Aiptasia Pollution Experiment: Lab Instructions
Jennifer Welborn
Learning Goals
 Use a digital camera and ADI software to observe and measure the effect of
crude oil and a chemical dispersant on a sea anemone, Aiptasia pallida
 Understand that Aiptasia pallida is used as a model for the effects of
environmental stressors---in this case crude oil (oil spills) and a chemical
dispersant (used to “clean up” the oil)-- on coral
 Understand that environmental stressors on coral lead to coral bleaching—which
happens when the coral kick out the symbiotic brown/green colored algae, which in
turn causes the coral to turn white. In time, this leads to coral death since the
algae provides food for the coral.
Materials Needed for the Crude Oil/Dispersant Lab Investigation (for 1 lab group)
1. 1” x 1” x 1” plastic magnifying box.
2. A tank with a culture of Aiptasia anemones living in it
3. Motor oil or vegetable oil
4. Liquid soap or liquid detergent designed to “cut grease”
5. 2 small cups/beakers – one with oil and one with dispersant/detergent
6. 3 eyedroppers or pipettes
7. pen or pencil
8. Index card
9. 8 1/2” x 11” white paper* If you use colored paper ( a color which is drastically
different from the Aiptasia as the background ) you may not need to have to use
color enhancement in ADI to get accurate masked pixels.
10. tripod or ring stand for photographing under constant distance, light conditions,
angle, etc.
11. Digital camera, of course.
12. Place for photographing with stable source of ambient light.
13. ADI software. Can be downloaded from: http://k12s.phast.umass.edu/digital/
Procedure --- Groups 1-5
1. Tape a piece of 8 ½” x 11” piece of white paper down onto the lab bench.
2. Set up a camera tripod/ringstand to take photos under consistent conditions.
3. Make three labels from the index card:
4.
Control
2 drops of oil
2 drops of Oil + 2 drops
of Dispersant
5. Obtain a small plastic magnifying box with a living Aiptasia anemone in it.
6. Place the box in the center of a piece of white paper. Let the anemone
relax for a while so that its tentacles are fully extended. This indicates a
contented (healthy and not stressed) anemone .
7. Take a control photograph of the anemone making sure that the Control label is
visible in the photo.
8. Using an eyedropper, add two drops of oil to the surface of the water in the
magnifying box.
9. Wait 2 minutes.
10. Take a picture of the Aiptasia which has been exposed to oil after 2 minutes. Make
sure the 2 drops of oil label is visible in the photo.
11. To get a clear photo of the Aiptasia which has been exposed to oil, you need to
clear the oil away. Do this by adding 2 drops of dispersant (detergent) to the
surface of the water then QUICKLY take a photo of the Aiptasia as soon as it is
visible.
12. If you did not already put 2 drops Put two drops of dispersant on top of the oil (if
you did not already do this in order to get a good image of the Aiptasia.)
13. After you take a quick photo of the Aiptasia which was exposed to oil, wait two
minutes again.
14. Again, take a photo being sure to include the 2 drops of oil + 2 drops of dispersant
label in the photo.
15. Use the third, clean eyedropper to carefully gather the anemone and return it to
the tank.
Procedure --- Groups 6-11
1. Follow the same steps outlined above but create 2 labels only. You will be testing
the effect of just the dispersant.
Control
2 drops of dispersant
USING THE ADI SOFTWARE—
The tools chosen for this analysis are Enhance Colors, Masked Colors and Spatial
Enhancement. Mask Colors enables you to find the area of an image because when
you identify which colors make up the pixels of the image, you can ask for pixels
with those color intensities to be made black. Then, the blackened pixels can be
counted and compared with other images to see change in area.
Enhance Colors tool is used because it enables you to isolate the image of choice
(the Aiptasia) from its surroundings by color. The Aiptasia is on white paper.
Therefore, the R, G, B values are equal in amount and intensity for the white.
However, the Aiptasia is sort of yellow which means that there is more red and
green in it than blue. By picking red vs blue normalized, you accentuate the red,
thereby isolating the critter from the white paper. Finally, the Polygon Tool in
Spatial Analysis enables you to isolate only the masked pixels in the image which are
representative of the image of choice, not pixels due to reflection, shadows, etc.
This gives you the area of only the selected image.
USING THE ADI SOFTWARE—
1. Download the images of the Aiptasia into your computer.
2. Click on Analyzing Digital Images
3. Click on Enhance Colors and open up the first photo you took of the Aiptasia (the
control).
4. Large image detected—choose yes
5. At Select trim option—choose Full Image at Selected resolution
6. Click on trim and use image
7. Select None
8. Note RGB histograms—the three tall RGB sections represent the white background.
The small (low intensity) bumps in the wings represent the Aiptasia
9. Select predefined enhancement
10. Click on drop down menu—red vs blue normalized—(this is chosen because in the
color histogram, red and green were similar to each other different from blue. In
this example red vs blue was chosen but you get similar results when green vs blue
is chosen.
11. Zoom in and pan around to have the critter be large and centered.
12. Click on Mask Colors.
13. Draw a box within the Aiptasia that covers a good sample of the variety of pixels.
14. Note—minimum and maximum values given for you
15. Click on Apply Mask Test 1. Compare the masked image with the original so see how
closely you have come to replicating the critter in black.
16. Adjust the min/max (play around with those values) and repeat Apply Mask Test 1
until you have closely replicated the critter in black.
17. Click on Spatial Analysis.
18. Click on Polygon Tool. Toggle back from the masked image to the original image.
19. Draw a polygon around the original image.
20. Toggle back to the masked image. Note the number of masked pixels inside the
polygon that you drew.
21. Repeat with the other photos and compare.
22. Compare the data you got with other groups who did the same experiment.
QUESTIONS TO CONSIDER
1. What do you notice about the effect of crude oil and dispersant on the Aiptasia?
2. What implications might this have for the effect of oil spills and dispersants on
bottom-dwelling organisms?