The pH of seawater is very weakly buffered. That is, it is easy to shift the pH either higher [basic] or lower [acidic] very easily. The major agent of buffering in seawater is bicarbonate, but it normally only at a concentration of 2 mM. By increasing the bicarbonate concentration we can increase the buffering capacity to help the pH stay on target for our experiment.

To 500ml of seawater add 1.05 grams [a little less than 1/4 tsp.] of citric acid. Bring the pH to 5.0 and pour off 50ml into a beaker. Adjust the pH to 5.5 and pour off 50ml and so on.

TUBE#	pH
1	8.0
2	7.5
3	7.0
4	6.5
5	6.0
6	5.5
7	5.0

The normal pH of seawater is 8-8.3 so the pH 8 tube acts as our control. Set up five test tubes with a half inch of each of the above solutions.

1. Add one drop of concentrated eggs to each.
2. Swirl and let sit for one minute.
3. Add one drop of diluted sperm suspension and swirl briefly.
4. Wait 5 minutes.
5. Score eggs for fertilization. If time permits can also score for later development [Next period see if divided. Next day see if there are swimming blastula.]