SCAEL Online Lab Notebook-Media

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

Prov Medium and Derivatives

(CCMP, see Andersen et al. 1997)

Prov medium and several derivatives (e.g., Blue Star agar, Prov50, Prov100) (see below) are based upon f/2-Si medium enriched with soil water extract. This is a variant of Erd-Schreiber's medium and many other soil-extract enriched media.

 

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Prov Medium

Prepare 1 L of f/2-Si medium (see below); add 15 mL of alkaline soil extract (see below) and autoclave.

 

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Alkaline Soil Extract

(Provasoli et al. 1957)

Combine two parts dH₂O with one part rich organic garden soil (containing no recent applications of chemical fertilizer or pesticides). Add 2-3 g NaOH/liter. Autoclave for 2 hours, cool and filter. This concentrated extract is then diluted 50:1 with dH₂O to make the final working stock.

 

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f/2-Si 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	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.

 

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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 and 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 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 1.0 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 1.0 mg of biotin, add 9.6 mL dH₂O).

 

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Prov Medium Derivatives

Prov50: Prepare 1 L of Prov medium, add 100 µL of a 500 mM NH₄Cl stock solution (26.7 g/liter in dH₂ O) and autoclave.

Prov100: Prepare 1 L of f/2-Si medium (see below), add 10 mL alkaline soil extract (see below), add 200  µL of a 500 mM NH₄ Cl stock solution (26.7 g/liter in dH₂O) and autoclave.

Blue Star agar: Prepare Prov medium and include Na₂SiO ₃ · 9H₂O (final medium concentration of 1.07 x 10 ^-4 M; use 1 mL of a 30 g/L dH₂O stock solution). 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 concentration can be varied to produce softer or firmer substrates.

Prov100 agar: Prepare 1 L of Prov100 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 concentration can be varied to produce softer or firmer substrates.

Prov modified (24 psu): For brackish water organisms. Mix 750 mL of filtered seawater, 245 mL of dH₂O, 15 mL of alkaline soil extract, and then add f/2-Si nutrients. Autoclave.

Prov50 + rice: Prepare Prov50 medium, dispense into test tubes or small flasks, and autoclave. Add a single dry rice grain to the medium when inoculating with cells. Note: This medium stimulates slow, controlled bacterial growth for algae that feed by phagocytosis. If the rice grain is autoclaved, it becomes too soft and too much organic is released into the medium, causing very dense bacterial growth (and often anoxia).

Prov50+NPM: Prepare the organic stock solution (see below). Prepare 975 mL of Prov50 medium, autoclave and cool. Aseptically, add 25 mL of the organic stock solution.

 

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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 Chanley, 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.

Provasoli, L., McLaughlin, J.J.A., and Droop, M.R. 1957. The development of artificial media for marine algae. Arch. Mikrobiol. 25: 392-428.