Middle School Labs

Diffusion and Cells: Investigating theMovement of Molecules Across Membranes

Introduction:

Most of you know that all living cells are surrounded by a structure called the plasma membrane, or more simply, the cell membrane. Since this structure is found in all cells, we might suspect that its functions are vital to life. We might also hypothesize that at least one of its functions involves regulating what goes into and out of the cell. But, if this is correct, how does the membrane do this? We will investigate this question in today's laboratory.

You will begin by investigating the properties of a synthetic, non-living membrane. This membrane is made of an organic polymer called cellophane. After investigating the ability of this cellophane membrane to regulate the movement of molecules, you will study the behavior of the membranes of sheep red blood cells and plant leaf cells in salt solutions and solutions of pure water.

Objectives:

1. To determine what factors control the movement of certain molecules across a cellophane membrane.

2. To investigate the ability to red blood cell membranes to regulate the movement of water and salt.

3. To investigate the ability of cells with cell walls to regulate the movement of water and salt and to account for any differences between such cells and those that lack walls.

Part 1: Molecular movement through a synthetic membrane

Materials:

Cellophane dialysis tubing 10-mL pipettesHemoglobin solution pi-pumpsRed dye solution250-ml beakers with de-ionized water

Procedure:

1. Prepare "bags" of cellophane dialysis tubing containing hemoglobin solution and red dye solution as instructed by your instructor; BE SURE THAT NEITHER OF THESE SOLUTIONS COMES IN CONTACT WITH THE OUTSIDE SURFACE OF THE "BAG".

2. Place the "bags" in 250 ml beakers that contain about 200 ml of de-ionized water. Set the beakers aside for 20-30 minutes and go on to the Procedure section of Part 2.

Part 2: Movement of water and salt through the red blood cell membranes

Materials:

Sheep blood on ice Microscopes, slides, and cover slipsDe-ionized water Containers for discarded serum0.9% NaCl Prepared slides of human blood15-ml centrifuge tubes5-ml pipettes with pi-pumpsPasteur pipettes with bulbs

Procedure:

1. Take a small drop of sheep blood, place it on a glass slide and add several drops of 0.9% NaCl. Add a cover slip and examine the slide using the compound microscope. Record your observations. Compare what you saw to a stained slide of human blood.

2. After you have completed your microscopic examination of both live and stained blood cells, your instructor will transfer 5 ml of sheep blood to each of 2, 15-ml conical tubes; mark both tubes so that they can be identified.

3. Making sure that your tubes are properly balanced, spin both tubes at about 3,500 rpm for 5 min in one of the low-speed centrifuges in the laboratory. If possible, combine your tubes with those of other pairs to make this step more efficient.

4. CAREFULLY remove the tubes from the centrifuge and observe the straw-colored supernatant (called blood serum) and the pellet of packed red blood cells. BE SURE THAT YOU DO NOT ALLOW THE PELLET AND THE SUPERNATANT TO BECOME MIXED AGAIN. Note that the red color of the pellet is due to the presence of the oxygen-binding protein, hemoglobin, inside the red blood cells.

5. Using a Pasteur pipette, carefully remove as much of the serum as possible from above each pellet in your two marked tubes and place the serum in the "discard" container.

6. To one of your 2 tubes containing the red cell pellet with the serum removed, add 5 ml of 0.9% NaCl. Quickly and thoroughly mix the pellet with the saline and mark this tube so you can identify it. Now add 5 ml of de-ionized water to your second tube with a second pipette and mix the contents as before. Mark this tube and place both tubes in the centrifuge for a second spin using the same speed and time as before.

7. After the spin is complete, carefully remove the two tubes and describe any differences between them. At this time, you should make slides from the two tubes and observe these slides under the microscope.

8. Your instructor will have centrifuged two additional tubes for your observations. One of these tubes contained a solution of hemoglobin dissolved in de-ionized water and the other tube contained a solution of hemoglobin dissolved in 0.9% NaCl. Observe the appearance of these two tubes and record this information.

