Appendix 9 Preparation of culture media

F/2 Nutrient solution
This solution was purchased from Aquatic Eco-systems. It is sold in two parts: Part A and Part B.
For 125 ml, 500 ml and 4 l cultures:

1. Mix equal volumes of each part into a 500 ml dark glass bottle (some of the chemicals are sensitive to light).
2. Cap the bottle with a cotton plug and autoclave according to manufacturer’s instructions.
3. Wrap bottle top with foil and autoclave.
4. Once autoclaved let solution cool.
5. Add vitamin stock solution using microbiological sterile techniques.
6. Close bottle with autoclaved top and label with date.
7. Store at -4 °C.
For 100 l cultures:
1. Use solutions directly from purchased containers.
2. Add equal parts of solutions directly to cultures using 25 ml graduated cylinder or 10 ml graduated pipettes.
3. Add algae in accordance with volume of culture. Instructions on volume of F/2 required per volume of culture are given on the purchased bottles.
Vitamin stock solution
Materials:
– 50 ml volumetric flask and top.
– Glass funnel.
– Q-water bottle.
– Vitamin B12.
– Vitamin B1.
– Biotin.

Method:
1. Autoclave the volumetric flask and the glass funnel.
2. Weigh out vitamins: B12 = 0.005 grams
B1 = 5 grams
Biotin = 0.02 grams
144 Installation and operation of a modular bivalve hatchery
3. Transfer vitamins into the flask.
4. Fill up the flask to total volume of 50 ml with Q-water.
5. Insert a magnetic stirrer and stir.
6. Keep the stock solution in the fridge in the algae container.
Vitamin solution is added to the autoclaved F/2 solution as follows:
1 ml vitamin solution per 1 liter F/2 solution
Sodium metasilicate solution (3 % w/v)
Materials:
– 500 ml dark glass bottle and top.
– Glass funnel.
– Q-water.
– Sodium metasilicate (Na2SiO3).
1. For a 300 ml volume, weigh 9 grams of sodium metasilicate.
2. Pour 300 ml of Q-water into a 500 ml graduated cylinder.
3. Fill a squeeze bottle with this water. In this way you’ll keep track of the volume of water added for dissolution.
4. Gradually transfer weighed sodium metasilicate powder into bottle, diluting solution with Q-water from your squeeze bottle progressively. Make sure to add a small amount of powder at a time otherwise it is difficult to dissolve.
5. Once diluted, cap bottle with cotton plug and autoclave.
6. Wrap glass top in foil and autoclave.
7. Once cool, store solution at -4 °C.
Note: If there is no dark glass bottle, use clear glass bottle wrapped in foil. Sodium metasilicate is sensitive to light.
Sodium metasilicate is added as a supplement to F/2 and vitamin solution for diatom species. Volume added is 2 ml of sodium metasilicate for 1 l of culture.
Convay medium
Preparation of Solution C for a 25 l volume:
KNO3 2 000 grams
K2HPO4 400 grams
EDTA 750 grams
H3BO3 50 grams
TMII 2 000 ml
SWII 125 ml
Vitamin Solution 25 ml
TMII Solution (25 l volume):
Fe2(SO4)3 395 grams
MnSO4.H2O 37.5 grams

ZnSO4.H2O 6.25 grams
CuSO4.5H2O 5.00 grams
CoSO4.7H2O 0.65 grams
NaMoO4.2H2O 0.35 grams
SWII Solution (10 l volume):
SrCl2:6H2O 13.00 grams
AlCl.6H2O 0.50 grams
RbCl.6H2O 0.20 grams
LiCl.H2O 0.10 grams
KI 0.05 grams
KBr 0.65 grams
Vitamin Stock Solution (1 l volume):
B12 0.10 grams
B1 100.00 grams
Biotin 0.40 grams
F/2 media
1. Nitrate NaNO3 75 g/l
2. Phosphate NaH2PO4.H2O 5 g/l
3. Silicate Na2SiO3.9H2O 30 g/l
4. Trace Metals FeCl3.6H2O 3.5 g
Na2EDTA 4.36 g
Dissolve in 900 ml distilled H2O.
Add 1 ml of each of the following trace metal solutions.
– CuSO4.5H2O 0.98 g/100 ml
– ZnSO4.7H2O 2.2 g/100 ml
– CoCl2.6H2O 1.0 g/100 ml
– MnCl2.4H2O 18.0 g/100 ml
– Na2MoO4.2H2O 0.63 g/100 ml
Bring solution to 1 l with distilled H2O (pH about 2.0).
Add 1 ml/l of filtered seawater of the above solution (#1-4).
5. Vitamins: Biotin 1.0 mg
B12 1.0 mg
Thiamine HCl 20.0 mg
Dissolve in 1 l distilled H2O. Store in refrigerator.
Add 0.5 ml of vitamin solution for every 1 l filtered seawater.