HEPES Medium



HEPES Medium Recipe

Directions 
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.

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)2·4H2O (Sigma C 5676) 1 mL/L 11.8 g/100 mL dH20 0.5 mM
2 MgSO4·7H2O (Sigma 230391) 1 mL/L 4 g/100 mL dH20 0.16 mM
3 Na2glycerophosphate.5H2O (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