Nutrient Medium Preparation – 3 Stock Method

Preparation of C-Fern Basic Nutrient Medium – 3 Stock Solution Method.

This method has been modified from the traditional 6-Stock Solution method to make final nutrient solution preparation easier.

Preparation of Stock Solutions

Materials

• Macro-, micronutrient and Fe salts (Table 1)
• 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)

Methods
All components of basic C-Fern nutrient medium should be prepared using very high quality distilled or deionized water.

Prepare macronutrient and micronutrient stock solutions separately by dissolving all listed quantities of components, individually, in listed sequence, 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 the Chelated Fe-EDTA stock solution by dissolving each component separately into c. 450 ml distilled water. On a hot plate, heat the 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

Materials

• Macro-, micronutrient and Fe stock solutions
• Volumetric glassware: 100 ml graduated cylinder, 10 ml pipet, 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 Basic C-Fern Nutrient Medium, add the appropriate volume of each stock solution (Table 1) TO about 800 ml distilled water in a volumetric flask and bring to a 1 L final volume. Between each addition, swirl flask to mix the solution.

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 the 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.

Table 1. Components and preparation of stock solutions and final nutrient solution of Basic C-Fern Medium

This formulation is based on a media described in;