Once you have completed steps 1-8 above, return to the beakers with the "bags" of hemoglobin and red dye that you set up in Part 1. Record the your observations of the results. Be prepared to discuss these results with your instructor and other students before proceeding to Part 3.

Part 3: Movement of water and salt through cell membranes of cells that have cell walls.

Materials:

Elodea plants De-ionized water0.9% NaCl6% NaCl solutionPaper towelsMicroscopes, glass slides, and cover slips Procedure:

1. Prepare a wet mount of Elodea leaf cells as instructed; cover the cells with de-ionized water and observe them under high power. Recall that Elodea cells have both a cell membrane and a cell wall.

2. Draw off most of the de-ionized water with a paper towel or piece of lens paper and replace this with 0.9% NaCl. Observe again under high power and note any differences that you can see within the individual cells.

3. Draw off most of the 0.9% NaCl and replace it with 6% NaCl. Observe under high power and describe any changes. What direct evidence can you now observe that supports the concept that these plant cells do have both a cell membrane and a cell wall?

4. Draw off most of the 6% NaCl and replace it with de-ionized water. Observe under high power and describe any changes. You will need to have complete records of your observations for discussion and your final report. You may want to make some diagrams to illustrate what you observed. You will need to be able to explain the differences between these results and those you observed with the red blood cells.

How Do We Detect Starch and Sugar?

One of the major components of many foods is starch. Another component of food is sugar. In this exercise, you will try to determine how to detect starch and sugar using simple chemical tests. To do this, you will examine a chemical reaction between starch and iodine which makes starch visible by changing its color. You will then examine a second chemical reaction between sugar and a liquid called Benedict’s solution that reacts with sugar when it is heated.

Materials:

Potato slicesApple slicesIodine solutionBenedict’s SolutionBoiling water bathMortars and pestles DroppersReaction tubes

Begin your investigation as follows. Potatoes are considered to be a good source of starch. Apples are believed to contain more sugar than starch. Obtain a slice of potato and a slice of apple. Place them in a dish or on a piece of paper towel. Using the dropper and the iodine solution, put a few drops of iodine solution on each slice. What do you observe?

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Assuming that potatoes really do contain more starch than apples, what does your observation tell you about the reaction of iodine with starch?

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Now, place the potato slice in a mortar along with enough water to cover it and grind it up with the pestle. Pour 1-2 ml of the mixture into a test tube, add about the same amount of Benedict’s solution and place the tube in a boiling water bath. Observe any color change and record the results below.

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Rinse the mortar you used to grind up the potato with water. Add a slice of apple to the mortar, cover it with water and grind it up. Repeat the test with Benedict’s solution using the apple-water mixture. Describe the results below.

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Assuming that apples really do contain more sugar than potatoes, what does your observation tell you about the reaction of Benedict’s solution with sugar?

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What Happens to StarchWhen It Is Mixed with Saliva?

Most of you probably know that saliva mixes with your food as you chew it. But what does saliva do to food? One of the major components of many foods is starch. In this exercise, you will try to determine what effect saliva has on starch. You will use the two chemical tests you learned about earlier.

Materials:

Iodine solutionBenedict’s solutionBoiling water bathDroppersReaction tubes2% Starch solution

Begin by adding the 2% starch solution to a reaction tube until the tube is filled up to the first mark. Add several drops of iodine solution to the tube. What do you observe?

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Now, prepare a second tube by adding the starch solution to the first mark as you did above. Carefully add some of your own saliva to the second tube containing the starch solution. After you have added the saliva to the second tube, gently swirl this tube for several minutes (you should swirl the tube for 5 to 10 minutes). Add the same number of drops of iodine solution to the second tube that you added to the first tube. What did you observe?

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Now, conduct the following procedure. Frist, add 2% starch solution to the first mark in each of 2 clean tubes. Add saliva to one of the tubes as

you did before and swirl it again. After 5-10 minutes, add Benedict’s solution to the second mark of both tubes and place them in the boiling water bath. Record your observations below.

