Phycology Media – Plant Tissue Culture Protocol

Phycology media can be prepared from concentrated solutions or from powdered salt mixtures. The concentrated solutions are complete, including vitamins, and should be stored frozen. The powdered media allow flexibiltiy in customizing the final media composition. The powders to prepare growth media for aquatic species consist of "enrichment" salt mixtures and salt mixtures for completely synthetic media. The powders or concentrated solutions are added to freshwater or seawater to prepare enriched freshwater or enriched marine media, respectively. To prepare synthetic marine growth media the powder is added to tissue culture grade freshwater. Published formulas of growth media for aquatic species often include sodium bicarbonate. Sodium bicarbonate is not included in SIGMA's powdered mixtures and should be added during medium preparation if required for your application.

Preparation from Packaged Powder

Powdered media are extremely hygroscopic and must be protected from atmospheric moisture. If possible the entire contents of each package should be used immediately after opening. Preparing the medium in a concentrated form is not recommended as some salt complexes may precipitate. Supplements that are added to the medium may affect shelf life and storage conditions. The basic steps for preparing the culture medium are listed below:

  1. Measure out approximately 90% of the final required volume of freshwater or seawater, e.g. 9 L for a final volume of 10 L. Select a container twice the size of the final volume.
  2. While stirring the water add the powdered medium and stir until completely dissolved. Heating may be required to bring powders into solution.
  3. Rinse the original container with a small volume of water to remove traces of the powder. Add to the solution in Step 2.
  4. If required for your application, add Sodium Bicarbonate (NaHCO3) or other buffer; stir to dissolve. Add desired heat stable supplements (e.g. sucrose, agar, vitamins, auxins, cytokinins, etc.)
  5. Add additional water to bring the medium to the final volume.
  6. If necessary, adjust medium to desired pH using NaOH, HCl or KOH.
  7. If a gelling agent is used, heat until the solution is clear.
  8. Dispense the medium into the culture vessels before (or after) autoclaving according to your application. Add heat labile constituents after autoclaving.
  9. Liquid medium can be filter sterilized using a 0.22 µm filter or sterilized in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C, for the time period described under Sterilization of Media Protocol.
  10. Allow medium to cool prior to use.

POWDERED MEDIA AND BASAL SALT MIXTURES ARE FOR LABORATORY USE ONLY. NOT FOR DRUG, HOUSEHOLD OR OTHER USES.

Materials Not Provided

  • Seawater, plant cell culture tested (Product. No. S9148)
  • Deionized tissue culture grade water (Product No. W3500)
  • 1 N Hydrochloric Acid (HCl) (Product No. H9892)
  • 1 N Sodium Hydroxide (NaOH) (Product No. S2770)
  • Sodium Bicarbonate, plant cell culture tested (Product No. S5761)

Auxins, cytokinins, carbohydrates, gelling agents, and other supplements listed in the Biochemicals section.

Storage

Store dry medium in a desiccator at 0-5 °C. Deterioration of powdered medium may be recognized by: 1) color change; 2) granulation, clumping, or particulate matter throughout the powder; 3) pH change; or 4) inability to promote growth when properly used.

Precipitation in Media

Precipitates are known to occur, with time, in plant tissue culture media. The precipitates have been analyzed (Sigma, unpublished data; Dalton, et al. 1983). They are composed of small, pale yellow-white particles. Analysis of precipitates indicated a predominance of iron, phosphate, and zinc. The probable cause of the precipitates is the inevitable oxidation of ferrous ions to ferric ions and the presence of unchelated ferric ions. When the solubility of ferric phosphate is exceeded precipitation occurs. There are no reports of detrimental effects on growth and development in plant tissue culture due to the precipitates.

Media Composition Table

B5282 – Bold modified basal freshwater nutrient solution
C3061 – Cyanobacteria BG-11 freshwater solution (50X)
G0154 – Guillard's (f/2) marine water enrichment solution
G9903 – Guillard's (f/2) marine water enrichment solution

 

Component (mg/L) B5282 C3061 G0154 G9903
Ammonium chloride        
Biotin     0.005 0.005
Boric acid 11.42 2.86    
Calcium chloride dihydrate 25.0 36.0    
Citric acid   6.0    
Cobalt chloride • 6H2O     0.01 0.01
Cobalt nitrate • 6H2O 0.49 0.0494    
Cobalt sulfate • H2O        
Cupric sulfate • 5H2O 1.57 0.079 0.01 0.01
EDTA (free acid) 50.0      
EDTA disodium • 2H2O     4.36 4.36
EDTA disodium magnesium   1.0    
EDTA ferric sodium        
Ferric ammonium citrate   6.0    
Ferric chloride anhydrous        
Ferric chloride • 6H2O     3.15 3.15
Ferrous sulfate • 7H2O 4.98      
ß-Glycerophosphate disodium        
Magnesium sulfate • 7H2O 75.0 75.0    
Manganese chloride • 4H2O 1.44 1.81 0.18 0.18
Molybdenum trioxide 0.71      
Nickel chloride • 6H2O 0.003      
Potassium hydroxide 31.0      
Potassium iodide 0.003      
Potassium phosphate monobasic 175.0      
Potassium phosphate dibasic 75.0 40.0    
Sodium carbonate   20.0    
Sodium chloride 25.0      
Sodium metasilicate • 9H2O       15.0
Sodium molybdate • 2H2O   0.39 0.006 0.006
Sodium nitrate 250.0 1500.0 75.0 75.0
Sodium phosphate monobasic     4.411 4.411
Sodium selenite 0.002      
Stannic chloride 0.001      
Thiamine • HCl     0.1 0.1
Trizma base (TRIS)        
Vanadium sulfate • 3H2O 0.0022      
Vitamin B12     0.005 0.005
Zinc sulfate • 7H2O 8.82 0.222 0.022 0.022
  B5282 C3061 G0154 G9903
Grams of powder to prepare 1L n/a n/a n/a n/a
*Weight after vacuum-drying.

Materials