Keller (K) Optimized Medium for Culturing Microalgae

Lynn Doran, Steven J Burgess, Roscoff Culture Collection

Published: 2022-04-18 DOI: 10.17504/protocols.io.36wgq76z3vk5/v1

Disclaimer

This protocol requires modifications for diatoms.

Abstract

Medium to grow most phytoplankton species in small eukaryotes (Mamiellophyceae, Pelagophyceae etc...).

Optimized for Ostreococcus tauri. Keller Optimized Medium was developed by Smallwood et al. (https://doi.org/10.1101/291211) which includes additions of 17 mM sodium nitrate and 0.37 mM NaH2PO4

Before start

Please refer to the Roscoff Culture Center general recommendations to grow cultures.

This protocol makes 1 L of Keller Medium in Artificial Sea Water and the minimum possible amount of each working and stock solution. To automatically adjust weights and volumes for a different amount of ASW in K Medium or the component working and stock solutions use the following Batch Size Workbook:

Batch Size Workbook_ ASW + K Medium.xlsx

*Protocols.io steps sections are color coded to match the color coding for the reagent prep in the Batch Size            

 Workbook.

It is best practice to keep a log book of all information used to make K Medium in Artificial Sea Water and the stock solutions. In the event that there is an issue with culture growth, it will be easier to find the problem if this data is recorded.

AgSynBio ASW + K Medium Prep Log Book.xlsx (Log in required) AgSynBio ASW + K Medium Prep Log Book.xlsx (Log in required)

Example Template for ASW + K Medium Prep Log Book.xlsx

Steps

Prepare Artificial Sea Water (ASW)

Prepare artificial sea water (ASW) (1L).

Note

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.

1.1.

Fill a 1L media storage bottle two-thirds full with 18.2 MΩ water (Milli-Q or Nanopure).

Note

18.2 MΩ water (Milli-Q or Nanopure) is not sterile unless autoclaved.

1.2.

Weigh the following chemicals using a spatula and weigh paper. Add them to the media storage bottle.

A	B
Chemical	Amount
Sodium chloride (NaCl)	24.55 g
Potassium chloride (KCl)	0.75 g
Magnesium chloride hexahydrate (MgCl2.6H2O)	4.07 g
Calcium chloride dihydrate (CaCl2.2H2O)	1.47 g
Magnesium sulfate heptahydrate (MgSO4.7H2O)	6.04 g

Chemical components and weights for 1 L of artificial sea water

Note

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).

Note

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.

1.3.

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.

A	B
Chemical	Amount
Sodium bicarbonate (NaHCO3)	0.21 g

Sodium bicarbonate weight for 1 L of artificial sea water

Note

Sodium bicarbonate is added last after all other components have dissolved to adjust pH.

1.4.

Bring to 1L in a graduated cylinder with 18.2 MΩ H2O (Milli-Q or Nanopure). Return solution to media storage bottle.

1.5.

Best practice would be to autoclave the artificial sea water before each use.

Note

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.

1.6.

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 K Medium Trace Metal Working Stock Solution (7mL).

2.1.

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.

A	B	C	D	E
Chemical	Volume prepared (mL)	Weight of chemical (mg)	Concentration (g/L)	Batches of Trace Metal Solution (7 mL) that can be prepared from this volume
Na2MoO4 • 2H2O	4	25.2	6.3	571
ZnSO4 • 7H2O (22.0 g/L)	1	22.0	22	143
CoCl2• 6H2O (10.0 g/L)	2	20.0	10	286
MnCl2 • 4H2O (180.0 g/L)	0.2	36.0	180	29
CuSO4• 5H2O (9.8 g/L)	2	19.6	9.8	286

Trace Metal stock solutions recipes

Safety information

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.

Note

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)

2.2.

Prepare the trace metal working stock solution by adding 5mL 18.2 MΩ water to a new 15 mL centrifuge.

2.3.

Weigh 0.29g EDTA Disodium Salt Dihydrate (Na₂EDTA.2H₂0) on an analytical balance using a spatula and weigh paper.

2.4.

Add the EDTA Disodium Salt Dihydrate to the trace metal working stock solution. Cap and shake until completely dissolved.

Note

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.

2.5.

Weigh 20mg of hexahydrated ferric chloride (FeCl₃.6H₂0) in a chemical fume hood on a microbalance using a spatula and weigh paper.

Safety information

Hexahydrated ferric chloride (FeCl₃.6H₂0) is acutely toxic. Review safety datasheet before handling.

2.6.

Add the hexahydrated ferric chloride to the trace metal working stock solution. Cap and shake until completely dissolved.

