burner). In this case, pick up the seeds and transfer them in the
Petri dishes by gently positioning the seeds on the surface of
the agarized medium. To facilitate the transfer, touch the
surface of the agarized medium with either the toothpick or
the glass Pasteur pipette before picking up the seeds.
12. To better synchronize seed germination, after sowing the
plates can be placed in the dark at 4
C for 2 days, before
moving them to the growth chamber (stratification).
13. The volume of the liquid cell culture medium is always
one-fifth of the flask total volume, in order to ensure a suitable
aeration of the cell suspension culture.
14. Forceps and scalpel need to be often re-sterilized during these
operations. To do this, soak the tip of the metal labware in a
tube containing 100% (v/v) ethanol, then pass it on the flame
of the Bunsen burner until it is red-hot. Cool it down before
use. Pay extreme attention when you work with ethanol close
to the Bunsen burner flame. Alternatively, a glassbeads sterilizer can be used, following manufacturer’s instructions.
15. While transferring the callus portions, adopt the maximal care
to maintain sterile conditions. To minimize the risk of contamination, avoid inserting the forceps in the flask, but rather drop
each callus portion from the top of the flask mouth. Sterilize
repeatedly the forceps and the scalpel, as described in Note 14.
16. Cut the aluminum foil lid so that about 1–2 cm of it are left
around the mouth of the flask. This allows a better exposition
of the cell cultures to the light regime of the growth chamber.
17. You can either buy sterile wide-bore pipettes or obtain them
from standard pipettes, by manually removing the tip. To do
so, simply break with your hands the tip while the pipette is still
inside its sterile package, paying attention to maintain sterility.
18. In our hands this procedure is the fastest way to measure and
transfer 1 mL PCV at every subculturing step. For less experienced researchers it may be easier to determine the PCV by
transferring 5 mL of the cell suspension culture into a sterile
15 mL tube and centrifuging at 500 g for 2 min. The new
subculture will be started by inoculating the appropriate volume of the 7-day-old suspension culture, corresponding to
1 mL PCV, into 20 mL fresh culture medium.
19. When large volumes of suspension cell cultures are needed,
subculturing can be performed in larger flasks, containing a
larger volume of cell culture medium (e.g., 250 mL flasks,
containing 50 mL culture medium; 500 mL flasks, containing
100 mL culture medium; 1 L flasks, containing 200 mL culture
medium). Remember to respect the rule of the inoculum size
(1 mL PCV in 20 mL fresh medium).
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183
Petri dishes by gently positioning the seeds on the surface of
the agarized medium. To facilitate the transfer, touch the
surface of the agarized medium with either the toothpick or
the glass Pasteur pipette before picking up the seeds.
12. To better synchronize seed germination, after sowing the
plates can be placed in the dark at 4
C for 2 days, before
moving them to the growth chamber (stratification).
13. The volume of the liquid cell culture medium is always
one-fifth of the flask total volume, in order to ensure a suitable
aeration of the cell suspension culture.
14. Forceps and scalpel need to be often re-sterilized during these
operations. To do this, soak the tip of the metal labware in a
tube containing 100% (v/v) ethanol, then pass it on the flame
of the Bunsen burner until it is red-hot. Cool it down before
use. Pay extreme attention when you work with ethanol close
to the Bunsen burner flame. Alternatively, a glassbeads sterilizer can be used, following manufacturer’s instructions.
15. While transferring the callus portions, adopt the maximal care
to maintain sterile conditions. To minimize the risk of contamination, avoid inserting the forceps in the flask, but rather drop
each callus portion from the top of the flask mouth. Sterilize
repeatedly the forceps and the scalpel, as described in Note 14.
16. Cut the aluminum foil lid so that about 1–2 cm of it are left
around the mouth of the flask. This allows a better exposition
of the cell cultures to the light regime of the growth chamber.
17. You can either buy sterile wide-bore pipettes or obtain them
from standard pipettes, by manually removing the tip. To do
so, simply break with your hands the tip while the pipette is still
inside its sterile package, paying attention to maintain sterility.
18. In our hands this procedure is the fastest way to measure and
transfer 1 mL PCV at every subculturing step. For less experienced researchers it may be easier to determine the PCV by
transferring 5 mL of the cell suspension culture into a sterile
15 mL tube and centrifuging at 500 g for 2 min. The new
subculture will be started by inoculating the appropriate volume of the 7-day-old suspension culture, corresponding to
1 mL PCV, into 20 mL fresh culture medium.
19. When large volumes of suspension cell cultures are needed,
subculturing can be performed in larger flasks, containing a
larger volume of cell culture medium (e.g., 250 mL flasks,
containing 50 mL culture medium; 500 mL flasks, containing
100 mL culture medium; 1 L flasks, containing 200 mL culture
medium). Remember to respect the rule of the inoculum size
(1 mL PCV in 20 mL fresh medium).
Cell Suspension Cultures
183