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. – 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).