The Effect of Cryonics on the Viability of Sea Urchin Gametes

By Stephanie A., 9^th grade

Contents:

Abstract	Project Log
Purpose	Background Report
Hypothesis	Results
Experiment Design	Conclusion
Materials	Bibliography
Procedures	Appendix

Abstract:

The purpose of this experiment was to determine whether cryogenic affects the fertilization of sea urchin gametes freezing them at different lengths of time and regenerating them in three different ways. I became interested in this idea when I was looking through the Internet. The information gained from this experiment could someday help people decide whether they should be frozen or not in case there is ever a cure for a certain decease they might have.

My hypothesis was that the slower I regenerated the eggs, the more chances there were that the gametes would die. Also, the longer I froze the eggs and sperm, the greater the chance that they would fertilize. I based my hypothesis on an Internet web site called “Cryobiology- The analysis of the effects of extremely low temperatures on things.” The web site stated that regenerating the cells quicker would increase the chance for the cells to survive.

The constants in this study were:

- the type of sea urchins

- the equipment

- the microscope objectives

- the amount of potassium chloride injected in the sea urchin

- the amount of sea urchin eggs being frozen (5ml)

- the amount of sea urchin sperm being frozen (one single drop)

The manipulated variable was the different lengths of time I let the gametes stay cryopreserved. Another manipulated variable was the three different temperatures at which I regenerated the eggs and sperm.

The responding variable was the percentage of eggs that were fertilized. To measure how many eggs were fertilized, I put the eggs under a microscope and randomly selected different sections of the petri dish and to zoom in on at 100x. Then I counted how many eggs there was in total and how many fertilized. Repeated this ten times.

The controlled group in this experiment was the eggs and sperm that were not frozen, but had been observed right after the sea urchin released their eggs or sperm.

The results of this experiment were that the longer I froze the gametes the greater percentage of them died and the slower I regenerated the eggs and sperm, the percentage of fertilized gametes increased. Therefore, the results indicate that my hypothesis should be rejected.

If I were to conduct this experiment again, I would get an earlier start of planning the whole project. I would make sure all my equipment is ordered and mailed to me long before I start the experiment. If I were to use sea urchins again, I would make certain the temperature of my aquarium is at a very low temperature.

Purpose:

The purpose of this experiment was to determine whether cryonics affects the fertilization of sea urchin eggs and sperms cryopreserving the eggs and sperm at different lengths of time and regenerating them in three different ways. I became interested in this idea when I was looking through the Internet for science project ideas. I came upon a web page that students all over the country had put their ideas into to share with the world. The information gained from this experiment could help people understand and learn more about what cryogenics is. Also, the results of this experiment may help some people to decide whether they should be frozen someday or not in case there is ever a cure for a certain decease they might have. Furthermore, I will determine the effects of cold storage on sex cells.

Hypothesis:

My hypothesis was that the slower I regenerated the gametes, the more chances there were that the eggs and sperm would die. Also, the longer I froze the eggs and sperm, the greater the chance that they would fertilize. I based my hypothesis on an Internet web site called "Cryobiology- The analysis of the effects of extremely low temperatures on things." The web site stated that regenerating the cells quicker would increase the chance for the cells to survive. Cooling rates are "slow" and "fast" depending on whether heat leaves the cell more slowly or more quickly than the water does. The extracellular solution almost always leaves first. The extracellular solutes are concentrated in the remaining unfrozen extracellular water, and, if cooling rates are slow enough, this dehydrates the cell by osmosis as water diffuses from the cytoplasm into the more concentrated external solution.

Experiment Design:

The constants in this study were:

• the same type of sea urchins
• the same liquid nitrogen
• the same equipment
• the same microscope (and objectives)
• the same atmospheric pressure
• the same artificial salt water
• the same laboratory
• the same amount of potassium chloride injected in the sea urchin
• the same amount and source of sea urchin eggs being frozen (5ml)
• the same amount and source of sea urchin sperm being frozen (one single drop)
• the same temperature of the refrigerator (4 degrees Celsius)

The manipulated variable was the different lengths of time I let the eggs and sperms stay cryopreserved in the liquid nitrogen. In addition, another manipulated variable was the three different temperatures at which I regenerated the eggs and sperm.

