|Nutrient Medium Preparation - Traditional Method|
Preparation of C-Fern Nutrient Medium Solution - Traditional - 6 Stock Solution - Method
Preparation of Stock Solutions
All components of basic C-Fern nutrient medium should be prepared using high quality distilled and/or deionized water.
Prepare individual macronutrient and micronutrient stock solutions separately by dissolving all listed quantities of components into about 800 ml distilled water then bring to a 1 l final volume. Macronutrient and micronutrient stock solutions can be autoclaved. Autoclaved stock solutions will keep for over 6 months and should be stored in glass at 4 C.
Prepare Chelated Fe-EDTA stock solution by dissolving each component separately into c. 450 ml water. On a hot plate, heat EDTA solution to boiling, add hot EDTA solution TO the FeSO4 solution. Boil combined solutions for 1 hour, cool completely, then bring to 1 liter volume. Store Chelated Fe-EDTA solution in glass at 4 C.
Preparation of Final Nutrient Medium
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 gtowth 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 youngstage. 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.
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