R Medium Recipe

1. Dissolve 9.11 g of KH2PO4 in 916 ml distilled water to make solution 1.
2. Adjust pH to 6.0-6.5.
3. Autoclave to sterilize.
4. Dissolve the following in 200 ml distilled water to make solution 2:
   • HCL 0.80 ml
   • FeSO₄ x 7 H₂O 0.40 g
   • MgSO₄ x 7 H₂O 16.00 g
5. Filter sterilize
6. Dissolve the following in 10 ml distilled water to make solution 3:
   • CaCl₂ 1.11 g
7. Filter sterilize
8. Dissolve the following in 10 ml distilled water to make solution 4:
   • Glucose 2.00 g
9. Filter sterilize
10. Dissolve the following in 10 ml methanol to make solution 5:
    • Atrazine 1.00 g
11. You may have to shake vigrously to dissolve the atrazine in methanol. Concentration needs to be at a minimum of 100 ug/ml.
12. Filter sterilize.
13. Dissolve the following in 1 L distilled water to make trace elements solution:
    • NiCl₂ x 6 H₂O 2.00 mg
    • Na₂MoO₄ x 2 H₂O 3.00 mg
    • CuCl₂ x 2 H₂O 1.00 mg
    • CoCl₂ x 6 H₂O 20.00 mg
    • H₃BO₃ 30.00 mg
    • MnCl₂ x 4 H₂O 3.00 mg
    • ZnSO₄ x 7 H₂O 10.00 mg
14. To make the final medium aseptically combine the following:
    • 916 ml Solution 1
    • Solution 2 5.00 ml
    • Solution 3 0.20 ml
    • Solution 4 10.00 ml
    • Solution 5 1.00 ml
    • Trace element solution 1.00 ml