May 30, 2023
SJB Artificial Seawater Medium Protocol
- 1University of Southern California
Protocol Citation: Shelby J Barnes, Michael Henson, Cameron Thrash 2023. SJB Artificial Seawater Medium Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzy615lx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 14, 2022
Last Modified: May 30, 2023
Protocol Integer ID: 60802
Keywords: Artificial Seawater
Abstract
SJB Artificial Seawater Medium Protocol
SJB Artificial Seawater Medium is an amended version of JW and MWH generations of Thrash Lab artificial seawater medium.
Materials
Sterilized Pyrex Bottle
0.2um PES filters
Base Salts
| A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | |
| Salinity | 1 | 2 | 3 | 4 | 4.25 | 4.5 | 5 | ||||||||||
| Base Salts | Chemical Formula | Formula Weight | g/L | Final M | g/L | Final M | g/L | Final M | g/L | Final M | g/L | Final M | g/L | Final M | g/L | Final M | |
| sodium chloride | NaCl | 58.443 | 23.5 | 0.402101 | 15.7 | 0.268067 | 7.83333 | 0.134033667 | 3.916666667 | 0.067016833 | 3.012820513 | 0.05155141 | 2.008547009 | 0.034367607 | 1.004273504 | 0.017183803 | |
| potassium chloride | KCl | 74.55 | 0.746 | 0.010007 | 0.497 | 0.006671333 | 0.248666667 | 0.003335667 | 0.124333333 | 0.001667833 | 0.095641026 | 0.001282949 | 0.063760684 | 0.000855299 | 0.031880342 | 0.00042765 | |
| sodium bicarbonate | NaHCO3 | 84.01 | 0.84 | 0.009999 | 0.84 | 0.006666 | 0.42 | 0.003333 | 0.21 | 0.0016665 | 0.161538462 | 0.001281923 | 0.107692308 | 0.000854615 | 0.053846154 | 0.000427308 | |
| sodium sulfate | Na2SO4 | 142.04 | 4.27 | 0.030062 | 2.85 | 0.020041333 | 1.423333333 | 0.010020667 | 0.711666667 | 0.005010333 | 0.547435897 | 0.003854103 | 0.364957265 | 0.002569402 | 0.182478632 | 0.001284701 | |
| sodium bromide | NaBr | 102.89 | 0.83 | 0.008067 | 0.55 | 0.005378 | 0.276666667 | 0.002689 | 0.138333333 | 0.0013445 | 0.106410256 | 0.001034231 | 0.070940171 | 0.000689487 | 0.035470085 | 0.000344744 | |
| boric acid | H3BO3 | 61.83 | 0.026 | 0.000421 | 0.017 | 0.000280667 | 0.008666667 | 0.000140333 | 0.004333333 | 7.01667E-05 | 0.003333333 | 5.39744E-05 | 0.002222222 | 3.59829E-05 | 0.001111111 | 1.79915E-05 | |
| strontium chloride | SrCl | 158.53 | 0.0142 | 0.00009 | 0.009467 | 0.00006 | 0.004733333 | 0.00003 | 0.002366667 | 0.000015 | 0.001820513 | 1.15385E-05 | 0.001213675 | 7.69231E-06 | 0.000606838 | 3.84615E-06 | |
| sodium flouride | NaF | 41.99 | 0.0023 | 0.000055 | 0.0015 | 3.66667E-05 | 0.000766667 | 1.83333E-05 | 0.000383333 | 9.16667E-06 | 0.000294872 | 7.05128E-06 | 0.000196581 | 4.70085E-06 | 9.82906E-05 | 2.35043E-06 | |
| magnesium chloride heptahydrate | MgCl2 x 6H2O | 203.3 | 10.6 | 0.05214 | 7.1 | 0.03476 | 3.533333333 | 0.01738 | 1.766666667 | 0.00869 | 1.358974359 | 0.006684615 | 0.905982906 | 0.00445641 | 0.452991453 | 0.002228205 | |
| calcium chloride dihydrate | CaCl2 x 2H2O | 147.01 | 1.52 | 0.010339 | 1.013 | 0.006892667 | 0.506666667 | 0.003446333 | 0.253333333 | 0.001723167 | 0.194871795 | 0.001325513 | 0.12991453 | 0.000883675 | 0.064957265 | 0.000441838 |
Table 1. Base Salts
The range of salinities are shown in order of highest (#1) to lowest (#5).
