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Bold 3N Medium

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Bold 3N Medium

Modification of Bold's recipe. General purpose freshwater medium used for xenic cultures, especially blue-greens and reds.


For 1 L Total

  1. To approximately 850 mL of dH2O, add each of the components in the order specified (except vitamins) while stirring continuously.
  2. Bring the total volume to 1 L with dH2O.
    • For 1.5% agar medium add 15 g of agar into the flask; do not mix.
  3. Cover and autoclave medium.
  4. When cooled add Vitamin B12.
    • For agar medium add vitamin, mix, and dispense before agar solidifies.
  5. Store at refrigerator temperature.

# Component Amount Stock Solution Concentration Final Concentration
1 NaNO3
(Fisher BP360-500)
30 mL/L 10 g/400mL dH2O 8.82 mM
2 CaCl22H2O
(Sigma C-3881)
10 mL/L 1 g/400mL dH2O 0.17 mM
3 MgSO47H2O
(Sigma 230391)
10 mL/L 3 g/400mL dH2O 0.3 mM
4 K2HPO4
(Sigma P 3786)
10 mL/L 3 g/400mL dH2O 0.43 mM
5 KH2PO4
(Sigma P 0662)
10 mL/L 7 g/400mL dH2O 1.29 mM
6 NaCl
(Fisher S271-500)
10 mL/L 1 g/400mL dH2O 0.43 mM
7 P-IV Metal Solution 6 mL/L
8 Soilwater: GR+ Medium 40 mL/L
9 Vitamin B12 1 mL/L

Important Notes To Consider:

  • The quality of water, including natural seawater, is important. The term 'dH2O' generally refers to distilled, deionized, distilled/deionized water, Milli-Q water (Millipore Corp.), etc. Natural seawater and natural freshwater should be obtained from a non-polluted source.
  • When dissolving chemicals, wait for the first component to dissolve before adding the second. Stirring, and sometimes the addition of heat, is often required to dissolve the chemicals efficiently. Preparation of stock solutions for frequently made algal culture media make preparing media convenient but also necessary to avoid errors from weighing very tiny amounts.
  • Attention should be given to the pH of the final medium. In most cases, if pH adjustment is required, this occurs before sterilization. Please note that autoclaving removes carbon dioxide from media lacking carbonate stabilizing buffers. This would make the medium very alkaline soon after removal from the autoclave. In these cases, you should wait approximately 24 hours for gaseous equilibrium before inoculating the medium.