To 1 L of supplemented seawater (60 ppt) aseptically add each of the sterile components in the order specificed. Vigorously swirl to mix thoroughly. Store at 4 C.

Supplemented seawater:

Pasteurized seawater 900 ml
NaCl 23 g
MgCl₂ 6H₂O 5 g
Na₂SO₄ 4 g

To approximately 900 ml of pasteurized seawater, add the components in the order specificed stirring cintinuously. Brign to 1 L with pasteurized seawater.

Pasteurized seawater:
Seawater with a salinity of at least 30 ppt is collected and pre-filtered. Seawater is diluted to 30 ppt with distilled water immediately prior to pasteurization. A batch of seawater in an Erlenmeyer flask (covered with a beaker) "pasteurized" in a steamer (over 95 C but less than 100 C) for 45 min. The seawater is allowed to cool and left undisturbed for 24 hr and then steam-pasteurized again for 45 min. The cooled medium is stored until used to prepare culture medium. The procedure should generally protect against precipitation and scratched or etched glassware should be avoided.

Soilwater: GR+ Medium:

Greenhouse soil 1 tsp
CaCO₃ 1 mg
Deionized water to 200 ml

Combine ingredients. Cover container and steam for 2 consecutive days, 3 h each day with gradually rising temperature to 95 C in 15 min, then to 98 C for 3 h. Cool at room temperature and store at 4 C.

P-IV metal solution:

Na₂EDTA 2H₂O 0.75 g
FeCl₃ 6H₂O 0.097 g
MnCl₂ 4H₂O 0.041 g
ZnCl₂ 0.005 g
CoCl₂ 6H₂O 0.002 g
Na₂MoO₄ 2H₂O 0.004 g
Deionized water to 1 L

To approximately 950 ml of dH₂O, add ingredients in order listed stirring continuously. The EDTA should be fully dissoled before adding other components. Bring to 1 L with dH₂O and store at 4 C.

Vitamin B₁₂ solution:

HEPES buffer 2.4 g
Vitamin B₁₂ 0.027 g
Deionized water to 200 ml

Prepare 200 ml of HEPES buffer at pH 7.8 and fully dissolve vitamin. Filter-sterilize and store in dark at -20 C.

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