Trace Metal Mix (100,000x Stock)
| A | B | C | D | E | |
| Compound | Chemical Formula | FW | g/100mL | Final M (nM) | |
| manganese dichloride tetrahydrate | MnCl2 x 4H2O | 197.91 | 0.018 | 9 | |
| zinc sulfate monohydrate | ZnSO4 X H2O | 179.47 | 0.002 | 1 | |
| cobalt(II) chloride | CoCl2 | 129.839 | 0.001 | 0.5 | |
| sodium molybdate | Na2MoO4 | 205.92 | 0.001 | 0.3 | |
| sodium selenite | Na2SeO3 | 172.94 | 0.002 | 1 | |
| nickel(II) chloride | NiCl2 | 129.5994 | 0.001 | 1 |
Vitamin Mix (100,000x)
| A | B | C | D | E | |
| Compound | Chemical Formula | FW | g/100mL | Final M (nM) | |
| B1/thiamine | C12H17ClN4OS · HCl | 337.27 | 1.69 | 500 nM | |
| B2/riboflavin | C17H20N4O6 | 376.36 | 0.0026 | 0.7 nM | |
| B3/niacin | C6H5NO2 | 123.12 | 0.985 | 800 nM | |
| B5/pantothenate | HOCH2C(CH3)2CH(OH)CONHCH2CH2CO2 ·1/2Ca | 238.27 | 1.013 | 425 nM | |
| B6/pyridoxine | C8H11NO3 · HCl | 205.64 | 1.028 | 500 nM | |
| B7/biotin | C10H16N2O3S | 244.31 | 0.0098 | 4 nM | |
| B9/folic Acid | C19H19N7O6 | 441.4 | 0.0177 | 4 nM | |
| B12/cyanocobalamin | C63H88CoN14O14P | 1355.37 | 0.0095 | 0.7 nM | |
| myo-inositol | C6H12O6 | 180.16 | 0.901 | 500 nM | |
| 4-aminobenzoic Acid | C7H7NO2 | 137.14 | 0.0823 | 60 nM |
Amino Acid Mix (5,000x)
MEM Amino Acids (50x) Solution Merck MilliporeSigma (Sigma-Aldrich)Catalog #M5550- 100mL
| A | B | C | D | E | |
| Compound | Chemical Formula | FW | g/100mL | Final M | |
| L-arginine x HCl | HN=C(NH2)NH(CH2)3CH(NH2)COOH · HCl | 210.7 | 0.632 | 0.0000005999050783 | |
| L-cystine x 2HCl | C6H12N2O4S2 · 2HCl | 313.22 | 0.1564 | 0.00000009986590895 | |
| L-histidine x HCl x H2O | C6H9N3O2 · HCl · H2O | 209.65 | 0.21 | 0.0000002003338898 | |
| L-Isoleucine | C2H5CH(CH3)CH(NH2)CO2H | 131.17 | 0.2625 | 0.0000004002439582 | |
| L-leucine | CH3)2CHCH2CH(NH2)CO2H | 131.17 | 0.262 | 0.0000003994815888 | |
| L-lysine x HCl | H2N(CH2)4CH(NH2)CO2H·HCl | 182.69 | 0.3625 | 0.0000003968471181 | |
| L-methionine | CH3SCH2CH2CH(NH2)CO2H | 149.21 | 0.0755 | 0.0000001011996515 | |
| L-phenylalanine | C6H5CH2CH(NH2)CO2H | 165.19 | 0.165 | 0.0000001997699619 | |
| L-threonine | CH3CH(OH)CH(NH2)CO2H | 119.12 | 0.238 | 0.000000399597045 | |
| L-tryptophan | C11H12N2O2 | 204.23 | 0.051 | 0.00000004994369094 | |
| L-tyrosine | 4-(HO)C6H4CH2CH(NH2)CO2H | 181.19 | 0.18 | 0.0000001986864617 | |
| L-valine | (CH3)2CHCH(NH2)CO2H | 117.15 | 0.234 | 0.0000003994878361 |
Miscellaneous Mix ( 20,000x)
| A | B | C | D | E | F | |
| Compound | Chemical Formula | FW | g/100mL | mL/100mL | Final M | |
| L-glutamine | C5H10N2O2 | 146.14 | 0.14614 | 0.0000005 | ||
| dextrose | C6H12O6 | 180.16 | 0.18016 | 0.0000005 | ||
| D-ribose | C5H10O5 | 150.13 | 0.15013 | 0.0000005 | ||
| sodium pyruvate | C3H3NaO3 | 110.04 | 0.11004 | 0.0000005 | ||
| sodium citrate | C6H5Na3O7 | 294.1 | 0.2941 | 0.0000005 | ||
| oxaloacetic acid | C4H4O5 | 132.07 | 0.13207 | 0.0000005 | ||
| sodium acetate | C2H3NaO2 | 82.03 | 0.08203 | 0.0000005 | ||
| sodium succinate dibasic hexahydrate | NaOOCCH2CH2COONa · 6H2O | 270.14 | 0.27014 | 0.0000005 | ||
