker at 37
C for 2 weeks (see Note 28).
14. Place roller bottles with 400 mL of GAS medium inside the
BSC (see Note 31).
15. Place one flask with 2 weeks of bacterial growth inside the BSC,
visually inspecting as before.
16. Swirl the flask gently to dislodge cells from the sides, and then
set the flask down to allow the cells to settle in the center.
17. Once the cells have settled, remove approximately 1/20 of
bacterial pellet and aseptically transfer it to one of the roller
bottles and tighten cap.
18. Repeat for remaining 19 bottles.
19. Place the inoculated roller bottles on roller bottle apparatus at
37
C for 2 weeks (see Note 28).
20. Once placed on the apparatus, the roller bottles should be
rotated at a slow speed to ensure proper aeration of the cells
while avoiding over-agitation.
21. To harvest cells, start by placing the flask cultures into the BSC
and allow cell pellets to settle.
22. Gently swirl the flasks to create a cell pellet in each. One flask
will be used to pool cell biomass (“collection flask”).
36
Elizabeth Wallace et al.