TITLE: Keller (K) Medium in Artificial Sea Water (ASW) for Culturing Microalgae (Ostreococcus tauri)
AUTHORS: Roscoff Culture Collection, Lynn  Doran, Steven Burgess

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Medium to grow most phytoplankton species in small eukaryotes (Mamiellophyceae, Pelagophyceae etc...).</div><div class = "text-block">Optimized for  Ostreococcus tauri.  </div></div>

[STEPS]
?. [Prepare Artificial Sea Water (ASW)]
Prepare artificial sea water (ASW) ().
1 L
Please note not all algae cultures tolerate artificial sea water and many culture stock centers will not guarantee culture growth in artificial seawater based media.To prepare natural seawater per the Roscoff Center: Collect seawater (salinity ca. 33‰);Leave the seawater (SW) to age for 2 months at room temperature in the darkness;
Note: This"aging" step is critical because during that period bacteria present in the sea water will degrade many organic molecules that are probably detrimental to algae.  The Roscoff Center's experince shows that medium made with 'fresh' seawater will in many cases prevent cells from growing.Filter through 0.22µM filters (Millipore filter GSWPO9000 plus Millipore prefilter AP1507500) and autoclave;Continue at step 2.
?. [Prepare Trace Metal Solutions]
Prepare K Medium Trace Metal Working Stock Solution ().
7 mL
?. [Prepare f/2 Vitamin Solutions]
Prepare K Medium f/2 Vitamin Working Stock Solution ().
100 mL
?. [Prepare Artificial Sea Water (ASW)]
Weigh the following chemicals using a spatula and  weigh paper.  Add them to the media storage bottle.ChemicalAmountSodium chloride (NaCl)24.55 gPotassium chloride (KCl)0.75 gMagnesium chloride hexahydrate (MgCl2.6H2O)4.07 gCalcium chloride dihydrate (CaCl2.2H2O)1.47 gMagnesium sulfate heptahydrate (MgSO4.7H2O)6.04 g
ChemicalAmountSodium chloride (NaCl)24.55 gPotassium chloride (KCl)0.75 gMagnesium chloride hexahydrate (MgCl2.6H2O)4.07 gCalcium chloride dihydrate (CaCl2.2H2O)1.47 gMagnesium sulfate heptahydrate (MgSO4.7H2O)6.04 g
Best practice is to use single use plastic utensils and weigh boats for weighing chemicals for ASW.  To date, we have not had any issues with stainless steel, washable utensils (2/9/2021).
Best practice is to dedicate chemical supplies for ASW and K medium; keep chemicals separate from general lab supply.  Trace amounts of cross contamination from other lab chemicals could kill algae cultures.
?. [Prepare Artificial Sea Water (ASW)]
Fill a   media storage bottle two-thirds full with 18.2 MΩ water (Milli-Q or Nanopure).
1 L
18.2 MΩ water (Milli-Q or Nanopure) is not sterile unless autoclaved.
?. [Prepare Artificial Sea Water (ASW)]
Swirl the bottle until all chemicals have dissolved. Weigh the following chemical using a spatula and  weigh paper.  Add them to the media storage bottle and swirl until dissolved.ChemicalAmountSodium bicarbonate (NaHCO3)0.21 g
ChemicalAmountSodium bicarbonate (NaHCO3)0.21 g
Sodium bicarbonate is added last after all other components have dissolved to adjust pH.
?. [Prepare Artificial Sea Water (ASW)]
Bring to   in a graduated cylinder with 18.2 MΩ H2O  (Milli-Q or Nanopure). Return solution to media storage bottle.
