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.

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 per liter of Media 1.0 ml 1.0 ml 1.0 ml 1.0 ml 0.5 ml

Compound
NaNO3 NaH2PO4.H2O Na2SiO3.9H2O f/2 Trace Metal Solution f/2 Vitamin Solution

Stock Concentration 75.0 g/L dH2O 5.0 g/L dH2O 30.0 g/L dH2O (see recipe below) Add to Media Autoclave separately Autoclave separately Autoclave separately

Filter sterilized (see recipe below) and added to 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. 3. NaH2PO4.H2O Na2SiO3.9H2O 5.0 g/L dH2O 30.0 g/L dH2O Autoclave separately Autoclave 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.

4. Trace Metal Solution To 950 mL distilled H2O add: Quantity 1.3 g 8.7 g 1.0 ml 1.0 ml 1.0 ml 1.0 ml 1.0 ml Compound FeCl3.6H2O CuSO4.5H2O ZnSO4.7H2O CoCl2.6H2O MnCl2.4H2O 980 mg / 100 ml dH20 2.2 g / 100 ml dH20 1.0 g / 100 ml dH20 18.0 g 100 ml dH20 Na2EDTA.2H2O Na2MoO4.2H2O 630 mg / 100 ml dH20 Stock Solution

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

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