1.

Algal Culture Media

In their natural habitats algae obtain all the nutrients, minerals and vitamins they
require from the water in which they live. To grow them in the lab however, you must
provide them with all of these essential resources i.e. you will need to make up some
growth media.

Algae media refers to the solution or culture in which algae grow. All the media have
several components in common: sources of nitrogen (in the from of nitrate, nitrite and
ammonia), phosphorus, vitamins and trace metals. However the specific types of
these nutrients, their concentrations and ratios vary between the media.

F/2 Medium There are many recipes for such algal growth media, F/2 medium
however, is the most common and widely used general enriched seawater medium
designed for growing marine algae used in the phycological and aquaculture studies.

Stock solutions and salts

In any recipes you will see two types of stocks “working stocks” and “primary
stocks”.
Working stocks are those whose aliquots contribute directly to making the final
media.
Primary stocks are normally made where several single substance solutions are then
combined to form the working stock.
Stock solutions are made up by accurately weighing the prescribed amount of nutrient
and dissolving in a specified volume of distilled water, if possible in a volumetric
flask. Some nutrients will readily dissolve, others need heat and stirring to fully
dissolve. In contrast vitamin stocks are heat sensitive and should not be subjected to
heat treatment and should also be kept in the dark. Failure to fully dissolve the
primary stocks of some nutrients such as EDTA can lead to gross precipitation when
these stocks are combined to make the media.
Nutrients come with different salts and hydration. For example, while copper and zinc
may be two desired active constituents they are readily obtained from suppliers with
either SO4 or Cl2 salts (ie CuSO4 or CuCl2 and ZnSO4 or ZnCl2). Some nutrients also
come with different hydrations, ie the .nH2O suffix. Substituting one form for another
may have no effect on the growth of some microalgae species, but it can lead to poor
growth in others and also lead to unwanted and time consuming precipitation
problems as the overall ratio of salts in the medium has changed. Therefore deviating
from the prescribed recipes is to be avoided and ordering the correct form is
recommended.

Seawater source and treatment

The marine microalgae species should be grown using unpolluted oceanic seawater or
artificial seawater. Artificial seawater media is composed of marine salts and
nutrients added to pure freshwater. Artificial seawater is only necessary where a clean
natural seawater source is unavailable or in particular research studies where the exact
composition needs to be controlled. Off-shore sites have very low concentrations of
metal and organic pollutants therefore it suitable as the base medium for a wide range
of marine microalgae species.

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The seawater should be collected in clean black polyethylene containers and then
stored until needed (preferred at 4 0C). Then it is treated using a filtration.

F/2 Medium Stock Solutions:

Quantity
Stock
per liter of Compound Concentration
Media
1.0 ml NaNO3 75.0 g/L dH2O Add to Media
Autoclave
1.0 ml NaH2PO4.H2O 5.0 g/L dH2O
separately
Autoclave
1.0 ml Na2SiO3.9H2O 30.0 g/L dH2O
separately
f/2 Trace Metal Autoclave
1.0 ml (see recipe below)
Solution separately
Filter sterilized
f/2 Vitamin
0.5 ml (see recipe below) and added to
Solution
autoclaved Media

Mineral salts working solution

1. NaNO3 75.0 g/L dH2O Add to Media

Add Nitrate directly to filtered seawater and autoclave.

Use: 1 ml per litre of seawater medium.

2. Autoclave
NaH2PO4.H2O 5.0 g/L dH2O
separately
3. Autoclave
Na2SiO3.9H2O 30.0 g/L dH2O
separately

Put the salts into one 1-I screw-capped oven-resistant glass bottle and fill with DW to
the mark. If not available, deionized water can also be employed. When fully
dissolved, autoclave separately aliquots of the phosphate and silicate. Store at ambient
temperature, avoiding direct light. Add these aseptically to your media once it has
cooled to room temperature. Store non-sterile stocks in the refrigerator.

Use: 1 ml per litre of sterilized seawater medium.

If the alga to be grown does not require silica e.g diatom, then it is recommended that
the silica be omitted.

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4. Trace Metal Solution

To 950 mL distilled H2O add:

Quantity Compound Stock Solution

1.3 g FeCl3.6H2O -
8.7 g Na2EDTA.2H2O -
1.0 ml CuSO4.5H2O 980 mg / 100 ml dH20
1.0 ml Na2MoO4.2H2O 630 mg / 100 ml dH20
1.0 ml ZnSO4.7H2O 2.2 g / 100 ml dH20
1.0 ml CoCl2.6H2O 1.0 g / 100 ml dH20
1.0 ml MnCl2.4H2O 18.0 g 100 ml dH20

Solution will initially be cloudy. Add 1N NaOH to adjust pH to about 4.5; solution
should clear unless too much NaOH has been added. Bring final volume to one liter.

Use: 1 ml per litre of sterilized seawater medium.

5. Vitamin Solution
First, prepare primary stock solutions for vitamin B12 and biotin (Vitamin H) according to the
proportions indicated below. To prepare final vitamin solution, begin with 950 mL of dH2O,
dissolve the thiamine, add the amounts of the primary stocks as indicated in the quantity
column below, and bring final volume to 1 liter with dH2O.

Warning: do not autoclave any vitamin solution but Filter sterilize.

Quantity Compound Stock Solution Tip

1.0 ml Vitamin B12 10 mg / 10 ml dH2O
Dissolve in a little 0.1N NaOH
1.0 ml Biotin 10 mg / 10 ml dH2O
first
200.0 mg Thiamine HCl -

Store primary stock solutions in freezer. Store completed vitamin solution in the
refrigerator or freeze small aliquots.

Use: 0.5 ml per litre of sterilized seawater medium.

Fig. Working solutions ready to use