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HEPES Medium



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HEPES Medium

Modification of the original recipe that changed the buffer of volvox medium to accommodate xenic cultures of LB 1662 Glaucosphaera vacuolata. Alkaline medium suitable for axenic and xenic cultures, especially LB 2411 Euglena sp. and LB 1333 Pithophora sp.

Directions

For 1 L Total
  1. To approximately 950 mL of dH2O, add each of the components in the order specified (except vitamins) while stirring continuously.
  2. Adjust the pH to 8.2.
  3. 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.
  4. Cover and autoclave medium.
  5. When cooled add vitamins.
    • For agar medium add vitamins, mix, and dispense before agar solidifies.
  6. Store at refrigerator temperature.

# Component Amount Stock Solution Concentration Final Concentration
1 Ca(NO3)24H2O
(Sigma C 5676)
1 mL/L 11.8 g/100 mL dH20 0.5 mM
2 MgSO47H2O
(Sigma 230391)
1 mL/L 4 g/100 mL dH20 0.16 mM
3 Na2glycerophosphate5H2O
(Sigma G 6501 )
1 mL/L 5 g/100 mL dH20 0.16 mM
4 KCl (Fisher P 217) 1 mL/L 5 g/100 mL dH20 0.67 mM
5 HEPES buffer
(Sigma H-3375)
0.94 g/L 3.9 mM
6 P-IV Metal Solution 6 mL/L
7 Vitamin B12 1 mL/L
8 Biotin Vitamin Solution 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.