Preparation of C-Fern Basic Nutrient Solution using Powdered Nutrient Medium.

Materials 

  • Powdered Basic C-Fem Medium
  • 1- L volumetric flask
  • Distilled or deionized water
  • 2-L Erlenmeyer Flask{s) (1 flask per L medium)
  • Agar (BactoAgar@)
  • Magnetic Stir Plate and Magnetic Stir Bar
  • pH Meter (for pH adjustment)
  • 0.1 M NaOH Solution
  • Foil (to cover flask during autoclaving)
  • Autoclave
  • Sterile Petri Dishes, 60 x 15 mm

Methods

To prepare 1 L of basic C-Fem medium, carefully open packet and gently add powder to about 800 mL distilled water in a volumetric flask. Rinse out the powder remaining in the packet with distilled water, stir until powder is completely dissolved, then bring to a final volume of 1 L.

For agar-solidified medium, transfer the solution to 2-L Erlenmeyer flask and add 10 g (1 % w/v) agar (BactoAgar@-Difco Laboratories). (Note: Some plant tissue culture grade agars and agar substitutes can result in inhibited or abnormal growth of gametophytes or sporophytes and should be avoided. )

Adjust nutrient medium to pH 6.0 using 0.1 M NaOH. Prior to autoclaving, cap Erlenmeyer flask with aluminum foil. Autoclave nutrient medium at 175° C/20 psi for 15 minutes. Dispense medium to petri dishes. Dishes should be about ¾ full, i.e. about 15 mL in a 60-mm diameter Petri dish and 40 mL in a 100-­mm diameter Petri dish. A large volume helps insure that the developing gametophytes have an adequate nutrient and water supply through to the young sporophyte stage. One liter of medium should pour about 55 60-mm dishes and 20 100-mm dishes. Allow dishes to cool completely undisturbed. Condensation on petri dish lids is minimized if dishes are poured and cooled in stacks.