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sterile) (Baker 3946) . 3. # Component Amount Stock Solution Final Concentration Concentration NaNO3 (Fisher BP360- 1 10 mL/L 30 g/200 mL dH2O 17. To approximately 400 mL of dH2O add the first 9 components in the order specified while stirring continuously.002 mM ED255) 8 Na2CO3 (Baker 3604) 10 mL/L 0.12 g/200 mL dH2O 0.03 mM A 104) Ammonium Ferric 6 10 mL/L 0.12 g/200 mL dH2O 0. To approximately 900 mL of dH2O add the first 9 components in the order specified while stirring continuously.03 mM 230391) CaCl2·2H2O (Sigma C- 4 10 mL/L 0. 2. 2. Bring total volume to 1 L with dH2O.4 g/200 mL dH2O 0.02 g/200 mL dH2O 0. 5. 4. Cover and autoclave both solutions.72 g/200 mL dH2O 0.6 mM 500) 2 K2HPO4 (Sigma P 3786) 10 mL/L 0.  Improved recipe as of March 2009 For 1 L Total Liquid media: 1. Combine both agar and liquid solutions.18 mM BG-11 Trace Metals 9 1 mL/L Solution Sodium Thiosulfate 10 Pentahydrate (agar media 24. In a separate container add 15 g of agar to 500 mL of dH2O.8 g/200 mL dH2O 0. Add sterile Sodium Thiosulfate to agar solution and mix well.2 mM 3881) Citric Acid·H2O (Fisher 5 10 mL/L 0. mix well. 4. Note the agar can solidify quickly.    1. Allow to cool then store at refrigerator temperature. 8. 3. Cover and autoclave medium. 6. 7.02 mM Citrate Na2EDTA·2H2O (Sigma 7 10 mL/L 0.22 mM MgSO4·7H2O (Sigma 3 10 mL/L 0.8 g/100 mL 1 mM only.15 g/200 mL dH2O 0. Allow to cool then store at refrigerator temperature. Bring total volume to 500 mL with dH2O. In a water bath allow both solutions to cool to 45-50°C.

5 M 230391) 3 KCl (Fisher P 217) 10 mL/L 6 g/100 mL dH20 8M NaNO3 (Fisher BP360- 4 10 mL/L 10 g/100 mL dH20 11. suitable for other axenic marine cultures (salinity = 30 ppt). 4.17 µM (Mallinckroft 4544) Artificial Seawater Medium Directions Modification of Brand's ASP2 medium for 2268 Agmenellum quadruplicatum.6 µM Baker 3764) CuSO4·5H2O (MCIB 5 0. 6.c  Trace Metals Solution Directions For 1 L Total 1. 4 0. *For agar medium: add vitamin.39 g/L 1. do not mix. 3. # Component Amount Stock Solution Final Concentration Concentration 1 NaCl (Fisher S271-500) 18 g/L 0.8 M 500) .22 g/L 0.4 mg/L 0.31 M MgSO4·7H2O (Sigma 2 10 mL/L 26 g/100 mL dH20 10.81 g/L 9 µM 2540) ZnSO4·7H2O (Sigma Z 3 0. 5. # Component Amount Stock Solution Final Concentration Concentration 1 H3BO3 (Baker 0084) 2. Adjust the pH to 8. Cover and autoclave medium.T. add each of the components in the order specified (except vitamins) while stirring continuously. *For 1. To approximately 850 mL of dH2O. Allow to cool and add vitamin B12. mix. For 1 L Total 1. Store at refrigerator temperature. 2. and dispense before agar solidifies.1. 2.77 µM 0251) Na2MoO4·2H2O (J. Store at refrigerator temperature.3 µM 3M11) Co(NO3)2·6H2O 6 49. Bring total volume to 1 L with dH2O. 3.86 g/L 46 µM MnCl2·4H2O (Baker 2 1.079 g/L 0. To approximately 900 mL of dH2O add the components in the order specified while stirring continuously.5% agar medium: add 15 g of agar to the flask. Bring the total volume to 1 L with dH2O.

