Basal Medium:
K2HPO4 .......................2.56 g
KH2PO4 .......................2.08 g
NH4Cl ........................1.0 g Mg
SO4 . 7H2O .................0.5 g
Agar (if needed)............20.0 g
Distilled water..............1.0 L
Adjust pH to 6.8. Filter-sterilize. If agar is desired, dissolve the salts in 500 ml water, adjust pH to 6.8 with NaOH and filter sterilize.
Dissolve agar in 500 ml water, boil to dissolve and autoclave at 121C for 15 minutes.
After cooling the agar solution to 56C and warming the salt solution to 56C, aseptically combine the two solutions to complete the basal medium.
Aseptically add Solutions A and B to the completed basal medium.
Solution A:
Ferric ammonium citrate......1.0 g
CaCl2 ........................0.1 g
Distilled water............100.0 ml
Filter-sterilize. Aseptically 5.0 ml to basal medium.
Solution B:
Succinic acid 1.0 M
Adjust pH to 6.0 with NaOH. Filter sterilize.
Aseptically add 15.0 ml to basal medium.