Contents:
Instructions for mediums
Instructions on how to make the black plexiglass boxes
Instructions on how to make a simple version of a hemocytometer
Suppliers

I. f/2 Media
The following are instructions for different kinds of mediums for the phytoplankton experiment.

1. Working nutrient stock

1. NaNO₃ 37.5 g/ 0.5 L dH₂O
2. NaH₂PO₄ . H₂O 2.5 g/ 0.5 L dH₂O
3. NaSiO₃ .9H2O 15,0 g/ 0.5 L dH₂O

a. add to 0.5 L dH₂O: 1.0 ml:
b. CuSO₄ . 5H₂O primary stock
c. ZnSO₄. 7H₂O primary stock
d. CoCl₂ . 6H₂O primary stock
e. MnCl₂. 4H₂O primary stock
f. NaMoO₄. 4H₂O primary stock
g. 2.18 g Na₂EDTA
h. 1.58 g FeCl₃ . 6H₂O

3. Working Vitamin Stock

a.Add to 0.5 L dH₂O (aliquot and freeze)
b.1.0 mL vitamin B12 stock
c. 10.0 mL Biotin
d. 200 mg Thiamine Hcl

4. Primary Metal Stock(should be frozen)

CuSO₄ . 5H₂O 0.490 g/ 50mL dH₂O
ZnSO₄ . 7H₂O
CoCl₂ . 6H₂O 0.500 g/ 50mL dH₂O
MnCl₂ . 4H₂O 0.900 g/ 50mL dH₂O
NaMoO_{4 .} 4H₂O 0.315 g/ 50mL dH₂O

5. Primary Vitamin Stock

Vitamin B12 1.0 mg/ 1.0 mL dH₂O
Biotin 1.0 mg/ 9.6 mL dH₂O (larger volumes are easier to measure)

6.To Make Culture Media

1. Filter and autoclave (or few drops of bleach) seawater (or f/2 media). (volumes larger than 8 L should be autoclave on the long cycle
2. Autoclave in separate Poly pro containers aliquotes of the nutrient and metal stock (#’s 1-4) making sure there is enough for 1 mL/1.0 L seawater
3. When the seawater and nutrients are at room or refrig temps (overnight) add 1-4 aseptically 1.0 mL/ 1.0 L of seawater. (It is not necessary to use different sterile tips for this)
4. Filter sterilize 1:1, v:v dH2O and vitamin and add 0.5 mL/1/0 liter seawater (NOT 1.0 ML AND DO NOT AUTOCLAVE)