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Based upon your observations, what effect do you think that saliva has on starch? Explain your answer.

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Integrating Your Knowledge

This morning, you observed some molecules in a cellophane bag. This afternoon, you investigated the reactions of starch and sugar in two chemical tests. Let’s see if we can take these two concepts and put them together in a simple system that models the process of digestion in our bodies.

Materials Available:

2% starch solutionCellophane tubing250 ml beakersIodine solution

Begin by preparing a “bag” of starch in the same manner as you did this morning with the hemoglobin and red dye solutions. Be careful not to get any starch solution on the outside of the bag. Now, place the bag in one of the beakers and add enough water to cover the bag. Finally, add several droppers of iodine solution to the water outside the bag (enough so that the solution turns yellow to light brown in color).

Answer the following questions:

What is your prediction about the result of this procedure?

Did your observations support your prediction?

How do you explain your observations in terms of the movement of molecules that were present in: 1) the bag; and 2) the water outside the bag?

Now, suppose you wanted to set up a demonstration to “model” what happens inside our bodies when saliva mixes with starch and passes through our digestive system. Assuming your students had the background knowledge that you now have about membranes, starch, sugar, iodine, and Benedict’s solution, describe your demonstration in enough detail so that someone else in our class could set it up. Explain how you might be able to use your demonstration to assess your students’ understanding of some of the concepts we have been investigating today.

Specialized Cells

Today, you will observe 4 different types of animal tissues with the microscope. The 4 types are listed below. The objective of your observations is to determine if you can see a relationship between the structure of the cells that make up each tissue and the function of that tissue.

Materials:

Prepared slides of intestinePrepared slides of cartilagePrepared slides of musclePrepared slides of nerves

Procedure:

1. Obtain a slide of each of the 4 tissue types. Carefully focus the slide, first under the 10X objective, then under the 40X objective.

2. For each slide, locate the boundaries of the individual cells that make up the tissue. Check with an instructor to see if the two of you agree about these boundaries. Look for familiar cell parts. Do these cells appear to have the same parts or different parts? Explain.

3. What are the unique features of each type of cell (i.e., how do they appear different from each other)?

4. Formulate hypotheses about the possible functions of each tissue based upon the appearance of the cells that make up that tissue.

5. Scientists who study tissues (histologists) generally recognize 4 basic types of tissues. These are: 1) epithelial tissue; 2) connective tissue; 3) contractile tissue; and 4) conductile tissue. Each type is represented by one of the slides you observed. Try to match the slide with the type of tissue. Explain your choices.

6. A fundamental concept in biology is that structure reflects function. How does this exercise illustrate this relationship?

Applying Your Knowledge

Blood is a special type of connective tissue. Yesterday, you looked at slide of normal human blood. These same slides are available again today. In addition, slides are available from individuals with two different blood diseases. Each team will be provided with: 1) a slide of normal human blood; 2) a slide from blood disease #1; and 3) a slide from blood disease #2. Your task is to prepare a written memo that describes what you think might be happening in each of the two diseases.

The Sheep Pluck:Integrating Circulation and Respiration

(This will need to be written unless we find a set of dissection instructions that is appropriate.)

Using Technology to Investigate Heart Rate

We will use the Vernier middle school lab for this laptop exercise. Here is the URL for the lab: http://www.vernier.com/middleschool/index.html click on “heart rate and body position” to download the PDF

What Happens When Carbon DioxideIs Bubbled Through BTB?

When vinegar is mixed with baking soda, a chemical reaction occurs and carbon dioxide gas is produced. One of your instructors will demonstrate how a simple apparatus can be used to collect the carbon dioxide produced by this reaction.

In this exercise, you will investigate what happens when the carbon dioxide produced by mixing baking soda with vinegar is bubbled through a solution of a blue compound called BTB.

Materials:

60-cc syringes1-hole rubber stoppersReaction tubesBaking sodaVinegarDistilled water30-cc cupsBTB solutionDroppersKimwipes

Procedure:

1. Get two 60-cc syringes, two rubber stoppers, and two reaction tubes. The reaction tubes each have the same amount of baking soda added to them.