To approximately 70 mL of dH2O.4 mg/100 mL 0.29 M Tricine (adjust to pH 8) 7 20 mL/L 22.002 mM 3169) Chelated ron Solution Directions This solution requires the combination of two liquid components: Component A: 1. Add 0. add each of the components in the order specified while stirring continuously. 2. Bring the total volume to 500 mL with dH2O.114 g/100 mL 1.5 M 500) 11 Vitamin B12 1 mL/L P Metal Solution Directions For  mL Total 1. Bring total volume to 100 mL with dH2O. # Component Amount Stock Solution Final Concentration Concentration Na2EDTA·2H2O (Sigma 1 0.1 g/100 mL 0. Note: CoCL2 can be interchancable with CoSO4. 2.0 g of Na2EDTA to the stirring beaker.2 mg/100 mL 0.4 g/100 mL dH20 25 M (Sigma T-5816-256) 8 P-II Metal Solution 10 mL/L 9 Chelated Iron Solution 1 mL/L NH4Cl (Fisher A 649- 10 1 mL/L 2. 3.6H2O to beaker.007 mM 0251) CoCl2·6H2O (Sigma C- 6 0.7 g/100 mL dH20 0. 3. Component c: 1. 3.5 g/100 mL dH20 0. 2.9 mg/100 mL 0. bring the total volume to 500 mL with dH2O.48 mg/100 mL 0. After the EDTA fully dissolves. Pour approximately 450 mL dH2O into a beaker. Pour approximately 450 mL 0.8 mM FeCl3·6H2O (Sigma 3 4.1 M HCL into a beaker. CaCl2·2H2O (Sigma C- 5 10 mL/L 3 g/100 mL dH20 2M 3881) 6 KH2PO4 (Sigma P 0662) 10 mL/L 0. .81 g of FeC3.018 mM 1513) MnSO4·H2O (Sigma 4 16.097 mM M8179) ZnSO4·7H2O (Sigma Z 5 2. Add 10.27 mM ED255) 2 H3BO3 (Baker 0084) 0. Store at refrigerator temperature. Heat the beaker in the microwave oven to near boiling.

** The amount of vitamins added can vary from medium to medium so the final concentration is not listed. (Sigma 0.027 g/200 mL dH2O V-6629) cristol Medium Directions H. For 1 L Total 1.17 mM . 4.1M HCL 450 mL 0. LDM. Prepare 200 mL of HEPES buffer (50 mM).1 mM) wait until fully dissolved. 2. Store at refrigerator temperature.5% agar medium add 15 g of agar into the flask.45 µm Millipore filter.94 mM 500) 2 CaCl2·2H2O (Sigma C. Soil extract. Bring total volume to 1 L with dH2O. Proteose. 10 mL/L 1 g/400mL dH2O 0. Cover and autoclave medium. 3. # Component Amount Stock Solution Final Concentration Concentration NaNO3 (Fisher BP360- 1 10 mL/L 10 g/400mL dH2O 2. Add Vitamin B12 (0. *For 1. # Component Amount Stock Solution Final Concentration Concentration Na2EDTA·2H2O (Sigma 1 10 g/500 mL 26 mM ED255) 2 0. 4. To approximately 900 mL of dH2O add each of the components in the order specified while stirring continuously. Bristol-NaCl. 2. and Trebouxia. Adjust the pH to 7. # Component Amount Stock Solution Final Concentration Concentration HEPES buffer pH 7.4 g/200 mL dH2O (Sigma H-3375) Vitamin B12 2 (cyanocobalamin. 3. Bold's modification of Bristol's recipe (Bold 1949).C. an essential component of other media--see Bold 1NV. Bold 3N.8 1 2. Store in dark at freezer temperature.81 g/500 mL 3 mM 1513) Vitamin c Directions For 200 mL Total 1. General purpose freshwater medium and as bristol's solution.8. do not mix.1 M FeCl3·6H2O (Sigma 3 0.Slowly add Component B to the heated Component A while stirring. Sterilize by 0.