The responding variable was the percentage of eggs that were fertilized after the cryopreservation. To measure how many eggs were fertilized after the cryopreservation, I put the eggs under a microscope and randomly selected different sections of the petri dish and to zoom in on at100x. Then I counted, in total, how many eggs there were. Next, I counted how many eggs were fertilized. I repeated this ten times.

The controlled group in this experiment was the eggs and sperm that were not frozen, but had been observed right after the sea urchin released their eggs or sperm.

Materials:

Quantity Item Description Cost to the experimenter

1	Dewer's Flask	$0.00
30.32L (8 gallon)	Liquid Nitrogen	$43.04
36	Cryovials	$102.00
50ml	Potassium Chloride	$0
10	Petri Dishes	$0
1	Microscope	$0
32	Sea Urchins	$220.00
2	Hot Plate	$0
2.2L (10gallons)	Artificial sea water	$16.99
3	Marine AQ. Gravel	$20.22
1	Hydrometer	$9.99
3	Glass pipette	$0
3	Syringe	$0
3	Stopwatch	$0
50ml	Ethanol	$0
1 (2.2L or 10gallon)	Aquarium	$0
1 (100ml)	Beaker	$0
1 (24 shots)	Film	$0
1	Tong	$0
1	Project board	$0
3	Thermometer	$0
1 pair	Gloves	$0
1	Goggle	$0
1	Stirring Rod	$0
3 (500ml)	Beakers	$0
2 (250ml)	Beakers	$0
5	slides	$0
10	Cover Slips	$0

Procedures:

The procedures I intend to use are:

1. Label each cryovial indicating how long each one should be cryopreserved and specifying how to regenerate the gametes. Label the cryovials as follows:

• 30 minutes (room temp, level 1, level 2), trial 1, 2, or 3
• 5 hours (room temp, level 1, level 2), trial 1, 2, or 3

- 1 day (room temp, level 1, level 2), trial 1, 2, or 3

• 3 days (room temp, level 1, level 2), trial 1, 2, or 3
• 1 week (room temp, level 1, level 2), trial 1, 2, or 3

2. Wear all safety equipments and double check if everything is set up right.
3. In a 250mL beaker filled with seawater, set the sea urchin on it.
4. Using a 3cc syringe with 0.5ml-1.5ml of potassium chloride is inserted into the sea urchin between the teeth and the hard outer shell.
5. A small amount of potassium chloride is injected into the sea urchin on both sides. This induces the sea urchin to spawn.

I. If the sea urchin is male:

a. After the male sea urchin has been injected, he is placed on a dry surface like a petri dish mouth side down.

b. After a minute or two you will start to see the white sperm appearing on the surface of the urchin.

c. Collect the sperm "dry" in a glass pipette or eyedropper and place it in a test tube. (Seawater "activates" the sperm reducing their life span from days to minutes. Keep concentrated until just before use.)

d. After you have collected all the sperm that you can, place the tube into a refrigerator if you don’t intend to use them immediately. Sperm will keep at 4C (39.2 F) for about 2-5 days.

e. Using the glass pipette put a single drop of sperm into each of the12 cryovials.

II. If the sea urchin is female:

a. After the female sea urchin has been injected, she is placed mouth side up over a beaker full of seawater. Eggs will not shed unless in contact with seawater. Beaker should be slightly smaller than the diameter of the sea urchin.

b. The eggs will be shed into the seawater and collected at the bottom of the beaker. This can take from 10-45 minutes for the female urchin to finish shedding the eggs.

c. It is best to keep the eggs and urchin at the same temperature.

d. Mix the eggs gently with a stirring rod and using a glass pipette put 5mL of eggs into each of the 12 cryovials.

1. Put the 24 cryovials (eggs and sperm) in the dewar’s flask containing the liquid nitrogen with tongs.
2. Start timing right when you put the cryovials in.

a. After 30 minutes:

1. Pre-heat the two hot plates and fill three beakers 2/3 full of water.

2. Take two beakers and put one on each of the hot plates.

3. Take out the 6 cryovials (3 eggs and 3 sperm) out of the dewer’s flask with tongs and wipe the cryovials with ethanol or isopropanal. Put one cryovial from each onto a beaker until it is completely melted.