2. Get two 30-cc cups. Fill one cup half full with distilled water and fill the other cup half full with vinegar.

3. Using two 60-cc syringes, fill one to the “5 cc” mark with distilled water and the other syringe to the “5 cc” mark with vinegar. Put a rubber stopper into each of your two reaction tube so that it fits snuggly. Insert the syringe with water

tightly into one stopper and the syringe with vinegar tightly into the other stopper.

4. Hold one tube as demonstrated earlier by your instructor and have someone else in your group hold the other tube. Push the distilled water and the vinegar into the reaction tubes at the same time. Describe any reaction that occurs.

5. Using the tube and syringe that had the water in it, pull up on the plunger until the amount of space in the syringe is about the same as the amount of space in the syringe that had vinegar in it. Fill one of the 30-cc cups with BTB solution and slowly bubble the air inside the syringe through the BTB solution. In the space below, describe the result:

6. Now remove the syringe from the tube that has the vinegar in it. The plunger should already have been pushed out by the carbon dioxide gas formed by the reaction of the vinegar with the baking soda. Using a Kimwipe carefully blot the tip to the syringe to make sure there is no vinegar on it. Fill another 30-cc cup with BTB and bubble the carbon dioxide in the syringe through the BTB, In the space below, describe the result:

7. What conclusion can you make about what happens when carbon dioxide is bubbled through BTB? How do you know that the change you observed is not just due to bubbling air through the BTB?

Concept Applications

Application 1

Most of you have heard Coke, Pepsi, 7-UP and other soft drinks described as “carbonated” beverages. The “fizz” in these drinks produced as a result of bottling the drinks with carbon dioxide under pressure. When you open the cap or tab, the pressure drops and the carbon dioxide is slowly (or sometimes rapidly) released.

Given a can of club soda, a syringe, a reaction tube, a stopper and a cup of BTB, how could you test the hypothesis that carbon dioxide is, indeed, being released when you open a club soda? Why don’t you try set up such a test?

Application 2

What do you think would happen if you were to blow your exhaled breath through a straw into a container with BTB solution in it (e.g., you might fill a reaction tube with 10 – 20 cc of BTB, stick a straw into the hole in the stopper and bubble away)? Try it. How do you explain what you observed?

If you did this as a demonstration (although it might be more fun if your students did it themselves), why might you also want to show them what happens when you repeatedly push room air from a syringe into the straw and bubble it through the BTB?

The Chemistry Behind the ColorChange of BTB

We will need to discuss the actual chemistry that causes BTB to change color when carbon dioxide dissolves in a BTB solution. Before we do that, try each of the following tests:

1. Fill a cup with BTB. Add a few drops of vinegar to this cup and observe what happens.

2. Now, using the same cup with the BTB + vinegar still in it, add some dilute ammonia solution until you observe another color change.

3. Fill another cup with BTB. Add a few drops of lemon juice until you observe a clear color change.

4. Now, obtain some ground up antacid tablets from one of your instructors and add these to the cup containing the BTB + lemon juice. Keep adding antacid until you see another color change.

Respiration: Investigating Factors that Affect the Production of Carbon Dioxide by Yeast

Yeast cells are used in baking and in manufacturing beer and wine. They are also found in our environment and some kinds of yeast can cause infections in animals including humans. Yeast are considered to be members of the kingdom, Fungi. In today's laboratory, you will use a solution of baker's yeast that is perfectly safe and will serve as a source for respiring yeast cells.

Today, you will design an experiment that you would do to find out how temperature affects respiration by yeast cells growing in a sugar solution. However, you will first need to become familiar with the equipment we have in our laboratory for measuring the rate of respiration in yeast cells. Yeast cells carry out both aerobic and anaerobic respiration (i.e., respiration with and without oxygen), but you will be investigating anaerobic respiration. In this reaction, oxygen is not used but carbon dioxide is released as a gas, creating an increase in gas pressure. This pressure change can be detected with a device that can measure changes in gas volume in a closed system. Your instructor will show you how to set up this device and you will need to conduct a short experiment using the device so that you can prepare a formal write-up describing a hypothetical experiment using the same device.

