Today we checked to see what development occurred since Wednesday’s strip spawn and KCl-treatment trials. There were so few eggs in the tripours to begin with, that there ended up being way too small of numbers of D-hinge larvae that I ended up dumping the trial and we’ll have to rethink this and re-do it. Details in post.
Counts using coulter counter
We started out using the coulter counter to see if it would work. We screened the tripours (used 31-33 because those are the extras) on 60micron screens. We realized quickly that there was a lot of larger junk that stayed in the sample, including pollen because the table wasn’t covered over the two days. We then swithed to screening with a 90micron screen into a 60 in order to catch the big stuff. I suspended them in 10ml seawater.
The first counts of the coulter counter were pretty high, but then the next two tripours were very low. Counts listed below:
Counts are #cells/ml in 10ml samples.
| Tripour No. | Trtmnt | Count 1 | Count 2 | Count 3 | Avg |
|---|---|---|---|---|---|
| 1 | 0mM KCl | 86 | 75 | 85 | 82 |
| 16 | 50mM KCl with hydration step | 13 | 12 | 11 | 12 |
| 25 | 80mM KCl | 29 | 43 | 34 | 36 |
The coulter counter was likely counting things that weren’t D-hinge.
Counts by hand
We then decided to do counts by hand. I screened them the same way and suspended them in 10ml seawter just like with the coulter counter.
I sampled out 1ml after mixing well by flipping contatiner upside-down a few times. I put the sample on a slide and put two drops of lugols in order to preserve the organisms so that they wouldn’t be moving around when I was trying to count them.
The counts were also very low. I didn’t count all tripours - I just started out by looking at a bunch of different treatments to see if it was worth me continuing and counting them all. Counts are below:
Counts are #D-hinge/1ml in 10ml samples
| Tripour No. | Trtmnt | Count/ml | Total No. |
|---|---|---|---|
| 2 | 0mM KCl | 0 | 0 |
| 4 | 10mM KCl | 4 | 40 |
| 8 | 20mM KCl | 6 | 60 |
| 17 | 50mM KCl + hydration step | 7 | 70 |
| 18 | 50mM KCl + hydration step | 10 | 100 |
| 19 | 60mM KCl | 3 | 30 |
| 26 | 80mM KCl | 0 | 0 |
| 27 | 80mM KCl | 3 | 30 |
| 29 | 50mM KCl without hydration | 3 | 30 |
The numbers are way too small to have too much meaning. Each tripour originally had ~10,000 eggs, so in terms of percentages, the number of eggs that made it to D-hinge range from 0%-0.01%.
Notes from this trial/things to change for next time
- Didn’t have a true “negative control”. A true negative control would have been having a tripour of just eggs (no sperm added to fertilize)
- 10,000 eggs/tripour is sticking with the 10eggs/ml rule that the hatchery has, but maybe it’s too small a scale for capturing any differences in this experiment. Maybe try putting more eggs in the tripours…. or using larger buckets?
- Keep male and female geoduck separate after biopsy punching gonads to determine sex - may be cause of early polar body sightings (see this post).
- Use less treatment groups, easier to manage and may be better to start on a much smaller scale, then get more complex as we learn what works