SCAEL Online Lab Notebook-Media

reprinted from the
Provasoli-Guillard National Center for
Culture of Marine Phytoplankton (CCMP)
http://ccmp.bigelow.org/

f/2 Medium and Derivatives
(Guillard & Ryther 1962, Guillard 1975)

Below are recipes for f/2 medium, its derivatives (e.g, f/2 agar, f/2-Si, f/2 + Se, f/4, f/50) and related media (e.g., Black Sea). F/2 is listed first, followed by derivatives of f/2.

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f/2 Medium

(Guillard & Ryther 1962, Guillard 1975)

To 950 mL filtered seawater add:

Quantity	Compound	Stock Solution	Molar Concentration in Final Medium
1 mL	NaNO3	75 g/L dH2O	8.83 x 10-4 M
1 mL	NaH2PO4 · H2O	5 g/L dH2O	3.63 x 10-5 M
1 mL *	Na2SiO3 · 9H2O*	30 g/L dH2O*	1.07 x 10-4 M*
1 mL	f/2 trace metal solution	(see recipe below)	-
0.5 mL	f/2 vitamin solution	(see recipe below)	-

Make final volume up to 1 L with filtered seawater and autoclave.
*Note: Autoclaved f/2 medium produces extensive silica precipitate. We delete silicate when it is not required by the alga (see f/2-Si medium below).

 

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f/2 Trace Metal Solution
(Guillard & Ryther 1962, Guillard 1975)

To 950 mL dH₂O add:

Quantity	Compound	Stock Solution	Molar Concentration in Final Medium
3.15 g	FeCl3 · 6H2O	-	1 x 10-5 M
4.36 g	Na2EDTA · 2H2O	-	1 x 10-5 M
1 mL	CuSO4 · 5H2O	9.8 g/L dH2O	4 x 10-8 M
1 mL	Na2MoO4 · 2H2O	6.3 g/L dH2O	3 x 10-8 M
1 mL	ZnSO4 · 7H2O	22.0 g/L dH2O	8 x 10-8 M
1 mL	CoCl2 · 6H2O	10.0 g/L dH2O	5 x 10-8 M
1 mL	MnCl2 · 4H2O	180.0 g/L dH2O	9 x 10-7 M

Make final volume up to 1 L with dH₂O. Autoclave.

 

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f/2 Vitamin Solution
(Guillard & Ryther 1962, Guillard 1975)

To 950 mL dH₂O add:

Quantity	Compound	Stock Solution	Molar Concentration in Final Medium
1 mL	Vitamin B12 (cyanocobalamin)	1.0 g/L dH2O	1 x 10-10 M
10 mL	Biotin	0.1 g/L dH2O	2 x 10-9 M
200 mg	Thiamine · HCl	-	3 x 10-7 M

Make final volume up to 1 L with dH₂O. Autoclave and store in refrigerator. Note: Vitamin B₁₂ and biotin are obtained in a crystalline form. When preparing the vitamin B₁₂ stock solution, allow for approximately 11% water of crystallization (for each 1 mg of Vitamin B_12, add 0.89 mL dH₂O). When preparing the biotin stock solution, allow for approximately 4% water of crystallization (for each 1 mg of biotin, add 9.6 mL dH₂O).

 

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f/2 Derivatives

Black Sea Medium: For brackish water organisms (16 psu, half-strength nutrients). Combine 500 mL f/2 medium and 500 mL dH₂O. Autoclave.

f/2 agar: Prepare 1 liter of f/2 medium and dissolve 9g Bacto-agar (heat and mix). For test tubes, dispense dissolved agar medium into tubes, autoclave, and then cool with tubes slanted at an angle. For Petri plates, autoclave in a flask, cool almost to the gelling point, and then aseptically dispense into sterile Petri plates. Note: Agar can be added to other media (e.g., f/50 agar), and agar concentration can be varied to produce softer or firmer substrates.

f/2-Si: Prepare as for f/2 medium but omit Na₂SiO ₃ · 9H₂O. This is preferred over f/2 medium for organisms with no silica requirement because less precipitation forms.

f/2 + Se: Extra silicon and selenium are beneficial to several diatom strains. Prepare 1 L of f/2 medium but use 2 mL of silicate stock, then add 1.0 mL of selenium stock solution (1.29 mg H₂SeO ₃ /L distilled H₂O). Autoclave.

f/2 (11 psu): For brackish water organisms. Mix 650 mL distilled H₂O and 350 mL filtered seawater. Add f/2 medium nutrients and autoclave.

f/2-Si (24 psu): Mix 750 mL distilled H₂O and 250 mL filtered seawater. Prepare as for f/2 medium but omit Na₂SiO ₃ · 9H₂O.

f/4: Add 500 mL f/2 medium to 500 mL filtered seawater, then autoclave.

f/4-Si: Autoclave 1 L of filtered seawater. When cool, aseptically add f/2-Si nutrients at half concentration (i.e., 0.5 mL).

f/20-Si: Autoclave 1 L of filtered seawater. When cool, aseptically add f/2-Si nutrients at one tenth concentration (i.e., 100 μL).

f/50-Si: This is more than a 1/25 dilution of f/2-Si medium. We autoclave 1 L of seawater in a Teflon-lined bottle. Wait for the autoclaved seawater to cool to room temperature (important). Aseptically add 40 μL of sterile f/2 nutrients (20 μL of vitamins).

f/50-Si + CCMP1320 as food: Prepare f/50 and aseptically add 50 μL of healthy, moderately dense culture of CCMP1320.

f/2m: To 1L f/2 medium add 1 g methylamine · HCl, mix until dissolved and autoclave. This medium is used to test for contamination by methylaminotrophic bacteria.

f/2p: To 1 L f/2 medium, add 1 g Bacto-peptone, mix until dissolves and autoclave. This medium is used to test for contamination by non- methylaminotrophic bacteria and fungi.

f/2pm: To 1L f/2 medium add 1 g Bacto-peptone and 1 g methylamine · HCl, mix until dissolved and autoclave. This general medium is used to test for contamination by bacteria and fungi.

f/2 + NPM: Add f/2 nutrients to 900 mL of seawater and autoclave. After cooling, aseptically add 100 mL of the following organic stock solution. Dispense aseptically into test tubes.

 

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Organics Stock Solution

(modified from Guillard 1960)

To 900 mL dH₂O add:

Quantity	Compound
1 g	sodium acetate
6 g	glucose
3 g	(di-) sodium succinate · 6H2O
4 g	neopeptone
1 g	Bacto-tryptone
100 mg	yeast extract

Bring up to 1 L with dH₂O. Dispense in small aliquots and autoclave.

 

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References

Guillard, R.R.L. 1960. A mutant of Chlamydomonas moewusii lacking contractile vacuoles. J. Protozool. 7: 262-268.

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

Guillard, R.R.L. and Ryther, J.H. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: 229-239.