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

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Author Contact Information

Frederick D. Atwood
Biology Teacher, Flint Hill School, Oakton, VA

TEA teacher Oct/Nov1998 at McMurdo Station, Ross I, Antarctica

In this activity students work in groups to design experiments to test the effects of cold on rate of diffusion, enzyme activity, nerve activity, dissolved oxygen, blood flow characteristics and lipid characteristics. After each group's plan is approved by the teacher, the group performs their experiment and reports their results to the rest of the class for class discussion.

Students will ...

  • design a controlled experiment
  • record their results in data tables and graphs
  • investigate the effects of cold on important physiological functions
  • report their results to the rest of the class
  • discuss the problems that must be dealt with by fishes that live in the extreme cold temperatures of polar regions
  • propose what adaptations polar fishes might have to deal with these problems

    Grade Level/Discipline
    High School Biology

    National Standards
    Teaching standards: A (inquiry-based), B (guide and facilitate learning), D (learning environment), E (community of science learners)

    Content Standards (9-12): A (inquiry based), B Physical Science (chemical reactions, interactions of energy and matter), C Life Science ( the cell; biological evolution; matter, energy, and organization in living systems)

    Pre-activity set-up
    Day before the lab. For class this day photocopy the Student Reproducible Master Polar Fish 1.1 and familiarize yourself with the possible procedures the students might be designing and with the types of data tables they will need to record their data. If students are designing their procedure it will help if you can provide them with some of the basic procedures that they can use for their activity (how to use Benedict's solution, how to set-up the CBL probe for their activity, etc). I give them a photocopy of this part of the procedure. They can use this information to design the rest.

    Mix the agar or gelatin if needed. Keep it in the refrigerator.

    Since all will be using cold water and ice make sure you put sufficient cold water in the refrigerator and bring a big enough bucket of crushed ice for each table so that all materials used for the cold set-ups can be kept cold. If you do not have access to a crushed ice machine (cafeteria, nurse's station or athletic department) it may take at least a day to make a sufficient number of ice cubes.

    If any other materials need to be frozen and chilled for the lab put these in the freezer and refrigerator.

    Day of the lab.

  • Photocopy the Student Reproducible Master 1.2 (Procedure) if the students didn't design their own procedure.

  • Assemble the materials they will need for their parcticular activity at their lab tables.

  • At least one half hour before class, nestle the materials that will be used for the ice-cold experiments in the bucket of crushed ice to give time for the temperature to stabilize.

  • Make sure each group has enough ice on hand to keep adding to their set-up to keep it cold as the ice melts.

  • Use a thermostatic hot plate to get a water bath going for each group. Do not let the temperature exceed 40 C. The enzyme activity will require two hot plates (35-40 C and 70-80C).

  • Put one set of equipment for each group in refrigerator.

    Since students are designing their own experiments it is difficult to predict exactly what they will need. However you will most probably need access to a refrigerator and a freezer, and the following materials should be available for each group:

  • bucket of crushed ice or ice cubes (crushed ice is preferred)
  • Four or five 1000 ml beakers: one for each temperature
  • Four or five thermometers: one for each temperature
  • Hotplate (preferably a thermostatic one)

    In addition you should have available the following materials for the different factors being studied. I have found it best not to tell the students any procedural information except for listing the materials that are available. After 10-15 minutes I generally go around to the different lab groups and pose leading questions to get them started or to get them to think about important factors or procedural problems that they have not considered.

    For Diffusion Study:

  • solidified agar, gelatin, or raw potato to serve as a diffusion medium
  • dark food coloring, potassium permanganate, or an undiluted dye which will diffuse through the diffusion medium
  • small metric ruler with mm divisions
  • watch or a good view of the classroom clock.

    For Enzyme Study:

  • A mammalian enzyme that is readily available. Two possible enzymes are....

