L1 Medium - IBEMicrobeHub/culturing GitHub Wiki

L1 Medium

To enriched seawater medium is based upon f/2 medium (Guillard & Ryther 1962) but as additional trace metals. It is a general marine medium for growing coastal algae.

Protocol: L1 _ Roscoff Culture Collection.pdf

BEAP lab tables [adapted to prepare less quantity of stocks (50 mL)]: L1 Medium.pdf

INGREDIENTS

  • Artificial/natural Seawater.
  • dH2O.
  • Filters 0,22 µm.

Table 1

Elements Stock Solution Quantity Molar Concentration in Final Medium
NaNO3 75 g/L 1 mL 8,82 x 10^-4 M
NaH2PO4·H2O 5 g/L 1 mL 3,62 x 10^-5 M
Na2SiO3·9H2O 30 g/L 1 mL 1,06 x 10^-4 M
Trace element solution See recipe below (1) 1 mL ---
Vitamin solution See recipe below (2) 0,5 mL ---

(1) L1 Trace Elements Solution:

Table 2

Elements Stock Solution Quantity Molar Concentration in Final Medium
Na2EDTA·2H2O --- 4,36 g 1,17 x 10^-5 M
FeCl3·6H2O --- 3,15 g 1,17 x 10^-5 M
MnCl2·4H2O 178,1 g/L dH2O 1 mL 9,09 x 10^-7 M
ZnSO4·7H2O 23 g/L dH2O 1 mL 8 x 10^-8 M
CoCl2·6H2O 11,9 g/L dH2O 1 mL 5 x 10^-8 M
CuSO4·5H2O 2,5 g/L dH2O 1 mL 1 x 10^-8 M
Na2MoO4·2H2O 19,9 g/L dH2O 1 mL 8,22 x 10^-8 M
H2SeO3 1,29 g/L dH2O 1 mL 1 x 10^-8 M
NiSO4·6H2O 2,63 g/L dH2O 1 mL 1 x 10^-8 M
Na3VO4 1,84 g/L dH2O 1 mL 1 x 10^-8 M
K2CrO4 1,94 g/L dH2O 1 mL 1 x 10^-8 M

(2) f/2 Vitamin Solution:

Table 3

Elements Primary Stock Solution Quantity Molar Concentration in Final Medium
Thiamine·HCL (Vit. B1) --- 200 mg 2,96 x 10^-7 M
Biotin (Vit. H) 0,1 g/L dH2O 10 mL 2,05 x 10^-9 M
Cyanocobalamin (Vit. B12) 1 g/L dH2O 1 mL 3,69 x 10^-10 M

METHOD

  1. Prepare the L1 trace element solution (table 2):
  • To 950 mL dH2O add the following components.
  • Bring the final volume to 1 L with dH2O.
  • Autoclave.
  1. Prepare the f/2 vitamin solution (table 3):
  • First, prepare the primary stocks solutions.
  • To prepare the final vitamin solution, begin with 950 mL of dH2O.
  • Dissolve the thiamine.
  • Add the amounts of primary stocks.
  • Bring the final volume to 1L with dH2O.

At National Center for Marine Algae and Microbiota (NCMA) autoclave the solution.

  • Store in refrigerator or freezer.
  1. Final L1 medium:
  • In a 950 mL of filtered artificial/natural seawater, add the quantity of each component (table 1).
  • Bring the final volume to 1 L using filtered artificial/natural seawater.
  • Autoclave 20 min at 100ºC.
  • Let it cool before adjusting the pH.

Final pH should be 8 to 8,2.

  • Filter (0,22 µm) under the hood.
  • Store in RT.

Culture List:

  • Aureococcus anophagefferens: 18ºC.
  • Bigelowiella natans: 23ºC.
  • Emiliania huxleyi: 19ºC.
  • Guillardia theta: 23ºC.
  • Symbiodinium kawagutii: 23ºC.
  • Thalassiosira pseudonana: 18ºC.