2.5@ Protonemata cultivation between two thin
layers of agar-gelatin in liquid medium for high quality microscope observation
1) Stainless-steel
wire
2) 6 cm Petri
dish@@@@@@@@@@ -> sterilize in dry oven
3) Coverslips
@@@@@@@@@@@@ -> sterilize in dry oven
4) 0.5% BACTO-agar
(w/v) and 0.05% gelatin (w/v) solution@ ->
Autoclave
5) BCDAT
liquid medium@@@ -> Autoclave
6) Electric
hot plate
7) Test tube
8) 3.5 cm
sterilized Petri dishes
1) Make a
loop (5cm diameter) and a handle with a stainless-steel wire. –
Flame-sterilize.
2) Melt
agar-gelatin solution with a microwave and pour into a 6 cm Petri dish on the
electric hot plate.
3) Dip the
stainless-steel loop into the 6 cm Petri dish and pull out slowly. Wait 10
seconds for a film of agar-gelatin to set.
4) Put the
agar-gelatin film on a coverslip on a test tube.
5) Put a
small piece of protonemal tissue on the coverslip.
6) Repeat 3
and cover the tissues with a new agar gelatin film.
7) To glue
the protonemal sandwich to the bottom of the Petri dish, drop agar-gelatin
solution (50 µl) on the bottom of a 3.5 cm Petri dish and place the
preparation on the agar-gelatin drop.@
Wait a few minutes for solidifying the agar-gelatin solution.
8) Pour 4 ml
BCDAT liquid medium into the 3.5 cm Petri dish slowly.
9) Under
white light, you will get regular protonemata that show branched growth. If you
prefer to grow protonemata without branching, cultivate for a week under
unilateral red light (0.5 –1 Wm-2).