      Catalase: an enzyme found in all cells, especially chicken liver, which produces bubbles as it breaks hydrogen peroxide into water and oxygen. Fresh liver can be used directly as a source of this enzyme. There is no need to extract the enzyme from the liver. You will also need a scalpel, 4% hydrogen peroxide (typical pharmacy concentration).

      Salivary Amylase: enzyme found in saliva that digests starch into sugar

  • An enzyme that works best in cold temperatures such as ....?
  • Two test tubes for each temperature and each enzyme (Control and variable)

    If amylase is used, a hot plate, 300 ml beaker (for a hot bath), Benedict's Solution (an indicator of monosaccharides), and several test tubes will be needed. If they do this experiment you will need to tell them how to use the Benedict's solution.

    For Nerve Activity:

  • Reaction Time Ruler (or meter stick)
  • blindfold
  • 50 pins in a petri dish
  • stopwatch or clock/watch with second hand
  • caliper
  • bucket large enough to submerge hand

    For Lipid Activity

  • Red Beets (if membranes are disrupted by freezing, red pigment will diffuse out)

  • corn oil, butter, other lipids
  • marble
  • your biggest test tubes that a marble will fit in easily
  • stopwatch
  • packaged pre-sliced salami
  • spring scale
  • CBL force detectors and related equipment
  • scalpel or pocket knife with sharp blade
  • access to a freezer

    For Blood Flow Activity

  • Animal Blood (preferred) from butcher?, or syrup or honey to serve as a model for blood
  • CBL apparatus with pressure probes
  • syringe
  • narrow plastic tubing
  • 100 ml graduated cylinder
  • glass tube that is taller than the graduated cylinder
  • stopwatch or watch with second hand
  • mm ruler

    For Dissolved Oxygen Activity

  • Dissolved Oxygen Test Kit (eg. Hach or Lamotte) or Probe (Vernier CBL or other)
  • 500 ml beaker of water at about 0 (ice water), 5 (refrigerator), 20 (room temp), and 35 C

    Time Frame
    3 periods of 45 minutes, one for introduction and planning, one for performing the experiments, and one for discussing the results. It might be best if there is a day between when they design the lab and when they perform it to give you time to review their procedures and get them back to the students for their modifications

    Engagement and Exploration (Student Inquiry Activity
    1. Ask students what they think the physical environment is like for fish living in Antarctica. List the correct descriptions on the board as they come up. Elaborate on these ideas explaining them in more depth. For instance if they say it is wicked cold, you can say yes it is so cold that it is about -2 C (28 F). Correct things they might come up with are:

  • it is very very cold
  • some of the fish swim among floating crystals of ice (this is actually a greater physiological problem than the temperature)
  • it is dark for 4 months and light for 4 months
  • the sea ice is often 2-3 meters thick
  • the sea ice melts in December or January and reappears in March(?)
  • under the ice there is a lot less light even when it is light all "night" long (only 1% of light hitting ice passes through it
  • there are no freshwater fish
  • etc etc.

    Ask the students what they think the amount of dissolved oxygen is like under the ice in the polar regions. Tell them that this factor is one of the ones that will be studied in this lab.

    2. Then ask students what questions this discussion has sparked in their own heads about the fish. What problems does this impose on the fishes? What would happen to us or to a goldfish in these conditions? If the students don't come up with the list of factors this activity studies, ask questions to lead them in this direction. Write their questions/problems on the board as they are volunteered by the students. I like to use arrows to link the questions with the specific observations that sparked them.

    Some questions the students might raise are:

  • Why don't they freeze?
  • How can the fish move in such cold water?
  • How can the fish find food and oxygen in the winter?
  • How can the fish get oxygen if the sea is separated from the air by 3 meters of ice?

    3. Break the students up into groups based on their interests and tell them to design a controlled experiment to test the effect of cold on that aspect of physiology. Refresh their memory about what a controlled experiment is and the basics of how they are done. Then give each group a list of materials that you have available for their use. Let them know that their material list does not have to be limited to these. These are just some ideas and they should let you know if they have need of anything else.