1 L
?. [Prepare Trace Metal Solutions]
Add the appropriate volume of the trace metal stock solutions to the trace metal working stock solution using a pipette.  AB1QuantityCompound27 ulSodium Molybdate Dihydrate (Na2MoO4.2H2O) Solution (6.3 g/L)37 ulZinc Sulfate Heptahydrate (ZnSO4.7H2O) Solution (22.0 g/L)
47 ulCobalt Chloride Hexahydrate (CoCl2.6H2O) Solution (10.0 g/L)
57 ulManganese (II) chloride, tetrahydrate (MnCl2.4H2O) Solution (180.0 g/L)
67 ulCopper(II) sulfate pentahydrate (Cu SO4.5H2O) Solution (4.9 g/L)
AB1QuantityCompound27 ulSodium Molybdate Dihydrate (Na2MoO4.2H2O) Solution (6.3 g/L)37 ulZinc Sulfate Heptahydrate (ZnSO4.7H2O) Solution (22.0 g/L)
47 ulCobalt Chloride Hexahydrate (CoCl2.6H2O) Solution (10.0 g/L)
57 ulManganese (II) chloride, tetrahydrate (MnCl2.4H2O) Solution (180.0 g/L)
67 ulCopper(II) sulfate pentahydrate (Cu SO4.5H2O) Solution (4.9 g/L)
?. [Prepare Artificial Sea Water (ASW)]
Best practice would be to autoclave the artificial sea water before each use.
Final ASW + K Medium solution will be sterilized by autoclave (sterile) or 0.22 um membrane filter (some bacteria and viruses can pass).  Because final solution will be sterilized, it is not necessary to sterilize the ASW only.  If ASW will be stored for a long time between uses, it is advisable to sterilize to inhibit any biological growth from potential contaminants.
?. [Prepare Artificial Sea Water (ASW)]
Label artificial sea water with analyst name, date, and the word sterile or non-sterile.  Fill out the ASW + K Medium Prep Log Book.
?. [Prepare Trace Metal Solutions]
Prepare each individual stock solution for Trace Metal Working Stock Solution. NOTE: Each row of the table below is a separate stock solution and should be prepared in its own container.  Do not mix the individual solutions at this time.Label a centrifuge tube (or microcentrifuge tube if applicable) for each individual stock solution.  Include the name, concentration, date, and preparer's initials.Fill a centrifuge tube (or microcentrifuge tube if applicable) with 2/3 of the required total volume with 18.2 MΩ water.Weigh the chemicals in a chemical fume hood on a micro analytical balance using a spatula and  weigh paper.  Add them to the appropriately labeled tube.        NOTE: Weights below are in milligrams.Mix by shaking until chemical is completely dissolved.Pour solution into a  graduated cylinder and bring to the total assigned volume with 18.2 MΩ water.Return to centrifuge tube.  Shake well.Store for future use.  Each individual working stock solution will make many batches of Trace Metal Working Solution.Fill out the ASW + K Medium Prep Log Book.Clean weighing utensils and graduated cylinder before preparing the next individual working stock solution.        NOTE: Weights below are in milligrams.Mix by shaking until chemical is completely dissolved.Pour solution into a  graduated cylinder and bring to the total assigned volume with 18.2 MΩ water.Return to centrifuge tube.  Shake well.Store for future use.  Each individual working stock solution will make many batches of Trace Metal Working Solution.Fill out the ASW + K Medium Prep Log Book.Clean weighing utensils and graduated cylinder before preparing the next individual working stock solution.ChemicalVolume prepared (mL)Weight of chemical (mg)Concentration (g/L)Batches of Trace Metal Solution (7 mL) that can be prepared from this volumeNa2MoO4 • 2H2O425.26.3571ZnSO4 • 7H2O (22.0 g/L)122.022143CoCl2• 6H2O (10.0 g/L)220.010286MnCl2 • 4H2O (180.0 g/L)0.236.018029CuSO4• 5H2O (9.8 g/L)219.69.8286
ChemicalVolume prepared (mL)Weight of chemical (mg)Concentration (g/L)Batches of Trace Metal Solution (7 mL) that can be prepared from this volumeNa2MoO4 • 2H2O425.26.3571ZnSO4 • 7H2O (22.0 g/L)122.022143CoCl2• 6H2O (10.0 g/L)220.010286MnCl2 • 4H2O (180.0 g/L)0.236.018029CuSO4• 5H2O (9.8 g/L)219.69.8286
Many of the chemicals used to prepare K Medium Trace Metal Stock Solutions are acutely toxic. Review all safety information and applicable Safety Data Sheets before handling.