Materials:

Reaction tubes Beakers or cups with sugarSyringes solutions and yeastRubber stoppers Beakers or cups with sugarA large container of sugar solution solutions of yeast that have Measuring containers been boiledGraph paper

Procedure:

1. Obtain 3 reaction tubes, 3 syringes, and 3 rubber stoppers.2. Add 10 ml of sugar solution to one tube, 10 ml of yeast in sugar

to the second tube, and 10 ml of boiled yeast and sugar solution

to the third tube.3. Insert the rubber stoppers into each tube; push the syringe

plungers all the way down in the syringes and insert each syringe tip into the stopper so that the system is tightly sealed.

4. Place all three tubes in the tube holders at your lab station; record the volume (in ml) of gas that is produced in each syringe at 5 minute intervals for at least 30 minutes. Agitate the tubes frequently so that the solution remained well mixed. (While you are conducting this experiment, make a wet mount from the yeast culture at the front of the lab and describe what you see when you examine this wet mount under high magnification.)

5. Using the graph paper available in the lab, construct a graph that accurately describes the results of your 30-minute experiment. Show this graph to your laboratory instructor before proceeding to the next part of this laboratory.

Experimental Design Assessment:

Each of you should now produce a write-up describing how you would conduct a controlled experiment to determine what effect temperature has on the rate of anaerobic respiration in yeast cells. To do this, you should use the same basic approach that you have just completed. Your write-up must contain the following elements: 1) a statement that clearly describes the purpose of your experiment; 2) a list of materials that you would need to conduct your experiment; 3) a section describing the procedure you would follow to conduct the experiment using the materials identified in #2; this procedure should be sufficiently detailed so that someone else could conduct the same experiment using your procedure; 4) a section that explains how you would present the results of your experiment and a description of the predicted results (i.e., describe what you expect to happen when you conduct this experiment); and 5) a discussion of factors that could potentially cause your experiment to turn out differently than you predict that it should.

Do Plants Produce Carbon Dioxide?

You may have learned that plants produce oxygen gas. But do plants also produce carbon dioxide? In science, the way to find the answer is by doing a good experiment. Today, you will need to design an experiment to test the possibility that plants produce carbon dioxide. As always, be sure your experiment has a control. Sometimes, it may be important to use more than one kind of control. Also recall that it usually takes seeds a day or two to begin to grow. Set up the experiment using the materials below. We will observe the results on Monday.

Materials:

Reaction tubes Paper towelsRubber stoppers Live radish seeds60-cc syringes Dead, sterile radish seedsPlastic cups BTB solutionWater

In the space below write down all the steps that you will do in your experiment. When the experiment is finished, you will need to describe and explain the results.

What Are Some properties of Chloroplasts?

Introduction and Background:

Most of you may know that plants are able to make their own food. You probably have learned that the process they use to do this is called photosynthesis. Photosynthesis uses carbon dioxide and water to a make a sugar called glucose and oxygen gas. Light energy is needed to "power" photosynthesis. The overall process of photosynthesis can be described with the following chemical equation:

6 CO2 + 6H2O + light energy C6H12O6 + 6O2

However, the actual process of photosynthesis is much more complicated than this equation suggests. It involves many separate chemical steps. These chemical reactions all take place inside a structure found in plant cells. This plant cell organelle is called a chloroplast. Last week, you had a chance to examine chloroplasts in the leaves of an Elodea plant and you determined the size of this organelle. In this laboratory you will investigate some properties of chloroplasts.

Materials:

compound microscopesglass slides and cover slipsextract of spinach leaves in ice-cold, 0.5 M sucroseextract of spinach leaves in ice-cold, 95% ethyl alcohol15-ml centrifuge tubescentrifuge

Procedure

In order to study parts of cells such as chloroplasts, biologists break the cells apart and separate the chloroplasts from the other cell parts. This procedure is called cell fractionation. One of your instructors will show you how biologists break up spinach leaves in order to get the chloroplasts out. The steps are written down below:1. The spinach leaves are trimmed to remove the large veins.

