F/2 Marine Algae Culture Medium Protocol

Method associated with the Master’s thesis of: Ben Berman

---

The f/2 medium is a commonly used medium for the culturing and growing of marine algae. The original “f medium” was developed in Guillard and Ryther (1962), and was subsequently modifed in Guillard (1975) by reducing the concentration of the original f medium to half, which was termed “f/2 medium”.

Materials & Steps

1. Trace Metal Stock Solution Preperation

• In an Erlenmeyer Flask containing 1 L of double distilled water (DDW), place on a stir plate with a stir bar, add the components in Table 1 weighed to the appropriate mass. Some ingredients could not be obtained and substitutions were performed with different hydrate concentrations yet metal concentration was maintained.
• After all ingredients have been combined and dissolved, remove the stir bar from the flask, and pour the solution through a sterile 0.22 um filter in a sterilized 1 L media storage bottle and seal tightly. Perform this step in a sterilized chemical hood.

If this stock solution is already prepared, please proceed to the next step.

Table 1: F/2 Medium Trace Metal Stock Solution Ingredients

Chemical Ingredient	Grams in 1 L of DDW	Chemical Substitution
EDTA-Na₂	4.36 g	4.83 g of EDTA-Na₂·2H₂O
FeCl₃·6H₂O	3.15 g	n/a
CuSO₄·4H₂O	0.01 g	0.01078 g of CuSO₄·5H₂O
ZnSO₄·H₂O	0.022 g	0.03525 g of ZnSO₄·7H₂O
CoCl₂·6H₂O	0.01 g	n/a
MnCl₂·4H₂O	0.18 g	n/a
Na₂MoO₄·2H₂O	0.006 g	n/a

*n/a = not applicable, no substitution made.

2. Vitamin Mix Stock Solution Preperation

• In an Erlenmeyer Flask containing 985 mL of DDW, place on a stir plate with a stir bar, and add the components in Table 2 measured to the appropriate volumes, obtaining a final solution volume of 1 L.
• After all ingredients are homogenized, remove the stir bar from the flask, and pour the solution through a sterile 0.22 um filter in a sterilized 1 L media storage bottle and seal tightly. Perform this step in a sterilized chemical hood.

If this stock solution is already prepared, please proceed to the next step.

Table 2: F/2 Medium Vitamin Mix Stock Solution Ingredients

Vitamin Component	Target Concentration in 1 L	Volume of Primary Stock Used	Concentration of Primary Stock
Cyanocobalamin (Vitamin B12)	0.0005 g/L	5 mL	0.1 mg/mL
Thiamine HCl (Vitamin B1)	0.1 g/L	5 mL	20 mg/mL
Biotin	0.0005 g/L	5 mL	0.1 mg/mL

3. Filtered Artificial Sea Water (FASW) Preperation

• In an Erlenmeyer Flask containing 1 L of DDW, place on a stir plate with a stir bar, add artificial sea salt mix (Red Sea Salt, Red Sea Ltd.) until a salinity between 38-40 has been reached.
• After the sea salt mixed has fully dissolved, remove the stir bar from the flask, and pour the solution through a sterile 0.22 um filter in a sterilized 1 L media storage bottle sealed tightly. Perform this step in a sterilized chemical hood.

If this solution is already prepared, please proceed to the next step.

4. F/2 Final Solution Preperation

The following steps should be carried out in a sterilized chemical hood.

• In a sterilized 1 L media storage bottle, add 950 mL of FASW (which can be poured through another 0.22 um filter if further sterilization of the solution is required).
• In an Erlenmeyer Flask, add 48 mL of FASW, then add the following chemical components and stock solutions in Table 3 using the appropriate masses and volumes. Mix the flask mannually, and all ingredients should dissolve.
• After all ingredients are combined in the flask, pour the solution through a sterile 0.22 um filter into the sterilized 1 L media storage bottle containing 950 mL of FASW.
• Seal this bottle tightly and genlty shake to mix the contents of the bottle combining both solutions, producing 1 L of the final F/2 medium.

Table 3: F/2 Medium Solution Components

Ingredient	Amount in 48 mL of FASW	Chemical Substitution
NaNO₃	0.075 g	n/a
NaH₂PO₄·2H₂O	0.00565 g	0.004997 g of NaH₂PO₄·H₂O
Trace metal stock solution	1 mL	n/a
Vitamin stock solution	1 mL	n/a

*n/a = not applicable, no substitution made.

Bibliography

• Guillard, R.R.L. (1975). Culture of Phytoplankton for Feeding Marine Invertebrates. In: Smith, W.L., Chanley, M.H. (eds) Culture of Marine Invertebrate Animals. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-8714-9_3
• Guillard, R. R., & Ryther, J. H. (1962). Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Canadian journal of microbiology, 8, 229–239. https://doi.org/10.1139/m62-029