4. Take another cryovial from each and expose both on a hot plate until it is completely melted.

5. Take the last two cryovials and put it under another hot plate but at a different temperature until it is completely melted.

6. Make sure to release the pressure building up inside the cryovials. Don’t open the cryovial all the way just halfway.

7. When the cryovials are completely melted, mix the eggs and the sperm together in one petri dish.

8. Wait for 2 minutes for the eggs to fertilize.

9. After 2 minutes, take the petri dish and put it under a microscope.

10. Once the microscope is focused, randomly select different sections of the petri dish and zoom in 100x.

11. Count how many, in total, eggs there are. Then count how many eggs there are fertilized and how many that is not fertilized.

12. Do the counting ten times.

b. After 5 hours:

1. Follow steps 1-12 in part ‘a’

c. After 1 day:

1. Follow steps 1-12 in part ‘a’.

d. After 3 days:

1. Follow steps 1-12 in part ‘a’.

e. After 1 week:

1. Follow steps 1-12 in part ‘a’.

1. Repeat steps 1- 7 two more times to make sure your data is accurate.

2. After you have done 3 trials of steps 1-7 do steps 2-5 for the controlled group.

3. In the controlled group you will take 5ml of eggs and a drop of sperm and put them in a petri dish.

4. Let it stand for about two minutes for it to fertilize.

5. Put it under a microscope and randomly select different sections of the petri dish and count how many eggs there are in total and how many are fertilized.

Do the counting ten times.

Project Log:

Date Time Procedures/Observations

11-16-98 55min. My teacher started to talk about science projects. He

gave out information on how the to do one. Afterwards I looked at other previous project boards.

30min. I went on the Internet and looked for science project ideas. I found one that looked interesting about cryogenic.

11-17-98 5min. I saw Mr. Brown in the hallway while going to the

library. I talked to him briefly about the topic cryogenic.

55min. I went on the Internet and looked for information about cryonics. I found none that fit my idea or any that I needed.

11-18-98 120min. I talked with Mr. Brown after school and talked

about my project. He made the idea more clear to

me.

11-19-98 50min. I went to the school library and looked for books

about cells. I found two that looked good and

checked it out.

15min. I called Mr. Brown after school and talked about

what kind of cells I should freeze.

40min. I went on the Internet and looked for subjects like

cryogenics, freezing cells, embryos, and in-vitro

fertilization. I wrote some information down that I

thought was interesting. I also thought of some

variable I could do on how and what to test. But I

got confused and stopped and decided to wait to

until I talked with Mr. Mcmillen.

11-20-98 10min. Talked with Mr. McMillen about my topic and

about the paperwork.

20min. Talked again with Mr. McMillen after school about

new ideas and how to get equipment and specimens.

11-21-98 60min. I went on the Internet and looked at the two sites

Mr. McMillen gave to me. I also, looked for other good sites that would fit my project. I found one

about in-vitro fertilization. It looked really interesting so I wrote down the web address and later I will put it in my bibliography.

11-24-98 30min. I went on the Internet and found one very good site.

It will help me a lot; wrote down the address. I read

some of it.

11-25-98 45min. I went to Mr. Brown and talked about whom to go

to, to find information and advance on my project. He said I should talk to any doctor who has connections with other doctors in the field of cryobiology.

11-30-98 15min. I called my doctor (Dr. Gondo) and asked if he

could help me with my project. Also, if I could use his lab and if he is willing to supervise me. He said yes but he needed more information.

12-1-98 10min. Worked on project.

12-2-98 20min. I went to the library and thought up of a good title.

I found none that I liked.

45min. I went to Mr. Brown after school and got my ISEF

forms. Talked more about my project.

60min. I talked with my uncle in San Francisco chemical engineer and talked about my project.

liquid nitrogen. I also made a rough draft of everything to show to Mr. Mcmillen. I made sure I have everything all set so when the sea urchins come in I will be ready.

