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Preparation of Agar-Solidified Basic C-Fern Nutrient Medium From Stock Solutions

Materials for Preparation of Stock Solutions:

• Macro-, micronutrient and Fe salts (Refer to Table 1 for listing)
• Distilled or deionized water
• 1 l volumetric flask(s)
• Microbalance
• Magnetic stir plate and stir bars
• For Fe stock solution: hot plate, 2 l erlenmeyer flasks, watch glass
• Storage bottle(s)

(Refer to Table 1 below) All components of basic C-Fern nutrient medium should be prepared using high quality distilled and/or deionized water. Prepare macronutrient and micronutrient stock solutions separately by dissolving all listed quantities of components , individually, in sequence, into about 800 ml distilled water; bring to a 1 l final volume. Both 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.

Materials for Preparation of Final Medium:

• Macro-, micronutrient and Fe stock solutions
• Volumetric glassware: 100 ml graduated cylinder, 10 ml pipet, 1 l volumetric flask
• Distilled or deionized water
• 2l erlenmeyer flask (per 1 l media)
• Agar P7-79-6200, P7-79-6201 -- BactoAgar
• For pH adjustment: pH meter, magnetic stir plate, magnetic stir bar, 0.1 N NaOH solution
• Foil (to cover flask during autoclaving)
• Autoclave
• Sterile Petri dishes, 60x15 mm P7-74-1346.

(Refer to Table 1 below) To prepare Basic C-Fern nutrient medium, add appropriate volume of each stock solution TO about 800 ml distilled water in a volumetric flask and bring to a 1 l final volume. For agar-solidified medium, transfer nutrient solution to 2 l Erlenmeyer flask, 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 therefore be avoided. Adjust nutrient medium to pH 6.0 using 0.1 M NaOH. Autoclave nutrient medium at 120 C / 20 psi for 15 minutes. Dispense medium to Petri dishes. Dishes should be about ¾ full -- about 15 ml in a 60x15 mm dish and 40 ml in 100 mm dish. This ensures an adequate nutrient and water supply through to the young sporophyte stage. One liter of nutrient medium should pour about 55 60x15 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.

Table 1. Composition of Basic C-Fern Medium Stock Solution
and Final Medium Nutrient   

This formulation is based on a media described in Klekowski 1969 (Botanical Journal of the Linnean Society 62: 361-377). Higher concentrations of macronutrients in the stock solution are unstable and may form precipitates as will most combinations of macronutrient, micronutrient and chelated iron stock solutions.