2.5　 Protonemata cultivation between two thin layers of agar-gelatin in liquid medium for high quality microscope observation

Yoshikatsu Sato

Materials

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

Procedure

1) Make a loop (5cm diameter) and a handle with a stainless-steel wire. ->sterilize with flames

2) Melt agar-gelatin solution with a microwave and pore into 6 cm Petri dish on the electric hot plate.

3) Dip the stainless loop into the 6 cm Petri dish and pull out slowly. Wait 10 second for setting a film of agar-gelatin.

4) Put the agar-gelatin film on a coverslip on a test tube.

5) Put a small piece of protonemal tissues on the coverslip.

6) Repeat 3 and cover the tissues with an new agar gelatin film.

7) To glue the protonema sandwich to a bottom of 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 with branching. If you prefer to grow protonemata without branching, cultivate for a week under unilateral red light (0.5 –1 Wm^-2).