1-4-99 10min. Mr.Mcmillen was gone so I couldn’t show him my

rough draft. After school I went to Ms. Gustin’s

				(title, procedure, graphing, everything) 20min. I sent an email to my doctor about the project in more detail. 12-3-98 45min. I went to Mr. Brown after school and I showed him an outline of my project. He approved. I tried looking for live sea urchins in his catalogs. I only found one. 5 urchins for $25.00. Still looking for more and cheaper kinds. 12-5-98 30min. I went on the Internet and looked for information to base my hypothesis on. I found it. Wrote down the address. 12-6-98 180min. I worked on my purpose, hypothesis, experiment design, procedure, materials, and the pre-experiment log. Typed some of it. I also went on the Internet and looked for information on sea urchins. Sent an email to Dr. Gondo asking if the x-ray viewing hot lamp has different settings and if he has microscopes there in the lab. 12-7-98 90 min. I went to Mr. Mcmillen’s room after school and talked about my project. I asked some questions and he told me exactly what to do. He told me to call some places to see if they can give me a procedure for sea urchin fertilization. Also, to call an artificial insemination place and ask where they get their liquid nitrogen. He made the idea a lot clearer. 12-8-98 40min. Talked about (in class) how to do the experiment design and procedure. 12-9-98 20min. Talked about (in class) materials and in more depth
			procedures. After, I asked him what I wanted to do for my controlled group. Drew me a picture of what I needed to do. Also, if I had to pay for the Dewar’s flask. No, I don’t. 30min. I called different people and stores if they had liquid nitrogen. None of them had any except for one: Pacific CA, but they will only sell 5 gallons or more. I only need about 4-5 liters (1 gallon). Decided to talk to Mr. Mcmillen first. 12-10-98 30min. I talked to Mr. Mcmillen about who I called for liquid nitrogen and artificial insemination. Told me to go to Ms. Gustin (a science teacher) to ask about artificial insemination. Also, he told me to talk to Mr. Durean (an agriculture science teacher). I went to his classroom but he wasn’t there. 12-11-98 30min. I went to the library and worked on project materials. Also looked for sea urchins in catalogs that were fertile. 12-14-98 35min. I went to the school library and started my procedures. I got the first part but I got confused at the middle and end. 12-15-98 10min. I went to Mr. Mcmillen after school and talked about my ISEF forms and how I won’t get them in until Friday because I was still struggling with my procedures. Okay with him. 150min. I worked on procedures and I finished but I’m not sure if it’s right. I also worked on experiment design.
		12-16-98 15min. I talked with Mr. Mcmillen during class. I showed him my procedures. He said it was great except that it was too long. I needed to shorten it up and take out a few variables. 30min. I went to Mr. Mcmillen’s room after school and talked about where I should get the vials that will not shatter when taken out of liquid nitrogen. Also, about where to get sea urchins. I took home his catalog. 20min. I called “Connecticut Valley Biological” and asked about their vials and sea urchins. I also called Petco to see if they had sea urchins there and the artificial salt water. They only had one sea urchin and so I asked if they could direct me to another place that might have sea urchins. 12-17-98 90min. I filled my ISEF forms and did my procedures again because I had lost it in my disk when the computer froze. I made sure I my purpose and hypothesis was just right. I had to change and take out some phrases and words to make it sound correct. 12-18-98 10min. I turned in my ISEF forms and asked last minute questions before going on winter break. 15min. I emailed Dr. Gondo and asked him some questions. I also gave him some information that he needed. And I sent him a copy of my procedures. 12-22-98 300min. I worked on my research report. I only got half way done when I stopped. After that, I started to work on my pre-experiment design, bibliography and experiment design. 12-28-98 90min. I called the “Connecticut Valley Biological” to
order the sea urchin. I asked some questions while I was talking to the salesperson. Then I called around to see who had cryovials. Someone finally directed me to “VWR Scientific Products.” The lady told me they had a lot of different kinds so she faxed me some of the different kinds of products. I went on their Internet web site and looked at the products more clearly. When I tried to call back they were closed. 12-29-98 30min. I called Mr. Mcmillen and told him that I had found a place where I could buy cryovials but I told him that the highest capacity for the cryovials were 5ml. He said it was great because that much could hold at least 500 eggs. So I ordered the cryovials and had to make it rush delivery. It will come in maybe January 5^th. 10min. I called Dr. Gondo and asked him if I could come to his office some time just to look at the place where I’m working at and to meet him. He said that I should come today because he had no patients and he’s not busy. 30min. I went to his office and met him. He showed me around and then showed me the place where I would be working at. It was really small and I told him that. It wasn’t what I expected. He suggested maybe that I could do the experiment at his house but I told him that would be too much trouble. So, I would make the best of my workspace. 45min. I went around the Internet to look for more information to add to my research report instead I found a better site. It included graphic animation’s on how to spawn sea urchins. It also said that a drop of sperm could fertilize 5ml of egg. I didn’t know that so I have to change everything on my procedure. I decided that I would call Mr. Mcmillen the next day to ask about this. In addition, I also
	tried to think of the quickest way to do this experiment in less than 5 weeks. 12-30-98 10min. I called Mr. Mcmillen and told him what I had found and asked him what I should do. He said that I should do a trial run before I start freezing the eggs and sperm. I also told him that this experiment would at least take 3 weeks and that we only had 5 weeks to finish the whole project. He said that he would let people by if they had any supply delay. And I also asked him other questions that I just needed to know. 65min. I worked on my procedures and changed almost everything using the guide of the Internet web site I had found the day before. I also added more to my research report about sea urchins while looking around the Internet. 1-1-99 30min. I worked on my research report and added liquid nitrogen as a subtitle. Wrote about what it’s made of and the gaseous effect. Also, the dangers of working with liquid nitrogen. 1-2-99 80min. I updated my Pre-Experiment Log and typed it on the computer. I also emailed Dr. Gondo about important information that he should know. 1-3-99 60min. I worked again on my research report including liquid nitrogen. I also made a rough draft of everything to show to Mr. Mcmillen. I made sure I have everything all set so when the sea urchins come in I will be ready. 1-4-99 10min. Mr.Mcmillen was gone so I couldn’t show him my