| a-ketoglutaric acid | C5H6O5 | 168.08 | 0.16808 | 0.0000005 | ||
| urea | CH4N2O | 60.6 | 0.606 | 0.000005 | ||
| gylcerol | C3H8O3 | 92.09 | 0.09209 | 0.0000005 | ||
| glycine betaine | C5H11NO2 | 153.61 | 0.15361 | 0.0000005 | ||
| choline | (CH3)3N(Cl)CH2CH2OH | 139.62 | 0.13962 | 0.0000005 | ||
| sodium thiosulfate | Na2S2O3 | 158.11 | 0.15811 | 0.0000005 | ||
| potassium cyanate | KOCN | 81.11 | 0.0032444 | 2.00E-08 | ||
| dmso | C2H6OS | 78.13 | 0.0062504 | 0.05682181818 | 4.00E-08 | |
| dmsp | C5H10O2S | 134.967 | 0.01079736 | 4.00E-08 | ||
| L-glycine | C2H5NO2 | 75.07 | 0.07507 | 0.0000005 | ||
| nucleotides (dNTPs) | - | - | - | 0.01 | 0.00000001 | |
| galactose | C6H12O6 | 180.16 | 0.18016 | 0.0000005 | ||
| lactose monohydrate | C12H24O12 | 360.31 | 0.36031 | 0.0000005 | ||
| glyoxylic acid sodium monohydrate | C2H3NaO4 | 114.03 | 0.11403 | 0.0000005 | ||
| tmao | C3H9NO | 75.11 | 0.07511 | 0.0000005 | ||
| L-alanine | C3H7NO2 | 89.09 | 0.08909 | 0.0000005 | ||
| L-asparagine | C4H8N2O3 | 132.12 | 0.13212 | 0.0000005 | ||
| L-aspartic acid | C4H7NO4 | 133.1 | 0.1331 | 0.0000005 | ||
| L-cysteine hydrochloride | C3H8ClNO2S | 157.62 | 0.17563 | 0.0000005 | ||
| L-glutamic acid | C5H9NO4 | 147.13 | 0.14713 | 0.0000005 | ||
| L-proline | C5H9NO2 | 115.13 | 0.11513 | 0.0000005 | ||
| L-serine | C3H7NO3 | 105.09 | 0.10509 | 0.0000005 | ||
| sodium alginate | C6H9NaO7 | 216.12 | 0.21612 | 0.0000005 | ||
| catalase | C9H10O3 | 2,000-5,000 U/mg | 0.000542857 | 10 U | ||
| glucosamine | C6H13NO5 | 221.21 | 0.22121 | 0.0000005 |
Phosphate Mix (1,000x)
| A | B | C | D | E | F | |
| Compound | Chemical Formula | FW | g/100mL | mL/100mL | Final M | |
| ortho phosphonate | C3H9O3P | 124.076 | 0.002167 | 0.0000002 | ||
| potassium phosphate monobasic | KH2PO4 | 136.09 | 0.068045 | 0.000005 |
Fatty Acid Mix (2,000,000x)
| A | B | C | D | E | F | |
| Compound | Chemical Formula | FW | g/100mL | mL/100mL | Final M | |
| octanoic acid | CH3(CH2)6COOH | 144.21 | 15.84725275 | 0.0000005 | ||
| decanoic acid | CH3(CH2)8COOH | 172.26 | 17.226 | 0.0000005 | ||
| isobutyric acid | (CH3)2CHCO2H | 88.11 | 9.274736842 | 0.0000005 | ||
| butyric acid | CH3CH2CH2COOH | 88.11 | 9.140041494 | 0.0000005 | ||
| valeric acid | C5H10O2 | 102.13 | 10.87646432 | 0.0000005 | ||
| ethanol | CH3CH2OH | 46.068 | 54.86150459 | 0.000004698025433 |
Inorganic Nitrogen (2,000x)
| A | B | C | D | E | |
| Compound | Chemical Formula | FW | g/100mL | Final M | |
| sodium nitrate | NaNO3 | 84.99 | 0.645924 | 0.000038 | |
| sodium nitrite | NaNO2 | 69 | 0.0276 | 0.000002 | |
| ammonium chloride | NH4Cl | 53.49 | 0.05349 | 0.000005 |
Iron and NTA Mix (1,000x)
| A | B | C | D | E | |
| Compound | Chemical Formula | FW | g/100mL | Final M (nM) | |
| iron (II) sulfate heptahydrate | FeSO4 x 7H2O | 278.01 | 0.0028 | 101 | |
| nitrilotriacetic acid disodium salt | NTA NA2 salt | 235.1 | 0.0081 | 345 |
Stock Preparation
Stock Preparation
Prepare the following stocks prior to medium assembly.
