TITLE: Keller (K) Optimized Medium for Culturing Microalgae
AUTHORS: Roscoff Culture Collection, Lynn    Doran, Steven J Burgess

[DESCRIPTION]
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:
  
    *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)

   


[GUIDELINES]
Not all algae cultures tolerate artificial sea water and many culture stock centers will not guarantee culture growth in artificial seawater based media.  We have had success with O. tauri using K medium made in artificial sea water.

Volumes of stock solutions and K medium are given for AgSynBio Lab's average consumption of ASW + K Medium.  Volumes may need to be scaled up or down depending on amount of algae being cultured weekly.

Reference

Guillard, R.R.L. 1975. Culture of phytoplankton for feeding marine invertebrates. pp 26- 60. In Smith W.L. and Chanley M.H (Eds.) Culture of Marine Invertebrate Animals. Plenum Press, New York, USA. 

Keller, M.D. and Guillard, R.R.L. 1985. Factors significant to marine diatom culture. pp. 113-6. In Anderson, D.M., White, A.W. and Baden, D.G. (eds.) Toxic Dinoflagellates. Elsevier, New York.

Keller, M.D., Selvin, R.C., Claus, W. & Guillard, R.R.L. 1987. Media for the culture of oceanic ultraphytoplankton. J. Phycol. 23:633–8.

Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. 

van Ooijen, G., Knox, K., Kis, K., Bouget, F. Y., & Millar, A. J. (2012). Genomic transformation of the picoeukaryote Ostreococcus tauri.Journal of visualized experiments : JoVE, (65), e4074. https://doi.org/10.3791/4074

Additional Information

K and K+Si medium, Roscoff Culture Collection

K Medium, Bigelow National Center for Microalgae and Microbiota

Keller (K) Medium in Artificial Seawater, Worden Lab at Monterey Bay Aquarium Research Institute

Making Media by Australian Algae National Culture Collection

[STEPS]
SECTION: Prepare Artificial Sea Water (ASW)
1. Prepare artificial sea water (ASW) (1 L).
 


SECTION: Prepare Trace Metal Solutions
2. Prepare K Medium Trace Metal Working Stock Solution (7 mL).
 
SECTION: Prepare f/2 Vitamin Solutions
3. Prepare K Medium f/2 Vitamin Working Stock Solution (100 mL).
SECTION: Prepare Artificial Sea Water (ASW)
1.2. Weigh the following chemicals using a spatula and  weigh paper.  Add them to the media storage bottle.

 

 

 

SECTION: Prepare Artificial Sea Water (ASW)
1.1. Fill a 1 L  media storage bottle two-thirds full with 18.2 MΩ water (Milli-Q or Nanopure).

 


SECTION: Prepare Artificial Sea Water (ASW)
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.

 

 

SECTION: Prepare Artificial Sea Water (ASW)
1.4. Bring to 1 L  in a graduated cylinder with 18.2 MΩ H2O  (Milli-Q or Nanopure). Return solution to media storage bottle.

SECTION: Prepare Trace Metal Solutions
2.7. Add the appropriate volume of the trace metal stock solutions to the trace metal working stock solution using a pipette.
 
 
SECTION: Prepare Artificial Sea Water (ASW)
1.5. Best practice would be to autoclave the artificial sea water before each use.

 

SECTION: Prepare Artificial Sea Water (ASW)
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.
SECTION: Prepare Trace Metal Solutions
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.
 
