Erd-Schreiber 2X Medium

Supplemented seawater 1 L
P-IV metal solution 12 ml
NaNO3 solution (0.7 M) 3.3 ml
Na2HPO4 7H2O (0.02 M) 3.3 ml
Soilwater: GR+ Medium 50 ml
Vitamin B12 solution 1 ml

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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