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Preparation of C-Fern Nutrient Medium Solution  - Traditional - 6 Stock Solution - Method

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 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

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 appropriate volume (Table 1) of each stock solution TO about 800 ml distilled water in a volumetric flask and bring to a 1 l final volume. (Mix in each stock solution after addition by swirling flask.)

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 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 Modified Parker/Thompson's Basal Nutrient Medium
Nutrient Components
Stock Solution
(g / L)
Final Medium
(mL Stock / L)
Final Medium
Composition (mg/L)
Macronutirents
1
NH4NO3
25
5
125
2
KH2PO4
20
25
500
3
MgSO4×7H20
10
12
120
4
CaCl2×2H2O
13
2
26
5
Micronutrients
10
MnSO4×H20
0.025
0.25
CuSO4×5H2O
0.037
0.37
ZnSO4×7H2O
0.052
0.52
H3BO3
0.186
1.86
(NH4)6Mo7O24×4 H20
0.0037
0.037
6
Chelated Iron Solution
10
FeSO4×7H20
2.78
27.8
Disodium EDTA×2H2O
3.73
37.3
This formulation is based on a media described in Klekowski 1969 (Botanical Journal of the Linnean Society 62: 361-377).
 
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Friday, 28 November 2014