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 or Nacalai
Tesque cat. no. 01028-85) |
8 g ( = 0.8%) |
|
|
Fill up to 1000 ml with H2O |
After
autoclaving, pour into 9 cm-petri dish.@
E Sterilized
water
E Cellophanes
(autoclave). Cut cellophanes to a little bit smaller than the size of a plastic
9 cm-petri dish (the same one as used for E.
coli culture). Pre-cut cellophane discs (Type 325P) are available in bulk
from AA Packaging Ltd., Liverpool Road, Walmer Bridge, Preston, Lancashire PR4
5 HY, England. Futamura Chemical Co., LTD
provides free cellophane and please contact to Mr. Koichi Murayama
(koichi.murayama@starch.futamura.co.jp), although we are not sure they can send
the cellophane abroad. Dip about 50 layers of the cut cellophanes into
MilliQ water in a glass petri dish, then autoclave. These cellophanes are
overlain on agar medium and prevent protonemata from growing into the medium.@
E Two pairs
of tweezers (autoclave)
E Yellow
tips and blue tips for pipetman (autoclave)
E 10% Sodium
Hypocholorite Solution (Antiformin): Do not autoclave.
@You
should perform the following steps in a Clean bench / Laminar flow cabinet.
One
sporangium contains thousands of spores. It is recommended to inspect the
sporangium with a microscope or magnifying glass to ensure 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 sporangia in a 1.5ml microtube, then add 1 ml of 10% Antiformin to
sterilize.@
3. Mix
gently for 5 min and discard supernatant using a pipetman. Usually the
sporangium sinks to the bottom of the tube, but be careful not to discard the
sporangium until step 7. Do not touch the sporangium with a tip of a pipetman,
otherwise the tiny spores are dispersed in the tube and it is not possible to
recover them.
4. Add 1ml of
sterilized water into the tube and mix gently for a few minutes.
5.
Discard supernatant with a pipetman.
6.
Repeat steps 4 and 5 four times.
7. Add 1ml
of sterilized water, crush the sporangium with the tip of a pipetman (yellow
tip or blue tip), and mix gently. When you crush the sporangium, you can see
the spores disperse in the tube.
8.
Pour 0.2ml of the spore suspension onto the center of the cellophane-overlaid
agar medium.
9. Add 1
ml of sterilized water to the droplet of spore suspension on the agar medium.
Spread the spore suspension 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.