    Give the students the reproducible master "Polar Fish 1.1" on which will they will record the question they are trying to answer, the variable, the conditions they will be keeping constant, their proposed procedure, a list of materials (and how many of each they will need) and their sample data table.

    4. After 10 minutes, start wandering through the class to get a feel for how each group is doing. Ask pointed questions to lead groups past specific barriers they are having or to point out certain factors they have not considered. If one group finishes early they can help another group with their plans. At some point during this time it might be useful to stop the class and have them ask questions to address certain problems that they are having. Then the class as a whole can provide suggestions. Or if you see a problem that needs to be addressed by most groups you can point it out to the whole class and have them propose ways to solve it.

    Some common problems/barriers are:

  • determining a way to measure the effect of the factor they are studying
  • coming up with a procedure if they have never used the materials before (You may need to give them specific procedures on using CBL probes, Benedict' solution, etc and let them design the rest)
  • making sure all variables are constant except for the one factor being studied
  • using dirty glassware
  • how to prevent contamination
  • how to keep the temperatures as constant as possible

    5. At the end of the period collect the proposals, review them and comment on them overnight, and return them to the teams for their modification the next day.

    Explanation (Discussing)
    On the next day they will perform their experiments. These are not the only possible procedures but the procedures they might use can be clicked on here:

  • Effect of Cold on Diffusion
  • Effect of Cold on Enzyme Activity
  • Effect of Cold on Lipids
  • Effect of Cold on Nerve Activity
  • Effect of Cold on Blood Flow
  • Effect of Cold on Dissolved Oxygen

    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.

    TEACHERS: The following two enzymes are readily available and work best at body temperature. It would be nice to also find an enzyme that will work best when cold and that can be easily tested. Any ideas? I have heard of enzymes in various laundry detergents that work best in cold water but I don't know anything about them yet. PLEASE PROVIDE FEEDBACK. (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 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. MORE DETAIL IS NEEDED HERE ON PROGRAM AND SETTINGS FOR THE CBL EQUIPMENT

    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 100 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.

    Explanation (Discussing) and Elaboration (Applications)

    Students will present their graphs and analyze their results to teach the rest of the class. The Student Reproducible Master Polar Fish 1.3 is a good guideline for what the students should include in their reports to the class. It is best if each student leaves the class at the end of the activity knowing exactly what his/her role will be in the next day's presentation.

    As the students are giving their explanations you will probably need to correct some misunderstandings, teach some terms, and use their data as an example to demonstrate some important principles. If you are unfamiliar with any of the following recommended topics to include in this discussion, you can find info about them and how they apply to polar fishes in the Background section of this activity. When I teach this I prefer to pose the question and ask the students to come up with answers. If they can't I lead them through it.

    What is diffusion?

    Why does decrease temperature decrease diffusion?

    How does life depend on diffusion?

    Why is it important for polar fish to conserve energy.

    The relationship between enzymes, chemical reactions, and temperature

    Enzyme structure

    How polar fish enzyme structure enables them to work at low temperature.

    Why don't polar penguins and seals have to worry about this?

    Cell membrane structure

    How does nerve activity depend on diffusion and lipid malleability? See Atwood journal Dec 5

    Why is dissolved oxygen important.

    Some fish in Antarctica don't have red blood cells. Relate to dissolved oxygen. Lead students (through questions) about how this ties in with lower number of red blood cells and the resulting affect on blood viscosity and why that is important in the cold.

    If anything comes up about why the fish don't freeze or the effect of salt on freezing point, you can continue this investigation using the next activity in the polar fish unit.

    Elaboration (Polar Applications)

    Exchange (Students Draw Conclusions)

    Evaluation (Assessing Student Performance)
    I grade Polar Fish 1.1 and Polar Fish 1.3 looking for clarity and thoroughness of explanation, proper labelling of graphs and tables, and accuracy and depth of understanding. I also ask questions on the concepts and terms described in the background section in my tests and quizzes.

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