Biology Reference
In-Depth Information
2
Materials
1. Biofoil, cut in rectangles of 24 mm × 30 mm and autoclaved in
a dry beaker covered with aluminum foil. Biofoil is a gas per-
meable foil. Currently it is available from Sarstedt (Lumox foil
25 305 mm × 5 m, product number 94.607317). Besides gas
permeability, Biofoil has several properties that make it well
suitable for microscopy; it is transparent for visible light, and it
is non-refl ective and not fl uorescent. As an alternative to
Biofoil, we have experimented with standard household foil
(cling fi lm), which has been working well in our trails.
2. Hoagland's medium (Hoagland modifi ed basal salt mixture;
PhytoTechnology Laboratories, KS, USA;
http://www.phy-
totechlab.com
) supplemented with 1 % v/w sucrose, pH 5.7,
and the same medium complemented with 0.7 % w/v agarose;
both autoclaved, the liquid medium at room temperature and
the medium with agarose at approximately 60 °C.
3. 70 % v/v ethanol in water.
4. 10-15 % household bleach (4 % sodium hypochlorite) in water
with 0.01 % detergent (both Triton X-100 and Tween 20
work fi ne), freshly prepared.
5. Sterile water.
6. Sterilized (autoclaved if possible) and air-dried (under sterile
conditions) consumables: coverslips (50 mm × 24 mm), Petri
dishes (110 mm diameter) or 50 ml Falcon tubes, sterile dis-
secting knife, blue (1.00 ml) pipette tips, Pasteur pipettes.
3
Methods
3.1 Seed
Sterilization
The method described below should be used for small amounts of
seeds only. The seed volume will increase during the procedure and
should not exceed one third of the total volume at any time. The
method can easily be scaled up using a larger container for the
seeds, for example, a 50 ml Falcon tube.
1. Place a small amount of seeds in an Eppendorf tube and fi ll the
tube with 70 % ethanol. Mix thoroughly but carefully; no air
bubbles should form. Allow the seeds to sediment and remove
the ethanol by pipetting within 1 min after application.
2. Immediately fi ll the tube with diluted bleach solution and mix
carefully. Incubate for maximally 5 min and rotate the
Eppendorf tube frequently. Ensure that there are no seeds
sticking to the tube surface above the bleach solution and
avoid bubble formation as much as possible. Allow the seeds
to sediment and remove the bleach solution by pipetting.
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