Note

If chemical won't dissolve after several minutes of vigorous shaking, indirectly heat solution in a water bath and continue intermittent shaking.

2.7.

Add the appropriate volume of the trace metal stock solutions to the trace metal working stock solution using a pipette.

A	B
Quantity	Compound
7 ul	Sodium Molybdate Dihydrate (Na2MoO4.2H2O) Solution (6.3 g/L)
7 ul	Zinc Sulfate Heptahydrate (ZnSO4.7H2O) Solution (22.0 g/L)
7 ul	Cobalt Chloride Hexahydrate (CoCl2.6H2O) Solution (10.0 g/L)
7 ul	Manganese (II) chloride, tetrahydrate (MnCl2.4H2O) Solution (180.0 g/L)
7 ul	Copper(II) sulfate pentahydrate (Cu SO4.5H2O) Solution (4.9 g/L)

Stock solution volumes for 7 mL of K Medium Trace Metal Solution.

2.8.

Bring final volume up to 7mL using 18.2 MΩ water. Shake to thoroughly mix all solutions. If any precipitate is visible, heat in a water bath.

Note

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.

2.9.

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 K Medium f/2 Vitamin Working Stock Solution (100mL).

3.1.

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.
Short term storage should be in the dark at 4°C , long term storage should be in the dark at -20°C . 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.

A	B	C	D	E
Chemical	Total volume (mL)	Solid weight (mg)	D	Batches of f/2 Vitamin Working Solution (100 mL) made from this volume
Biotin	100	10.0	0.1	100
Vitamin B12	20	20.0	1	200

f/2 stock solution recipes

3.2.

Prepare the f/2 Vitamin working stock solution by adding 99mL 18.2 MΩ water to a new media storage bottle.

3.3.

Add the appropriate volume of the f/2 vitamin stock solutions to the f/2 Vitamin working stock solution using a pipette.

A	B
Quantity	Compound
100 ul	Vitamin B12 Solution (1 g/L)
1000 ul	Biotin Solution (0.1 g/L)

Stock solution volumes for 100 mL of f/2 Vitamin Working Stock Solution

3.4.

Mix f/2 vitamin working stock solution by inverting several times.

3.5.

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.

Note

Limit freeze/thaw of f/2 vitamin working stock solutions to three cycles.

Prepare Keller Medium in ASW

Prepare Keller Medium in artificial seawater (1000mL).

4.1.

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.

Prepare each individual stock solution for Keller Medium according to the table below.

A	B	C	D	E
Chemical	Total volume (mL)	Solid (mg)	Concentration in final solution (g/L)	Batches of Keller Medium (1 L) made from this volume
NH4Cl	8	21.4	2.68	8
β-glycerophosphate	10	21.6	2.16	10
H2SeO3	1000	1.29	0.00129	1000
Tris-base(pH 7.2)	1	121.1	121.1	1

Keller Medium stock solution recipes

4.2.

Prepare Keller Medium by measuring 993.5mL of artificial sea water into a graduated cylinder.

4.3.

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.

A	B
Quantity	Stock Solution
1 mL	NH4Cl (2.68 g/L)
1 mL	β-glycerophosphate (2.16 g/L)
1 mL	H2SeO3 (0.00129 g/L)
1 mL	Tris-base(pH 7.2) (121.1 g/L)
1 mL	K trace metal solution
0.5 mL	f/2 vitamin solution
1.44 g	NaNO3 (17 mM final)
1 mL	NaH2PO4 stock

Stock and working stock volumes for 1 L of Keller Medium in Artificial Seawater

4.4.

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 22mL of 18.2 MΩ water into a centrifuge tube.
In a chemical fume hood, add 8mL 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 20mL of 18.2 MΩ water into a centrifuge tube.
Weigh 4.8g 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.
Safety information
Strong acids and Strong acids and bases can cause burns. can cause burns.

4.5.

Filter sterilize the Keller Medium in artificial seawater into a sterile media storage bottle using a 0.22 micron membrane filter.

Note

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.

4.6.

Label Keller Medium with analyst name, date, and the word sterile. Fill out the ASW + K Medium Prep Log Book.

4.7.

Store Keller Medium in the dark at 4°C for up to 2 weeks. Only open Keller Medium in a sterile, laminar flow hood.

Note

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.

Keller (K) Optimized Medium for Culturing Microalgae

Disclaimer

Abstract

Before start

Steps

Prepare Artificial Sea Water (ASW)

Prepare Trace Metal Solutions

Prepare f/2 Vitamin Solutions

Prepare Keller Medium in ASW

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