Live Cell Imaging of Arabidopsis Root Hairs - Plant Cell Morphogenesis: Methods and Protocols - page 190

3. Immediately add sterile water and wash the seeds 3 times as
described above ( see Note 1 ). After the last wash, the seeds are
stored in sterile water.
4. If your seed stock requires so, seeds can be stratifi ed in the
dark at 4 °C before use. Sterilized seeds can be stored for at
least 2 weeks under these conditions.

3.2 Biofoil Sandwich
Preparation

Work under sterile conditions in a laminar air fl ow hood.

1. Place coverslips on the lid of a Petri dish and distribute approx-
imately 0.7 ml warm Hoagland's medium containing agarose
over the coverslip such that an area of at least 35 mm × 24 mm
is completely covered by the medium.
2. Once the medium has gellifi ed, place a sheet of Biofoil
(24 mm × 30 mm) on top of the medium so that the short side
aligns with the short side (24 mm) of the coverslip that has
been covered with medium.
3. With a sterile dissecting knife, cut off the medium that is not
covered by the Biofoil straight along the edge of the Biofoil
and remove the cutoff part. The Biofoil slide is now ready for
application of sterilized seeds.

3.3 Fixing the Biofoil
Sandwich in a Petri
Dish or Falcon Tube

Biofoil sandwiches can be placed either in a 110 mm Petri dish or
in a 50 ml Falcon tube.
1. In a Petri dish, make a support by sliding two 1 ml pipette tips
into each other. By cutting the pointed tip, the length of the
support is adjusted such that it is as wide as the Petri dish. The
Biofoil sandwich is then placed with the end not covered by
medium on the support so that its angle is approximately 20°
from the horizontal axis, and fi xed with an excess of warm, still
liquid Hoagland's medium with agarose. This prevents the
Biofoil sandwich from drying out and fi xes its position.
2. In a 50 ml Falcon tube, the Biofoil sandwich is simply inserted
with the end not covered with medium towards the top of the
tube. Due to the tube diameter, the angle of the Biofoil sand-
wich is approximately 70° from the horizontal axis. The bot-
tom of the tube is fi lled with liquid Hoagland's medium so
that the bottom of the Biofoil sandwich is just covered with
the medium when the tube is standing up straight.

3.4 Seed Application

Sterilized seeds are carefully placed on the cutoff edge of the
medium using a sterile Pasteur pipette. We use approximately ten
seeds per slide since in our hands root hair growth is better when
the seed density is not too low. Excess water is removed using a
Pasteur pipette.