Preparation of C-Fern Basic Nutrient Solution using Powdered Nutrient Medium.
Materials
- Powdered Basic C-Fem Medium
- 1 Liter volumetric flask
- Distilled or deionized water
- 2 Liter Erlenmeyer Flask{s) (1 flask per liter of 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 the packet and gently add the 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). 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 ensure that the developing gametophytes have an adequate nutrient and water supply through to the young sporophyte stage. One liter of the 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.