2. Twenty-five grams (g) of trimmed spinach leaves are weighed out and placed in a blender.

3. The spinach leaves are then covered with 100 milliliters (mL) of ice-cold fractionation solution. The procedure will be done twice, using a 0.5 M sucrose solution first and a 95% ethyl alcohol solution the second time.

4. Once the leaves are covered with the fractionation solution, the blender is turned on for three, 10 second time periods.

5. The blended spinach leaves are then passed through 2 layers of cheesecloth to remove any pieces that were not blended.

6. The filtered leaf mixtures are poured into glass containers which are placed on ice.

7. Each lab group should bring two clean glass slides to the front of the lab; mark one slide with an "S" (for sucrose) and the other slide with an "E" (for ethyl alcohol); place a drop of each solution on the properly-marked slide and place a cover slip over each drop.

8. Examine the wet mount of each solution and answer the following questions:

A. Describe what you see on each slide; draw if necessary.

B. What has happened to the plant leaf cells? Explain.

C. Can you still measure the chloroplasts? If so, what is their diameter on each slide?

D. What difference(s), if any, do you see between the sucrose extract and the ethyl alcohol extract?

9. Once you have answered these questions, you should find two, 15-mL centrifuge tubes at each station. Mark one tube with an "S" and one with an "E" and bring the two tubes to the front of the lab. You will then add 2 mL of each extract to the properly-marked tubes.

10. Once an instructor has made sure that your tubes are properly balanced, the tubes will be placed in a centrifuge. When the centrifuge is ready, the tubes will be spun at 5000 rpm for 10 minutes.

11. While the tubes are spinning, discuss this procedure with members of your lab group and predict what you think will happen. Write your prediction in the space below.

12. Obtain your two tubes are carefully observe them. DO NOT SHAKE OR MIX THE CONTENTS OF THE TUBES. In the space below, describe how the appearance of the two tubes differs.

13. How do you explain what you see? Write your explanation below and discuss it with other groups and with your instructor.

What Causes the DCIP to Lose Its Color?

Introduction and Background:

Think about what you have just discovered about chloroplasts. Remember that you were able to break spinach leaves apart in order to get out the chloroplasts. The chloroplasts are the parts of plant cells where photosynthesis takes place.

Now you will investigate a reaction which takes place when a spinach leaf extract is added to a solution of a blue dye called dichlorophenolindophenol. We usually use the abbreviation, DCIP, when we talk about this dye.

Materials:

The following materials will be provided to your group at each lab station:

A test tube rack with 20 clean, glass test tubesTransfer pipettesA plastic 5-mL pipetteA pipette pumpA container with iceA spinach leaf/ 0.5 M sucrose extract on iceA spinach leaf/ 95% ethyl alcohol extract on ice0.5 M sucrose on iceA lamp with a 100-watt bulbA marking pen100 mL of DCIP solution in Hill* bufferAluminum foil

*Hill buffer is a standard solution that biochemists use in photosynthesis experiments.

Procedure:

All teams should conduct the following short experiment:

1. Add 4 ml of The DCIP solution to each of 2 clean test tubes.

2. Turn on the 100 watt bulb.

3. Using a transfer pipette, add 3-5 drops of ice-cold 0.5 M sucrose solution to one of the tubes.

4. Now add 3-5 drops of the spinach extract in 0.5 M sucrose to the second tube; hold both tubes in front of the bulb and gently swirl the contents of the tubes.

5. Continue holding the tubes in the light for 2-5 minutes or until you see a clear change in one or both tubes.

6. Describe your observations. Before continuing, the class will discuss these results.

What do you think will happen if we repeat this experiment, but add 3-5 drops of the spinach/sucrose extract to one tube of DCIP and 3-5 drops of the spinach/ethyl alcohol extract in the other tube of DCIP? (Note: What you think will happen is not a hypothesis, it is a prediction; why you think it will happen is a hypothesis – i.e., a causal explanation.)

