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(Larsen et al. 1994)

This medium is a modification of an Erd-Schreiber medium. Originally we used it as described by Throndsen (1969, 1978), hence the designation T-medium, but later again we made our own formula. The variations are subtle and without great importance. The TL-medium differs by the addition of L1 trace metals.

We use TL-medium as standard media for isolation and maintenance of marine autotrophic flagellates; because this medium is not autoclaved, it is not suitable for axenic cultures. Our (rather comprehensive) experiments have shown that many marine autotrophic flagellates grow better TL- medium than in many other standard media, probably because of the lack of autoclaving.

The figure following the letter(s) indicates the salinity of the seawater used (e.g. T20 = T-medium based on 20 ‰ filtered seawater).

The use of L1 trace metals in the modified Erd-Schreiber medium has improved the growth (and often the durability) of many cultures of Dinophyceae, Chrysochromulina and other Prymnesiophyceae. Our cultures of Micromonas pusilla also grow better after addition of the L-solution.

Many benthic algae grow well in TL medium, sometimes better than in the standard medium for these types of algae (MV), probably because of the content of TRIS in the MV-medium.

1. Stock solutions for major elements

NaNO3 10g/100 mL
Na2HPO4•12 H2O 2g/100 mL

Store the solutions refrigerated.

2. Primary stock solutions for L1 trace elements

MnCl2•4H2O 18.0 g/100 mL
ZnSO4•7H2O* 2.3 g/100 mL
CoCl2•6H2O* 1.19 g/100 mL
CuSO4•5H2O 0.245 g/100 mL
Na2MoO4•2H2O 1.99 g/100 mL
H2SeO3 0.13 g/100 mL
NiSO4•6H2O 0.27 g/100 mL
Na3VO4 0.184 g/100 mL
K2CrO4 0.194 g/100 mL

Notice: 2.2g and 1.1 g/100mL are given in the orig. recipe, but the here used concentrations give a final concentration as specified in the orig. recipe (see Guillard & Hargraves 1993).

3. Trace metal working stock solution
1) Dissolve 4.36 g Na2EDTA•2H2O and 3.15 g FeCl3 6 H2O in ca. 900 mL dH2O in a 1000-mL volumetric flask.
2) Add 1 mL of each trace metal primary stock solution.
3) Bring to 1000 mL with dH2O.
4) Autoclave

4. Vitamin stock solution

Thiamine HCL (B1) 200 mg
Biotin 1 mg
Cyanocobalamine (B12) 1 mg
dH2O to 1000 mL

The vitamin stock solution is dispensed to appropriate volumes and kept frozen in vials of polyethylene until use.

5. Soil extract
500 mL dry good quality but non-fertilized garden soil is placed in a large Erlenmeyer flask and wet with 1L glass distilled water. Approx. 0.5g CaCO3 (the amount is not critical) is added and the mixture is autoclaved. After autoclaving, let the mixture stand ca. 24h and filter through a normal laboratory paper filter. The filtration process easily takes one day. After filtration, the filtrate is filtered again, divided into lots of about 100 mL, autoclaved, and stored in the refrigerator. We do not use the extract before it has become clear. It usually takes several weeks. Be careful not stir up the sediment when using the extract.

6. Final preparation of T and TL
To each liter of seawater, adjusted to the desired salinity, add:
1 mL NaNO3 stock solution
1 mL Na2HPO4·12H2O stock solution
3 mL Soil Extract
1 mL L trace metal working stock solution

either 0.25 mL NaFe EDTA stock solution (for T-medium)
or 1 mL L trace metal working stock solution (for TL-medium)
The medium is heated in a waterbath to 80°C for 15 minutes and then cooled as fast as possible to avoid precipitation.
After cooling, 2 mL of the non sterile vitamin stock solution is added, using a disposable syringe and a disposable, sterile, 0.2-µm membrane filter.

Larsen, N.H., Moestrup, Ø. & Pedersen, P.M. 1994. Catalogue 1994. Scandinavian Culture Centre for Algae & Protozoa. Department of Phycology. Botanical Institute. University of Copenhagen.
Throndsen, J. 1969. Flagellates of Norwegian Coastal Waters. Nytt Mag. Bot. 16(3-4), 161-216.
Throndsen, J. 1978. The dilution-culture method. In Sournia, A. (Ed.) Phytoplankton manual. UNESCO. Paris 337 pp.