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Activity 1: The Effect of Cold on Characteristics Important to Fishes

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POLAR FISHES 1.1 Experimental Design for the Effect of Cold On .....

As you think about your experimental design for your parcticular problem the following questions should help guide you through the process.

1. Names of scientists in the team:

2. What factor are you studying? The effect of cold on _____________________

3. What is your variable?

4. What temperatures will you try to study? (Enzyme researchers should have 5 temperatures. Others can do 4 temperatures.)

5. How will you maintain these temperatures?

4. What conditions will you need to keep constant and how?

5. How are you measuring (quantifying) the effect of cold on the factor you are studying?

6. What materials will you require? Include how many of each you will need. Item

  • Number Needed
  • What it is needed for.

    7. Describe your proposed procedure in "cookbook format" so that a 7th grader would be able to follow it easily. If it would help, include a sketch.

    8. Show the data table you will record your data in. Label it properly and include units.




    1. Fill a small beaker (100 ml) half way with undiluted dark food coloring, potassium permanganate solution, or dark hair dye. Be careful because potassium permanganate stains clothes and skin.

    2. Use the crushed ice, refrigerator, room temperature and hot plate to bring each beaker of dye to one of 4 different temperatures. If you are using gelatin make sure the temperature does not go above 30? C so it doesn't melt. While you are waiting for the temperature to adjust do step 3. (Alternatively, instead of gelatin or agar, will potato cubes work?)

    3. Your teacher has filled a Petri dish to a depth of about 1 cm with agar or gelatin. Use a scalpel to cut out 10 one cm (1 X 1 X 1 cm) cubes. Put 2 of these in each of four 50 ml beaker and put one beaker next to each dye-filled beaker at each temperature. Wait 10 minutes for the temperatures to stabilize.

    4. Record the temperature for each beaker of dye.

    5. At each temperature set-up pour the dye into the beaker of agar cubes. Make sure the cubes are totally covered with dye and that the cubes are not touching each other.

    6. After 25(?) minutes dump out the dye and rinse the cubes gently with water.

    7. Cut each cube in half and use a mm ruler to measure how far the dye diffused into the agar in 25 minutes.

    8. Record the maximum diffusion distance at each temperature in a table that will show the relationship between temperature on distance diffused.


    (Option A: Catalase)

    1. Cut 5 equal pea-sized portions of fresh chicken-liver (about 1 cm X 1cm X 1cm)

    2. Place each piece of liver in a small test tube and put one tube in each of the 5 temperature set-ups: ice bath, refrigerator, room temperature, and 1 warm and 1 hot temperature maintained by a water bath on a thermostatic hot plate. About 35-40 C and about 70- 80 C are two good temperatures to try for. The exact temperature is not important as long as it is stable.

    3. Put 3ml of 4% hydrogen peroxide (typical pharmacy concentrations) in each of 5 separate small test tubes. Place one hydrogen peroxide tube in each of the temperature set-ups.

    4. Allow the temperature to stabilize for about 10 minutes.

    5. After 10 minutes, record the temperature of each set-up.

    6. Pour the room temperature hydrogen peroxide into the room temperature liver tube. The catalase enzyme in the liver will convert the hydrogen peroxide into oxygen gas and water, causing oxygen bubbles to fizz out of the mixture. Rate this amount of bubbles as a 2 on a scale of 0-3, with 3 being the maximum and 0 being no bubbles.

    7. Combine the H202 and liver at each of the other temperatures. With the amount of bubbling seen at room temperature a 2 on a scale of 0-3, rate the amount of bubbling produced by the catalase at each temperature. The amount of bubbling is an indication of the enzyme's ability to catalyze the reaction.

    (Option B: Amylase) This activity will probably require more than 45 minutes because of the need to perform multiple Benedict's tests.

    1. Put 5 ml of starch suspension in each of 10 clean and dry small test-tubes.

    2. Put 2 of these test tubes at each of the 5 temperature set-ups: ice bath, refrigerator, room temperature, and 1 warm and 1 hot temperature maintained by a water bath on a thermostatic hot plate. About 35-40 C and about 80 C are two good temperatures to try for. The exact temperature is not important as long as it is stable.