For long term storage, some labs have found in beneficial to store these solutions in the freezer if they will not be used in totality the day of preparation.  AgSynBio lab currently stores them at room temperature with minimal issues. (2-22-2021)
?. [Prepare Trace Metal Solutions]
Prepare the trace metal working stock solution by adding  18.2 MΩ water to a new 15 mL centrifuge.
5 mL
?. [Prepare Trace Metal Solutions]
Weigh  EDTA Disodium Salt Dihydrate (Na2EDTA.2H20) on an analytical balance using a spatula and weigh paper.
0.29 g
?. [Prepare Trace Metal Solutions]
Add the EDTA Disodium Salt Dihydrate to the trace metal working stock solution.  Cap and shake until completely dissolved.
EDTA must be added first and completely dissolved before the addition of the remaining chemicals in order to chelate the metal ions and prevent them from precipitating out of solution.
?. [Prepare Trace Metal Solutions]
Weigh  of hexahydrated ferric chloride (FeCl3.6H20) in a chemical fume hood on a microbalance using a spatula and weigh paper.
20 mg
Hexahydrated ferric chloride (FeCl3.6H20) is acutely toxic.  Review safety datasheet before handling.
?. [Prepare Trace Metal Solutions]
Add the hexahydrated ferric chloride  to the trace metal working stock solution.  Cap and shake until completely dissolved.
If chemical won't dissolve after several minutes of vigorous shaking, indirectly heat solution in a water bath and continue intermittent shaking.
?. [Prepare f/2 Vitamin Solutions]
Add the appropriate volume of the f/2 vitamin stock solutions to the f/2 Vitamin working stock solution using a pipette.QuantityCompound100 ulVitamin B12 Solution (1 g/L)1000 ulBiotin Solution (0.1 g/L)
QuantityCompound100 ulVitamin B12 Solution (1 g/L)1000 ulBiotin Solution (0.1 g/L)
?. [Prepare Trace Metal Solutions]
Bring final volume up to   using 18.2 MΩ water. Shake to thoroughly mix all solutions.  If any precipitate is visible, heat in a water bath.
7 mL
The metals are reactive species.  If disturbed they will form complexes and precipitate out.  Precipitate can usually be re-solublized with indirect heating.  If heating, doesn't cause re-solubilization than a pH adjustment may be necessary.Autoclaving, freezing, photodegradation, changes in pH, and long term storage may cause precipitate to form.
?. [Prepare Trace Metal Solutions]
Label trace metal working stock with analyst name and date. Fill out the ASW + K Medium Prep Log Book.  Any trace metal working stock solution that will not be used the same day should be aliquoted and stored at .
-20 °C
?. [Prepare f/2 Vitamin Solutions]
Prepare each individual stock solution for f/2 Vitamin Working Stock Solution. NOTE: Each row of the table below is a separate stock solution and should be prepared in its own container.  Do not mix the individual solutions at this time.Label a centrifuge tube (or media storage bottle if applicable) for each individual stock solution.  Include the name, concentration, date, and preparer's initials.Fill a centrifuge tube (or media storage bottle if applicable) with 2/3 of the required total volume with 18.2 MΩ water.Weigh the chemical on a micro analytical balance using a spatula and  weigh paper.  Add them to the appropriately labeled tube.        NOTE: Weights below are in milligrams.        NOTE: If the desired weight is the exact amount that comes in the reagent bottle, rinse the chemical directly from           reagent bottle into the stock solution using 18.2 MΩ water.Mix by shaking until chemical is completely dissolved.Pour solution into a  graduated cylinder and bring to the total assigned volume with 18.2 MΩ water.Return to centrifuge tube or media bottle.  Shake well.Store for future use.  Each individual working stock solution will make many batches of f/2 Vitamin Working Solution.Fill out the ASW + K Medium Prep Log Book.Clean weighing utensils and graduated cylinder before preparing the next individual working stock solution.        NOTE: Weights below are in milligrams.        