		room and asked if the dewar’s flask had come in. It was in so I took it home with me. I got home and the cryovials had also arrived this morning. 1-5-99 5min. In class I showed Mr.Mcmillen my materials that I needed to borrow from the school. He looked over it and I told him that I would come back after school to pick the stuff up. 120min. I went back in his class after school and again showed him my list. We went around all the science rooms and borrowed the equipment that Mr. Mcmillen didn’t have. I talked to him about my project and how I was unsure about everything. He also suggested that it would be nice if I had a laptop with me at the competition to show judges animations on how the urchin eggs fertilizing, etc. 270min. I went to my doctors office and I did some trial runs before I would start the real experiment. I did the right procedures but apparently the first sea urchin is not shedding eggs. It is really weak with little movement. I think its going to die before it releases any eggs. It’s been almost 25 minutes and it has done nothing. After 30min., I observed two orange patches that I believe are to be the eggs. I thought that it would be more than that. I’m not sure what to do. After an hour my doctor added more KCl to the urchin. It released tons of eggs but we thought it might have been the KCl because it was the same color. I put the beaker with eggs under a dissecting microscope and examined the water. It was eggs. So I took a pipette and put the eggs in the cryovials. I took another urchin and it was male. The sperm only came out around the tentacles so I couldn’t get it. I waited for 45min. to see if it would release more sperm. Nothing happened so I thought I had
	made a mistake and took the urchin and put it under water as if it was a female urchin. Right when I exposed it to water all the sperm came out. Since the sperm was supposed to be collected dry, the trial experiment was all-wrong. So I just put the sperm and eggs together and had it fertilize. 1-6-99 10min. I told Mr. Mcmillen about the test run experiment and how it went. I also asked for KCl because the KCl my doctor had was wrong. So measured and mixed the KCl with water and asked for two more beakers to borrow. 1-7-99 10min. I went to the Yakima Welders Supply to buy liquid nitrogen. It didn’t cost as much as I thought it would have. I need to pick up my dewar’s flask tomorrow. I got home and I tried contacting my doctor but he was busy so I left a message. 1-8-99 180min. I went home and I was told that all my sea urchins had dies while I was at school. So now I can’t do my experiment until I get more urchins. I have to reorder and have the urchins be here next. I’ll be set back another week. I called the Yakima Welders Supply and told them that I wouldn’t be able to pick up my liquid nitrogen. He said okay but I would have to pay again when I pick it up. I thought about how long it would take to thaw the cryovials and so I thought why don’t I just pick up the liquid nitrogen and put the vials I have in my doctor’s office in the liquid nitrogen and test run how long it would take to thaw. So I called the Yakima Welders Supply again and told them that I will be picking up the dewar’s flask. I went to my doctors office and did the test run. It took less than 10min. for all three
variable to reach room temperature. 1-9-98 45min. I worked on my pre-experiment log and updated everything and typed more. The sea urchins will come in next Wednesday. 1-13-99 10min. I emailed Chris Patton at Stanford University to see if I could use the animations he designed on a web page I found. I told him that I would credit his work in my project. 1-15-99 5min. I checked my email and Chris emailed me back saying that I could use the animations and that all I have to do is download it and save right on the web site. I plan to use these animations and save them on a laptop, which I plan to take with me to Regional and State Science Fair. 30min. I went to the Yakima Library and looked for information on sea urchins but didn’t check anything out and made notes instead. 1-17-99 20min. I updated all my work and made sure I’m ready to go when I get my sea urchins. I also made sure that Dr. Gondo knew that I would come in on Wednesday to start the experiment.