    3. One person should drool 30 ml of saliva into a beaker. This person must rinse his/her mouth out with water, must not hock a mucus-filled loogy, and must not chew gum or anything else to produce the saliva since all of these can contaminate the results.

    4. Put 5 ml of saliva into 4 small clean and dry test tubes and place two test tubes at each temperature.

    5. Put 5 ml of water in each of 4 test tubes and put one water tube at each temperature.

    6. After allowing temperatures to stabilize for 10 minutes, combine a saliva tube with a starch tube and a starch tube with a water tube at each temperature. Place back in the temperature bath.

    7. After every 2 minutes swirl each tube and do a Benedict's test for sugar on 1 milliliter of solution from each tube to see if the starch has been digested into sugar by the amylase. Always use the same eyedropper for a given tube. Mixing eyedroppers can cause contamination and give misleading results. Here is how to do the Benedict's test:

    Squeeze about 1 ml (5? drops) of blue Benedict's Solution from the dropper bottle into a small test tube.

    Use an eyedropper (pipette) to add about 1 ml of the solution to be tested (5? drops) to the same test tube as the Benedict's.

    Label this tube with a number so you don't mix it up with others.

    Put this tube in your hottest water bath and let it sit for 3 minutes.

    If the Benedict's turns yellow, green, orange or brown there is sugar present and the amylase has done its job.

    8. For each temperature situation, record how long the amylase had been working when the Benedict's indicated that sugar was present.

    9. If after 20 minutes no sugar is detected in any of the temperatures, you may stop your experiment and just note that at 20 minutes no sugar was detected.


    1. Measure reaction time for a hand at body temperature (37 C) according to the instructions that accompany the reaction time ruler. Alternatively use a meter stick as a measure of reaction time in the following way. The person testing reaction time is sitting. A partner stands next to him and holds the ruler out at arm's length. The sitting person reaches out as if to grab the meter stick but leaves his thumb separated from his fingers by about 2 inches. The standing partner holds the ruler so the sitting person's thumb is at the 10 cm line of the meter stick and the meter stick is held between the sitting person's thumb and fingers without touching them. Then without warning the standing person drops the meter stick. As soon as the sitting partner sees that the meter stick is falling he must grab the meter stick by closing his hand on it without moving his arm. Do this 5 times and record the average. The number of cm the ruler fell before the sitting person grabbed it should be recorded. This is not an actual reaction time but it will serve as an indication of reaction time. Reaction time is the time it takes for your nerves to cause your finger muscles to react .

    2. Meanwhile another pair of will test manual dexterity and touch sensitivity as follows.

    Place 50 pins in a Petri dish

    A person wearing a blindfold will feel for the pins in the Petri dish. While her partner times how long it takes, the blindfolded person will pick up the pins one by one, and remove them from the Petri dish until all 50 have been removed. This time should be recorded.

    3. Meanwhile another pair of students will measure pain sensitivity in the following? way.

    Tighten a metal caliper on the fleshy part of the blindfolded volunteer's finger. As soon as the volunteer first mentions that he/she feels pain, the partner should stop tightening the caliper and record the number indicated on the caliper.

    4. Now the people who did #1 and #2 and #3 must submerge their hands in a bucket of crushed ice or ice water for 5? minutes, until their fingers get numb.

    4. Then they will repeat the same activity they did before, in the same way, but this time with their numb fingers. The measurements before and after the ice-treatment should be compared.

    5. If time allows, other members of the team should repeat these procedures and record the results for a better average.


    1. What happens to cell membranes when they freeze? (Remember that cell membranes are made out of lipids.)

    Slice a red beet into thin slices. Rinse it off fresh water until no more red pigments diffuse out of the cells.

    Place 2 slices in the freezer. Leave 2 slices in a Petri dish of water.

    After the beet is frozen solid, place them in a Petri dish of water. As the frozen beet thaws do you see any red pigment coloring the water around the beet?

    2. While waiting for the red beet to freeze, study the effect of cold on viscosity of lipids. If a lipid is more viscous it is less flexible, and harder to move or change shape. Remembering how proteins and lipids are oriented in a cell membrane and the importance of shape change in protein function, why is it important to study lipid viscosity?