Cover and autoclave medium. mix well. # Component Amount Stock Solution Final Concentration Concentration 1 NaCl (Fisher S271-500) 18 g/L 0. In a separate container add 15 g of agar to 500 mL of dH2O (final 1. 2. 4. To approximately 390 mL of dH2O add the first 8 components in the order specified while stirring continuously. Cover and autoclave both solutions. 3881) MgSO4·7H2O (Sigma 3 10 mL/L 3 g/400mL dH2O 0. Add sterile Sodium Thiosulfate to agar solution and mix well.308 M MgSO4·7H2O (Sigma 2 5 g/L 0.29 mM 6 NaCl (Fisher S271-500) 10 mL/L 1 g/400mL dH2O 0.3 mM 230391) 4 K2HPO4 (Sigma P 3786) 10 mL/L 3 g/400mL dH2O 0.3 g/100 mL 80. 5.5% w/v).5 g/100 mL 0. 9.05 mM CaCl2·2H2O (Sigma C- 5 10 mL/L 3.02 M 230391) Na2EDTA·2H2O (Sigma 3 10 mL/L 0. 2.43 mM A+ Medium Directions For 1 L Total Liquid media: 1. 6. Combine both agar and liquid solutions.2 10 mL/L 10 g/100 mL 8. Bring total volume to 500 mL with dH2O.7 g/100 mL 3. To approximately 900 mL of dH2O add the first 8 components in the order specified while stirring continuously. 3.8 mM 500) 7 KH2PO4 (Sigma P 0662) 10 mL/L 0. 4. 5.43 mM 5 KH2PO4 (Sigma P 0662) 10 mL/L 7 g/400mL dH2O 1.26 mM A+ Trace Components 9 10 mL/L (sterilize before adding) .6 µM ED255) 4 KCl (Fisher P 217) 10 mL/L 6 g/100 mL 8. Bring total volume to 1 L with dH2O. Add filter sterilized A+ Trace Components. Allow to cool then store at refrigerator temperature.33 mM 3881) NaNO3 (Fisher BP360- 6 10 mL/L 10 g/100 mL 11. 3.36 mM 8 Trizma Base pH 8. 7. Allow to cool then store at refrigerator temperature. Add filter sterilized A+ Trace Components. Note the agar can solidify quickly. 8. Agar media: 1. In a water bath allow both solutions to cool to 45-50 °C.

2 µM 2540) CuSO4·5H2O (MCIB 7 1 mL/L 0. Sodium Thiosulfate 10 Pentahydrate (agar media 1 mL/L 24.55 mM 2 ZnCl2 (Sigma Z-0152) 0.sterile) (Baker 3946) A+ Trace Components (sterilize before adding) Directions For 1 L Total 1.3 g/100 mL 34.012 g/100 mL 9.89 g/100 mL 24 µM 1513) MnCl2·4H2O (Baker 6 1 mL/L 4. away from bright light.4 nM 3169) .8 nM 3M11) CoCl2·6H2O (Sigma C- 8 1 mL/L 0.003 g/100 mL 18. 2. # Component Amount Stock Solution Final Concentration Concentration 1 H3BO3 (Baker 0084) 3.03 mg/L 0. To approximately 900 mL of dH2O add the components and stock solutions in the order specified while stirring continuously.819 g/100 mL 1 µM only.95 nM V-6629) FeCl3·6H2O (Sigma 5 1 mL 3.17 µM Vitamin B12 4 (cyanocobalamin.315 mg/L 2. (Sigma 0.43 g/L 0.004 mg/L 2.31 µM 3 MoO3(85%) 0. Bring total volume to 1 L with dH2O at refrigerator temperature.