CFM Preparation

CFM Preparation – 4 Liters

1.     Make 4 Liters of FM following the FM Preparation protocol.

2.     Obtain 4 – 2800 mL fernbachs that have been acid treated.

3.     Place 1 liter of FM in each of the fernbachs.

4.     Place a foam stopper in the top and 3 pieces of aluminum foil over each fernbach.

5.     Place fernbachs in the autoclave in the sub-basement for 30 minutes at 121°C.

6.     Remove fernbachs and let cool to room temperature.

7.     Sterilize 200 mg of HER spores and place 50 mg in each fernbach.

8.     Grow these spores in the growth chambers located in room B014 for about a month.

9.     Filter the gamedophytes out with a large filter flask and a Büchner funnel.

10.  Transfer the filtrate from all 4 fernbachs to a 4 liter container.

11.  Add the following to the 4 liter container:

a. 2 package of MS salt.

b. 2 mL of Gamborgs Vitamin Soltuion 1000x

c. Large magnetic stir bar.

12.  Stir the solution with a magnetic stirrer until all of the salts are dissolved.

13.  Using a non-FM pH electrode, bring the solution up to 6.5 using 1N KOH.

14.  Pour the CFM into orange topped 1 liter bottles.

15.  Place the 1 liter bottles into the autoclave and autoclave for 30 minutes at 121°C.