NOTE: If the desired weight is the exact amount that comes in the reagent bottle, rinse the chemical directly from           reagent bottle into the stock solution using 18.2 MΩ water.Mix by shaking until chemical is completely dissolved.Pour solution into a  graduated cylinder and bring to the total assigned volume with 18.2 MΩ water.Return to centrifuge tube or media bottle.  Shake well.Store for future use.  Each individual working stock solution will make many batches of f/2 Vitamin Working Solution.Fill out the ASW + K Medium Prep Log Book.Clean weighing utensils and graduated cylinder before preparing the next individual working stock solution.Short term storage should be in the dark at  , long term storage should be in the dark at  .  Limit freeze/thaw of vitamin stock solutions in long term storage to three cycles.Prepare each individual stock solution for f/2 Vitamin Working Stock Solution. ChemicalTotal volume (mL)Solid weight (mg)DBatches of f/2 Vitamin Working Solution (100 mL) made from this volumeBiotin 10010.00.1100Vitamin B12 2020.01200
4 °C
-20 °C
ChemicalTotal volume (mL)Solid weight (mg)DBatches of f/2 Vitamin Working Solution (100 mL) made from this volumeBiotin 10010.00.1100Vitamin B12 2020.01200
?. [Prepare f/2 Vitamin Solutions]
Prepare the f/2  Vitamin working stock solution by adding  18.2 MΩ water to a new media storage bottle.
99 mL
?. [Prepare f/2 Vitamin Solutions]
Mix f/2 vitamin working stock solution by inverting several times.
?. [Prepare f/2 Vitamin Solutions]
Label f/2 vitamin working stock with analyst name and date. Fill out the ASW + K Medium Prep Log Book.  Any f/2 vitamin working stock solution that will not be used the same day should be aliquoted and stored in the dark at .
-20 °C
Limit freeze/thaw of f/2 vitamin working stock solutions to three cycles.
?. [Prepare Keller Medium in ASW]
Prepare Keller Medium by measuring  of artificial sea water into a graduated cylinder.
993.5 mL
?. [Prepare Keller Medium in ASW]
Prepare each individual stock solution for Keller Medium. NOTE: Each row of the table below is a separate stock solution and should be prepared in its own container.  Do not mix the individual solutions at this time.Label a centrifuge tube (or media storage bottle if applicable) for each individual stock solution.  Include the name, concentration, date, and preparer's initials.Fill a centrifuge tube (or media storage bottle if applicable) with 2/3 of the required total volume with 18.2 MΩ water.Weigh the chemical on a micro analytical balance using a spatula and  weigh paper.  Add them to the appropriately labeled tube.        NOTE: Weights below are in milligrams.Mix by shaking until chemical is completely dissolved.Pour solution into a  graduated cylinder and bring to the total assigned volume with 18.2 MΩ water.Return to centrifuge tube or media bottle.  Shake well.Store for future use.  Each individual working stock solution will make many batches of Keller Medium.Fill out the ASW + K Medium Prep Log Book.Clean weighing utensils and graduated cylinder before preparing the next individual working stock solution.        NOTE: Weights below are in milligrams.Mix by shaking until chemical is completely dissolved.Pour solution into a  graduated cylinder and bring to the total assigned volume with 18.2 MΩ water.Return to centrifuge tube or media bottle.  Shake well.Store for future use.  Each individual working stock solution will make many batches of Keller Medium.Fill out the ASW + K Medium Prep Log Book.Clean weighing utensils and graduated cylinder before preparing the next individual working stock solution.Prepare each individual stock solution for Keller Medium according to the table below. ChemicalTotal volume (mL)Solid (mg)Concentration in final solution (g/L)Batches of Keller Medium (1 L) made from this volumeNaNO3&nbsp;175.0751NH4Cl821.42.688β-glycerophosphate&nbsp;1021.62.1610H2SeO3&nbsp;10001.290.001291000Tris-base(pH 7.2)&nbsp;1121.1121.11
ChemicalTotal volume (mL)Solid (mg)Concentration in final solution (g/L)Batches of Keller Medium (1 L) made from this volumeNaNO3&nbsp;175.0751NH4Cl821.42.688β-glycerophosphate&nbsp;1021.62.1610H2SeO3&nbsp;10001.290.001291000Tris-base(pH 7.2)&nbsp;1121.1121.11
?. [Prepare Keller Medium in ASW]
Prepare Keller Medium in artificial seawater ().
