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
- Macro-, micronutrient and Fe salts (Table 1)
- Distilled or deionized water
- 1 L volumetric flask(s)
- Magnetic stir plate and stir bars
- For Fe stock solution: hot plate, 2 L erlenmeyer flasks, watch glass
- Storage bottle(s)
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 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
- 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 (BactoAgarÂ®)
- Magnetic Stir Plate and Magnetic Stir Bar
- pH Meter (for pH adjustment)
- 0.1 M NaOH Solution
- Foil (to cover flask during autoclaving)
- Sterile Petri Dishes, 60 x 15 mm
To prepare Basic C-Fern nutrient medium, add 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.
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 175Â° C/20 psi for 15 minutes. Dispense medium to petri dishes. Dishes should be about Â¾ full, i.e. about 15 mL in a 60-mm diameter Petri dish and 40 mL in a 100-Âmm 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.
|Table 1. Components and preparation of stock solutions and final nutrient solution of Basic C-Fern Medium|
(g / L)
(ml Stock / L)
|1 -- 10 X MACRONUTRIENTS||100|
|MgSO4 X 7H20||1.20||120|
|CaCl2 X 2H20||0.26||26|
|2 -- 200 X MICRONUTRIENTS||5|
|MnSO4 X H20||0.0500||0.25|
|CuSO4 X 5H20||0.0740||0.37|
|ZnSO4 X 7H20||0.1040||0.52|
|(NH4)6Mo7O24 X 4H20||0.0074||0.037|
|3 -- 100 X CHELATED IRON SOLUTION||10|
|FeSO4 X 7H20||2.78||27.8|
|Disodium EDTA X 2H20||3.37||37.3|
|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.|