1-19-99 180min. The Sea Urchins came early so I had to rush

to do the experiment because I didn’t want the sea urchins to die again. I went and got the liquid nitrogen and then went to Dr. Gondo’s office. Before I started the experiment I forgot to bring salt water so I had to go back home and get salt water for the female urchin. The first sea urchin I injected was a female and it was releasing its eggs for about 45 minutes before it stopped. Next I got a male. Not a lot of sperm came out so I had to put more potassium chloride each time it stopped releasing. I did the controlled group then put the eggs and sperm in cryovials. I put the cryovials in the liquid nitrogen. I didn’t have time to do the 15-minute trial so I changed that to a one-day trial.

1-20-99 180min. After school I went and got the liquid

nitrogen and then went to Dr. Gondo’s office to do the experiment. I brought five urchins with me today and I didn’t get either egg or sperm in all of them. Most were already weak when I brought them. After waiting an hour to see if any will release just in case I next took out the one day trial cryovials out of the dewer’s flask. I regenerated the eggs and sperm three different ways. I mixed the eggs and sperm and got the results.

1-21-99 130min. Again after school I went and got the liquid

nitrogen and went to Dr. Gondo’s office to do the experiment. I again tried to do the second trial experiment. This time it released a lot of eggs but not much sperm. I did get enough for the experiment though. I put them all in the dewer’s flask. I didn’t regenerate anything today. I also started my third trial today.

1-22-99 150min. I went and got the liquid nitrogen at the

Yakima Welders Supply. When I to my doctors office I started the experiment once I got all my equipment ready. I took out of the flask the three-day cryovials trial one. I also regenerated second and third trials for the one-day experiment. I regenerated the eggs and sperm in three different ways and mixed the eggs and sperm together. I put drops of it into a slide and looked at it under the microscope and wrote down the results.

30min. After I finished my experiment I made sure I cleaned up everything. Then I edited my procedures and typed more in my

experiment log and pre-experiment log.

1-24-99 180min. I got ready to go get my liquid nitrogen and

then go to my doctor’s office to do the experiment. Today I regenerated trial two and three for the three-day trial. There were some eggs that fertilized and that was amazing because I didn’t expect that. But there was very little. The eggs doesn’t look like the eggs when they were fresh but it is more very light yellow and it looks as if it’s

cracked in the inside. The ring around the eggs was not easily visible but can still see.

45min. I tried adding more information to my

research report but I was really tired so I just typed more in my experiment log and again changed my procedures because today I decided that I will continue the 30 minute trial and the 5 hour trial. I did this part at home.

1-25-99 210min. After school I went to go get my liquid

nitrogen and then went to Dr. Gondo’s office. I started my 30 minute and 5 hour trials. It took about an hour and a half for each trial to regenerate. I again found eggs that were alive in both 15 minute and 5 hour trials. This time there was more eggs that were fertilized.

