Yuji Hiwatashi
A sporangium
contains thousands of spores. A solid medium containing 4-10 mM Ca is used for
spore germination.@@
EBCD+10 mM Ca (germination medium)F1000 ml
H2O |
900 ml |
Stock B |
10 ml |
Stock C |
10 ml |
Stock D |
10 ml |
Alternative
TES |
1 ml |
500mM Ammounim
tartrate |
10ml (= 5
mM) |
CaCl2
2H2O |
1.5g ( = 10
mM) |
Agar (Sigma
A6924) |
8 g ( =
0.8%) |
|
Fill up to 1000
ml with H2O |
After autoclaved,
pour into 9 cm-Petri dish.@
E Sterilized water
E Cellophane (autoclave)
– Wash as the follows
1) Cut
cellophanes to a little bit smaller than the size of a 9 cm-dish.
2) Place
cellophane in a glass Petri dish and then add 5 mM EDTA solution (pH8.0).
Autoclave.
3) Wash with
MilliQ water several times.
4) Add MilliQ
water in the glass plate, then autoclave
E Two tweezers (autoclave)
E Yellow tips and blue
tips for pipetman (autoclave)
E 10% Sodium Hypocholorite
Solution (Antiformin): Do not autoclave.
See 2.2 for the
Stock solutions.
@You should perform the following steps in
a cleanbench.
One
sporangium contains approximately thousands of spores. It may be better to see
the sporangium by microscope or magnifying glass to confirm it is not empty.
1. Overlay agar
medium with a sheet of cellophane. Do not include air between media and
cellophane.
2. Put one or
two sporangium in a 1.5ml microtube, then pour 1 ml of 10% Antiformin to
sterilize.@
3. Mix gently
for 5 min and discard supernatant using a pipetman. Usually the sporangium sink
at the bottom of tube, but be careful not to discard the sporangium until step
7. Do not touch the sporangium with a tip of a pipettman.@ Otherwise, tiny spores are dispersed in a
tube and it is not possible to recover.
4. Add 1ml of
sterilized water into the tube and mix gently for few minutes.
5. Discard
supernatant with a pipetman.
6. Repeat
step 4 and 5 four times.
7. Add 1ml of
sterilized water, crush the sporangium with the tip of a pipetman (yellow-tip),
and mix gently. When you crush the sporangium, you can see spores disperse in
the tube.
8. Pour 0.2ml
of the spore-solution at the center of the agar medium, which had been
overlayed with a cellophane.
9. Add 1 ml
of sterilized water to the drop of the spore-solution on the agar medium.
Spread the spore solution over the agar medium by gently shaking the plate.
10. Incubate
at 25˚C under continuous light.
11. Spores will germinate in a couple of days
and protonemata start to grow. After about 15 days, gametophores start to grow.