Autoclave after all additions.

Alkaline Soil Extract:
In a flask add two parts by volume distilled water (dH₂O) to one part rich organic garden soil (containing no recent applications of chemical fertilizer or pesticides). Add 2-3 g NaOH/liter. Autoclave for 2 hours, and filter when cool. This concentrated extract is diluted 1:50 in dH₂O for the final working stock. (Seawater may substituted for dH₂O if the extract is to be used for marine species).

f/2 Trace Metal Solution:
FeCl₃ 6H₂O 3.15 g
Na₂EDTA 2H₂O 4.36 g
CuSO₄ 5H₂O (9.8 g/L dH₂O) 1.0 ml
Na₂MoO₄ 2H₂O (6.3 g/L dH₂O) 1.0 ml
ZnSO₄ 7H₂O (22.0 g/L dH₂O) 1.0 ml
CoCl₂ 6H₂O (10.0 g/L dH₂O) 1.0 ml
MnCl₂ 4H₂O (180.0 g/L dH₂O) 1.0 ml
Distilled water to 1.0 L

Autoclave.

f/2 Vitamin Solution:
Vitamin B₁₂ (1.0 g/L dH₂O) 1.0 ml
Biotin (0.1 g/L dH₂O) 10.0 ml
Thiamine HCl 200.0 mg
Distilled water to 1.0 L
Filter sterilize into plastic vials and store in refrigerator.

Note: Vitamin B₁₂ and Biotin are obtained in a crystalline form. When preparing the Vitamin B₁₂ Stock Solution allow for approximately 11% water of crystallization (For each 1.0 mg of Vitamin B₁₂ add 0.89 ml dH₂O). When preparing the Biotin Stock Solution allow for approximately 4% water of crystallization (For each 1.0 mg of Biotin add 9.6 ml dH₂O).

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