SJB Artificial Seawater Medium Protocol

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.

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.

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.