1-26-99 85min. After school I went to Mr. Mcmillen’s room

to discuss about some questions that I had. I asked him about the digital camera and when I could use it. He said to come in on Thursday after school. We hooked up the digital camera just to make sure it would work when I am ready to use it. I also asked about what my title should be because I’m still not sure which one to use. I also told him that I needed about 2 weeks after the due date for the project. It’s okay with him.

60min. I went to the Yakima Welder’s Supply and

got the liquid nitrogen then went to my doctor’s office to do the last trial for my trial one. None of the eggs were alive and that

didn’t really surprise me because the

percentage of live eggs kept going down as the legnth of time they were in the flask continued. I cleaned up and went home.

180min. I updated my experiment log and typed

more. I started to graph my results on the computer but it doesn’t look right so I figure I will have Mr. Mcmillen look at it on Thursday.

1-27-98 45min. I went to Yakima Welder’s Supply and got

my flask re-filled with liquid nitrogen. I graphed more of my results. It really takes a long time to make the graphs. I figured that I’d have around forty-five graphs and data to go with each one of them. I don’t know how I’ll be able to fit it all on my board.

1-28-99 60min. After school I went to Mr. Mcmillens room

and asked him what time I could come in to use his digital camera to take pictures of dead urchin eggs. He said to come in around six o’clock. I regenerated the gametes at around four o’clock and then got ready to go to Mr. Mcmillen.

140min. I went to Mr. Mcmillens room and we

started to set up the equipment. We didn’t see any of the eggs fertilizing so we didn’t get any pictures of the eggs fertilizing after being frozen. We only got to take still pictures of dead urchin eggs. What we did find was that there was living protist swimming around eating the dead eggs. They must have survived the freezing temperatures. When we tried to look for more of the protists in the eggs only vile, we

couldn’t find any so I assume they came from the sperm. But I can’t be sure of that since I didn’t look at the sperm only because I didn’t have any extra vials that had sperm only.

2-2-99 120min. I continued on making my graphs. I finished

all of trial one and I started trial two.

2-5-99 45min. In class, Mr. Mcmillen talked about what we should

have in our Pre-experiment log, Post-experiment log, Experiment log, Conclusion, and Scientific Error section. We took notes. After that, we were put into different groups and we would exchange journals of what we had and edit each other’s. During that time, I asked him about the laptop I’m going to borrow. He said that he has to check them out of the library and see if it worked.

2-6-99 190min. I finished making the rest of the graphs but I still

need to edit it before I print it all out. I also have to show it to Mr. Mcmillen to see if it’s all right.

2-7-99 60min. I started to edit all the work in my journal. I also

started to create my title, abstract, results, conclusion, and appendix. I don’t have my science binder with me which included the instructions on how to do the results and conclusion so I can’t continue far with that part of the journal.

2-9-99 45min. I started to analyze my results. I wrote part my

results and conclusion. I printed two graphs but decided that I had to make changes. It didn’t look right and I really have to go and show Mr. Mcmillen this sometime.

2-10-99 30min. I went in to see Mr. Mcmillen today after school

and I asked him about the laptop. He said that I should start creating it two weeks before I go to

check says that its wrong and I have to ask. I also printed what I can for my board. First, I need to get a board though. I updated my bibliography and added more.

2-15-99 5min. I printed off my abstract to show to Mr. Mcmillen

because its over 250 words and I need to make sure that that’s ok with him and that he won’t down grade me on it. I changed it from a thousand and more words from around four hundred words. It’s really short now.

2-16-99 30min. I went in to Mr. Mcmillen’s room after school to

show him the graphs that I made. He said that I should change it so that the graph will be over a period of time and not at the length of time I had them in. I also showed him my abstract to see if it was ok with him because it was way over 250 words. I asked him if I could print the pictures that I took with the digital camera. There were people on the computers so I had to wait. I didn’t get to print anything off except the dead eggs but that wasn’t any good so were going to do it again.

30min. I tried to change the graphs like what Mr. Mcmillen

sai