                  Na2MoO4 • 2H2O (6.3 g/L)  4  0.0252  N.A.  6.3  571  :Batches of Trace Metals Solution prepared from Na2MoO4 • 2H2O (6.3 g/L)    ZnSO4 • 7H2O (22.0 g/L)  1  0.0220  N.A.  22  143  :Batches of Trace Metals Solution prepared from ZnSO4 • 7H2O (22.0 g/L)    CoCl2• 6H2O (10.0 g/L)  2  0.0200  N.A.  10  286  :Batches of Trace Metals Solution prepared from CoCl2• 6H2O (10.0 g/L)    MnCl2 • 4H2O (180.0 g/L)  0.2  0.0360  N.A.  180  29  :Batches of Trace Metals Solution prepared from MnCl2 • 4H2O (180.0 g/L)    CuSO4• 5H2O (9.8 g/L)  2  0.0196  N.A.  9.8  286  :Batches of Trace Metals Solution prepared from CuSO4• 5H2O (9.8 g/L)  

 

 

SECTION: Prepare Trace Metal Solutions
2.2. Prepare the trace metal working stock solution by adding 5 mL 18.2 MΩ water to a new 15 mL centrifuge.
SECTION: Prepare Trace Metal Solutions
2.3. Weigh 0.29 g EDTA Disodium Salt Dihydrate (Na2EDTA.2H20) on an analytical balance using a spatula and weigh paper.

SECTION: Prepare Trace Metal Solutions
2.4. Add the EDTA Disodium Salt Dihydrate to the trace metal working stock solution.  Cap and shake until completely dissolved.
 

SECTION: Prepare Trace Metal Solutions
2.5. Weigh 20 mg of hexahydrated ferric chloride (FeCl3.6H20) in a chemical fume hood on a microbalance using a spatula and weigh paper.

 

SECTION: Prepare Trace Metal Solutions
2.6. Add the hexahydrated ferric chloride  to the trace metal working stock solution.  Cap and shake until completely dissolved.
 

SECTION: Prepare f/2 Vitamin Solutions
3.3. Add the appropriate volume of the f/2 vitamin stock solutions to the f/2 Vitamin working stock solution using a pipette.

 

SECTION: Prepare Trace Metal Solutions
2.8. 
Bring final volume up to 7 mL  using 18.2 MΩ water. Shake to thoroughly mix all solutions.  If any precipitate is visible, heat in a water bath.  
 

SECTION: Prepare Trace Metal Solutions
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.
SECTION: Prepare f/2 Vitamin Solutions
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. 
              Chemical  Volume Desired (mL)  Solid (g)  Volume (mL)  Concentration in final solution (g/L)  

SECTION: Prepare f/2 Vitamin Solutions
3.2. Prepare the f/2  Vitamin working stock solution by adding 99 mL 18.2 MΩ water to a new media storage bottle.
SECTION: Prepare f/2 Vitamin Solutions
3.4. Mix f/2 vitamin working stock solution by inverting several times.  
SECTION: Prepare f/2 Vitamin Solutions
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.
 

SECTION: Prepare Keller Medium in ASW
4.2. Prepare Keller Medium by measuring 993.5 mL of artificial sea water into a graduated cylinder.

SECTION: Prepare Keller Medium in ASW
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. 
 
                NaNO3   1  0.0750  N.A.  75  1    NH4Cl  8  0.0214  N.A.  2.68  8    β-glycerophosphate   10  0.0216  N.A.  2.16  10    H2SeO3   1000  0.0013  N.A.  0.00129  1000    Tris-base(pH 7.2)   1  0.1211  N.A.  121.1  1  

SECTION: Prepare Keller Medium in ASW
4. 
Prepare Keller Medium in artificial seawater (1000 mL).

SECTION: Prepare Keller Medium in ASW
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.
 
      993.5  Volume of ASW    1  NaNO3 (75 g/L)    1  NH4Cl (2.68 g/L)    1  β-glycerophosphate (2.16 g/L)    1  H2SeO3 (0.00129 g/L)    1  Tris-base(pH 7.2) (121.1 g/L)    1  K trace metal solution    0.5  f/2 vitamin solution  

 
 

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

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


SECTION: Prepare Keller Medium in ASW
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 22 mL of 18.2 MΩ water into a centrifuge tube.
In a chemical fume hood, add 8 mL 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 20 mL of 18.2 MΩ water into a centrifuge tube.
Weigh 4.8 g 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.