    Put a marble in each of 8 very large test tubes.

    Fill 4 test tubes with corn oil ( a lipid) and 4 with peanut oil, melted butter, or some other fluid lipid. Put a stopper on each tube and put one of each lipid tube in each of your temperature situations.

    After 25 minutes use a stopwatch to time how long it takes for the marble to sink through the lipid in each temperature situation.

    3. While waiting for the above experiment to adjust its temperature, assemble the following set-up to measure the effect of temperature on flexibility.

    Since a slice of salami has lots of fat in it we can use it to get an idea of the role of lipids in tissue flexibility in the following way.

    Take 4 pieces of packaged pre-sliced salami out of the package and put a slice of salami in each of the 4 temperature situations. While waiting for the salami to adjust temperatures (about 25? minutes), do the following test on the room temperature salami.

    Hitch a CBL force sensor to a spring scale and attach the spring scale to the edge of the salami which has been clamped in place. Pull with a gentle, even motion on the spring scale until the slice of salami has been bent down at a 90 degree angle.

    Repeat the same procedure with salami at each of the other temperatures. Try to keep the salami in the temperature environment as you do the experiment.


    TEACHERS: This idea is purely in my head. I am sure it needs lots of refinement at this point.

    1. Put ?ml of "blood" in each of your different temperature set-ups to allow the temperature to adjust. (Ice bath, refrigerator, room temperature, warm water bath(35-40 C) using hot plate.

    2. Set up the apparatus as shown in the diagram with a syringe to apply pressure at one end, the CBL pressure sensor apparatus on the other end, a reservoir to hold the "blood" , and a thin tube (representing the fish's blood vessels) for the blood to flow through.

    3. Use the syringe to apply pressure on the blood at each temperature set-up. Using the stopwatch, measure how long it takes the blood to move a set distance in the tube. This will give an idea of the effect of temperature on blood velocity (because of greater viscosity). Also use the CBL probe to measure the pressure in the system. This will give an idea of the effect of temperature on the amount of pressure needed to pump more viscous blood through the blood vessels.

    4. Another way to measure effect of temperature on the velocity of blood flow due to its change in viscosity is the following. Fill a 100 ml graduated cylinder with the blood at the right temperature. Stick a long narrow glass tube into the cylinder and put your thumb over the top. Remove the glass tube, hold the tube over a beaker and when your partner is ready, remove your thumb. Your partner will time how long it takes for the blood to flow out.

    5. If you do not have CBL apparatus, the following procedure can be used to study the effect of temperature on the amount of pressure needed to pump the blood due to its greater viscosity. (TEACHERS: Please send me any ideas you have.)


    1. Put 500 ml of tapwater in each of 5 beakers and place these in the different temperature situations (ice bath, refrigerator, room temperature, and hotplate). Keep a thermometer in each beaker and check the temperature every few minutes. When the temperatures become stable record the temperature of each.

    2. Follow directions in the test kit to test for dissolved oxygen. Be careful not to cause any stirring of the water or trap any air in the sample bottles.

    Discussions Questions/Extensions ......
    POLAR FISH 1.3 Class presentation about "The Effect of Cold on...."

    Your presentation to the class should have each of the following components. Before you leave class on the day of your experiment you should know specifically what your role will be in the class presentation tomorrow.

    1. What question were you trying to answer?

    2. What conditions did you keep constant in your different experimental groups and why?

    3. How did you do the experiment?

    4. A Data Table large enough for the class to see. This can be put on a large poster, the overhead, in a powerpoint presentation, or in a handout on the same sheet as the graph.

    5. A graph that shows your results. Make sure you label the graph completely and follow the other rules about proper graphing. This can be presented as described in #4.

    6. Discuss what this data shows about the effect of temperature on the factor you studied.

    7. Why does cold affect the factor you studied in this way?

    8. Would this effect be a problem or a benefit for an organism? Why?

    9. Polar fishes survive well in this situation. If this effect is a problem for most forms of life, propose some ideas about HOW your group thinks the fishes might be able to thrive in this situation (eg. what adaptations might it have?) If it is a beneficial effect how might this have helped

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