Iron mix (1,000x)
Trace Metals (100,000x)
Vitamins (100,000x)
AA mix (500x)
Misc. mix (20,000x)
Phosphate mix (1000x)
Inorganic Nitrogen mix (2,000x)
Fatty Acid mix (2,000,000x)
Filter sterilize stocks using 0.2 µM PES filter.
Base Salts
Base Salts
Using an acid-washed and autoclaved one Liter screw-top Pyrex bottle, combine basic salts in 997.4095 mL MilliQ-filtered water. (stock volumes subtracted)
Mix continuously with stir bar or invert the tightly capped bottle.
Each chemical should be fully dissolved before next addition.
Stock Addition
Stock Addition
Stocks should be added in a Biosafety cabinet or hood with appropriate ventilation.
Swirl or invert media between stock additions.
Add (per Liter):
10 μL Trace Metal Mix
10µL Vitamin Mix
20 µL Amino Acid Mix
50 µL Misc. Mix
500 µL Inorganic Nitrogen Mix
0.5 µL Fatty Acid Mix
1mL Iron Mix
1mL Phosphate Mix
When in the biosafety cabinet or laminar fume hood filter-sterilize medium using a 0.2 µm filter into another sterilized one Liter Pyrex bottle.
pH and record.
Media can be stored at room temp (~25 C°) in the tightly closed Pyrex bottle.
Protocol references
BS, Mg/Ca- Kester et al. 1967; Giovannoi & Stingl 2007 Fe- Carini et al. 2013; Giovannoni & Stingl 2007 P- Justic et al. 1995; Dagg et al. 2004 TM/Vit- w/ modification from Carini et al., 2013; also see Moore et al. 2007; Dagg et al. 2004; Rabalais et al. 1996; Henson et al. 2016; S Belviso, SK Kim, and F Rassoulzadegan, Production of dimethylsulfonium propionate (DMSP) and dimethylsulfide (DMS) by a microbial food web, Limnology and (1990). 3. Zindler-Schlundt, C, H Lutterbeck, and S Endres, Environmental control of dimethylsulfoxide (DMSO) cycling under ocean acidification, Environmental (2015). 4. Y Cai, L Guo, X Wang, and AK Mojzis, The source and distribution of dissolved and particulate organic matter in the bay of st. louis, northern gulf of mexico, Estuarine (2012). 5. Motard-Cˆot´e, J, DJ Kieber, A Rellinger, and RP Kiene, Influence of the mississippi river plume and non-bioavailable DMSP on dissolved DMSP turnover in the northern gulf of mexico, Environmental Chemistry (2015). 6. NA Kamennaya, M Chernihovsky, and AF Post, The cyanate utilization capacity of marine unicellular cyanobacteria, Limnology and Oceanography (2008). 7. Z Liu and S Liu, High phosphate concentrations accelerate bacterial peptide decomposition in hypoxic bottom waters of the northern gulf of mexico, Environmental science & technology (2015). 8. RR Malmstrom and RP Kiene, Identification and enumeration of bacteria assimilating dimethylsulfoniopropionate (DMSP) in the north atlantic and gulf of mexico, Limnology and (2004). 9. RR Malmstrom, RP Kiene, and MT Cottrell, Contribution of SAR11 bacteria to dissolved dimethylsulfoniopropionate and amino acid uptake in the north atlantic ocean, Applied and (2004). 10. R Sim´o, Pedr´os-Ali´o, C, and G Malin, Biological turnover of DMS, DMSP and DMSO in contrasting open-sea waters, Marine Ecology Progress (2000). 11. RE Turner, NN Rabalais, RB Alexander, McIsaac, G, and RW Howarth, Characterization of nutrient, organic carbon, and sediment loads and concentrations from the mississippi river into the northern gulf of mexico, 30 (2007), no. 5, 773–790.