Shewanella algae Simidu et al. emend. Nozue

1. Open vial according to enclosed instructions or visit www.atcc.org for instructions.
2. Rehydrate the entire pellet with approximately 0.5 mL of #2 broth. Aseptically transfer the entire contents to a 5-6 mL tube of #2 broth. Additional test tubes can be inoculated by transferring 0.5 mL of the primary broth tube to these secondary tubes.
3. Use several drops of the primary broth tube to inoculate a #2 plate and/or #2 agar slant.
4. Incubate at 30°C for 24 hours.

To demonstrate anaerobic growth by dissimilatory iron (III) reduction, use ATCC Medium #1931 broth.

1931 Broth:  Ferric citrate Medium

Ferric citrate, 13.7 g

NaHCO₃, 2.5 g

KCl, 0.1 g

NH₄Cl, 1.5 g

NaH₂PO₄, 0.6 g

Wolfe's Vitamin Solution (see below), 10.0 mL

Wolfe's Mineral Solution (see below), 10.0 mL

Sodium lactate, 30.0 mM

Distilled water, 1.0 L

Boil ferric citrate in distilled water to dissolve; cool to room temperature.  Adjust to pH 6.6 with 10N NaOH.  Add remaining components.  Equilibrate medium under 80% N₂, 20% CO₂.  Dispense medium anaerobically under same gas phase.  Autoclave anaerobically at 121^oC for 15 minutes.  Final pH 7.0.

Wolfe's Vitamin Solution:

Biotin, 2.0 mg

Folic acid, 2.0 mg

Pyridoxine HCl, 10.0 mg

Thiamine HCl, 5.0 mg

Riboflavin, 5.0 mg

Nicotinic acid, 5.0 mg

Calcium D-(+)-pantothenate, 5.0 mg

Vitamin B₁₂, 0.1 mg

p-Aminobenzoic acid, 5.0 mg

Thioctic acid, 5.0 mg

Distilled water, 1.0 L

Wolfe's Mineral Solution:

Nitrilotriacetic acid, 1.5 g

MgSO₄^.7H₂O, 3.0 g

MnSO₄^.H₂O, 0.5 g

NaCl, 1.0 g

FeSO₄^.7H₂O, 0.1 g

CoCl₂^.6H₂O, 0.1 g

CaCl₂, 0.1 g

ZnSO₄^.7H₂O, 0.1 g

CuSO₄^.5H₂O, 0.01 g

AlK(SO₄)₂^.12H₂O, 0.01 g

H₃BO₃, 0.01 g

Na₂MoO₄^.2H₂O, 0.01 g

Distilled water, 1.0 L

Add nitrilotriacetic acid to approximately 500 mL of water and adjust to pH 6.5 with KOH to dissolve the compound. Bring volume to 1.0 L with remaining water and add remaining compounds one at a time.

Additional information on this culture is available on the ATCC web site at www.atcc.org.

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