1000 mL
?. [Prepare Keller Medium in ASW]
Add the appropriate volume of the Keller medium stock solutions, trace metal working stock solution, and the f/2 Vitamin working stock solution to the Keller Medium in the graduated cylinder using a pipette.QuantityStock Solution1 mLNaNO3 (75 g/L)1 mLNH4Cl (2.68 g/L)1 mLβ-glycerophosphate (2.16 g/L)1 mLH2SeO3 (0.00129 g/L)1 mLTris-base(pH 7.2) (121.1 g/L)1 mLK trace metal solution0.5 mLf/2 vitamin solution
QuantityStock Solution1 mLNaNO3 (75 g/L)1 mLNH4Cl (2.68 g/L)1 mLβ-glycerophosphate (2.16 g/L)1 mLH2SeO3 (0.00129 g/L)1 mLTris-base(pH 7.2) (121.1 g/L)1 mLK trace metal solution0.5 mLf/2 vitamin solution
?. [Prepare Keller Medium in ASW]
Filter sterilize the Keller Medium in artificial seawater into a sterile media storage bottle using a 0.22 micron membrane filter.
Filter sterilization will not remove 100% of bacteria or viruses.  If culture is axenic or is not a robust culture, add everything but the f/2 vitamin  and trace metals solutions, autoclave, and filter sterilize the f/2 vitamin and trace metals solutions into the finished medium.If zero tolerance for biological contamination is required, autoclave the complete keller medium.  Some vitamin efficacy will be lost during autoclaving as f/2 vitamin solution is heat-labile and white precipitate may form due to the reactivity of the trace metals under heat and pressure.
?. [Prepare Keller Medium in ASW]
Label Keller Medium with analyst name, date, and the word sterile.  Fill out the ASW + K Medium Prep Log Book.
?. [Prepare Keller Medium in ASW]
Store Keller Medium in the dark at  for up to  2 weeks.  Only open Keller Medium in a sterile, laminar flow hood.
4 °C
f/2 vitamin stability limits the shelf life for Keller Medium.  B1 and B12 alone are shelf stable but when in formulation together can lose up to 20% efficacy in 5 days, unless frozen.
?. [Prepare Keller Medium in ASW]
pH Keller Medium and adjust pH to 8.1-8.2 using 10% hydrochloric acid (HCl) and 4 M sodium hydroxide (NaOH).To make 10% HCl:Measure  of 18.2 MΩ water into a centrifuge tube.In a chemical fume hood, add  of 37% concentracted hydrochloric acid. Always ADD ACID.Cap and shake until mixed.Label with chemical name, analyst initials, date, and concentration.Fill out the ASW + K Medium Prep Log Book.To make 4 M NaOH:Measure   of 18.2 MΩ water into a centrifuge tube.Weigh   of sodium hydroxide pellets onto weigh paper using a spatula.In a chemical fume hood, slowly add the sodium hydroxide to the water.  Exothermic reaction.  Add pellets slowly and allow solution to cool to handling temperature before adding more pellets.Cap and shake until mixed.Label with chemical name, analyst initials, date, and concentration.Fill out the ASW + K Medium Prep Log Book.To make 4 M NaOH:Measure  of 18.2 MΩ water into a centrifuge tube.Weigh  of sodium hydroxide pellets onto weigh paper using a spatula.In a chemical fume hood, slowly add the sodium hydroxide to the water.  Exothermic reaction.  Add pellets slowly and allow solution to cool to handling temperature before adding more pellets.Cap and shake until mixed.Label with chemical name, analyst initials, date, and concentration.Fill out the ASW + K Medium Prep Log Book.
22 mL
8 mL
20 mL
4.8 g
Strong acids and bases can cause burns.