SCAEL Online Lab Notebook-Media

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

K Medium and Derivatives
(Keller et al. 1987)

K Medium was developed for open ocean phytoplankton. It is highly cheleated to reduce metal toxicity, and it contains ammonia an an organic phosphate (Keller et al. 1987).

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K Medium
(Keller et al. 1987)

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	NH4Cl	2.68 g/L dH2O	3.63 x 10-5 M
1 mL	beta-glycerophosphate	2.16 g/L dH2O	1 x 10-5 M
1 mL*	Na2SiO3 · 9H2O*	30 g/L dH2O*	1.07 x 10-4 M*
1 mL	H2SeO3	1.29 mg/L dH2O	1 x 10-8 M
1 mL	Tris-base (pH 7.2)	121.1 g/L dH2O	1 x 10-3 M
1 mL	K 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: We do not add the silicate to our normal K medium because it causes extensive precipitation. If we add silicate, the recipe symbol is K + Si. K medium also has substantial precipitation without silicate.

 

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K Trace Metal Solution
(Keller et al. 1987)

To 900 mL dH₂O add:

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

Make final volume up to 1 L using dH₂O. Heat to dissolve.

 

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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	1 x 10-9 M
200 mg	Thiamine · HCl	-	1 x 10-7 M

Make final volume up to 1 L with dH₂O. Filter sterilize into plastic vials and store in refrigerator.

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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K Medium Derivatives

K/4: Prepare with ¼ nutrients (e.g., 250 µ L NaNO₃).

K/20: Prepare with one twentieth nutrients (e.g., 50 µL NaNO₃ ).

K + Si: At the CCMP, we routinely prepare K medium without silica. If silica is required, we refer to that medium as K + Si in our database.

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

K + rice: Prepare K 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).

 

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References

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.

Keller, M.D., Selvin, R.C., Claus, W. and Guillard, R.R.L. 1987. Media for the culture of oceanic ultraphytoplankton. J. Phycol. 23: 633-638