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A+ Medium



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A+ Medium

Directions

For 1 L Total

Liquid media:
  1. To approximately 900 mL of dH2O add the first 8 components in the order specified while stirring continuously.
  2. Bring total volume to 1 L with dH2O.
  3. Cover and autoclave medium.
  4. Add filter sterilized A+ Trace Components.
  5. Allow to cool then store at refrigerator temperature.
Agar media:
  1. To approximately 390 mL of dH2O add the first 8 components in the order specified while stirring continuously.
  2. Bring total volume to 500 mL with dH2O.
  3. In a separate container add 15 g of agar to 500 mL of dH2O (Final 1.5% w/v).
  4. Cover and autoclave both solutions.
  5. In a water bath allow both solutions to cool to 45-50 °C.
  6. Add sterile Sodium Thiosulfate to agar solution and mix well.
  7. Add filter sterilized A+ Trace Components.
  8. Combine both agar and liquid solutions, mix well. Note the agar can solidify quickly.
  9. Allow to cool then store at refrigerator temperature.

# Component Amount Stock Solution Concentration Final Concentration
1 NaCl
(Fisher S271-500)
18 g/L 0.308 M
2 MgSO47H2O
(Sigma 230391)
5 g/L 0.02 M
3 Na2EDTA•2H2O
(Sigma ED255)
10 mL/L 0.3 g/100 mL 0.08 mM
4 KCl
(Fisher P 217)
10 mL/L 6 g/100 mL 8.05 mM
5 CaCl22H2O
(Sigma C-3881)
10 mL/L 3.7 g/100 mL 2.52 mM
6 NaNO3
(Fisher BP360-500)
10 mL/L 10 g/100 mL 11.8 mM
7 KH2PO4
(Sigma P 0662)
10 mL/L 0.5 g/100 mL 0.37 mM
8 Trizma Base pH 8.2 10 mL/L 10 g/100 mL 8.26 mM
9 A+ Trace Components (sterilize before adding) 10 mL/L
10 Sodium Thiosulfate Pentahydrate (agar media only,sterile)
(Baker 3946)
1 mL/L 24.819 g/100 mL 1 µM

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.