Now, conduct the experiment. Describe the result.

So was your prediction correct? How do you know? Now, why do you think that the result you observed occurred (i.e., what is your hypothesis)?

Consider the following hypotheses:

In order to cause DCIP to lose its color, the chloroplast must be intact.

Chlorophyll is necessary but not sufficient for DCIP color loss.

Have you tested these hypotheses? Explain.

What about this hypothesis?

Light is necessary for DCIP color loss.

Design an experiment that would test this hypothesis using materials provided and try it.

Cell Division: I think we should use some version of Lawson’s learning cycle lab: How Do Multicellular Organisms Grow?

The BioPhoto sheets of human karyotypes will provide the “raw materials” for the afternoon activities. We may or may not want an additional write-up besides what comes with the sheets. There would be 6-7 sheets and lots of cutting out photos of chromosomes. Linda, you may want to contribute to this since it’s basically human genetics.

Human genetic traits – a class survey

We will be using Carolina BioPhoto sheets again along with PTC paper. The idea is to survey the traits pictured on the sheets along with tasting the PTC paper and record the genetic diversity of the entire group.

This one is not the lab we should use, but it will give you an idea about the content of the kit; the exercise in the kit is simpler – it does not include the “Rh” factor, just the “A”, “B”, “AB” and “O” blood groups; the Whose Baby BioKit from Carolina will have a write-up with it.

Genetics: Investigating the Rules of Heredity

Why do we look like our parents? This question has intrigued humans for ages. The question has been answered differently as our understanding of biology has changed and – we hope – improved. One of the earliest successful scientific investigations of heredity was conduction in the 1860s by the head of an Austrian monastery named Gregor Mendel. Mendel investigated the patterns of inheritance of 7 pairs of traits in garden peas (actually, he picked these 7 pairs of traits because they showed a pattern of inheritance that was not obvious in other traits). Mendel’s results described inheritance of these pairs of traits in precise, quantitative terms. Nevertheless, Mendel died having never understood the physical basis of the quantitative rules that governed the patterns of inheritance of his pairs of traits.

Today, we believe that we do understand the physical basis of trait inheritance. Traits are determined by pairs of factors located on chromosomes found within the nuclei of living cells. These factors are known as genes. Genes, in turn, are believed to be composed of DNA. Next week, you will investigate the way in which the arrangement of the building blocks of the DNA molecule (called nucleotides) function to

encode genetic information. This week, you will investigate the rules of inheritance that were originally discovered by Mendel.

Relating Chromosomes and Genes

Last week, you investigated cell division. You should have observed chromosomes in both plant and animal cells. One of the most important conclusions reached by early investigators of heredity was the realization that Mendel “factors” (i.e., genes) were physically located on chromosomes. Each of the traits Mendel studied in peas was determined by a pair of factors, or genes. Once genes were located on chromosomes, it became clear that chromosomes also came in pairs and that any given pair of genes was located on a pair of chromosomes. During meiosis, it was observed that the pairs of chromosomes separated from each other. This immediately explained why Mendel had deduced that his “factors” became separated from each other during the formation of sex cells (i.e., gametes). The pairs of genes that were carried on the chromosomes were separated when the paired chromosomes separated during meiosis. In other words, the physical behavior of chromosomes during mitosis and meiosis explained the quantitative behavior of genes.

Inheritance of Human Blood Antigens

Humans have a number of genes that affect what is commonly called blood type. Indeed, the actual blood type of an individual is determined by the number and kinds of antigens found on red blood cells. Blood type antigens are molecules with unique shapes that project from the cell surface of the red cells. Each unique antigen can cause the immune system to form antibodies (i.e., specific proteins) that will recognize and bind to the antigen provided that the antigen is not normally found on the surface of the red cells of the person who is making the antibodies. Thus, blood from an individual with a type “A” antigen that is transfused into an individual who lacks the “A” antigen will cause the immune cells of that individual to form antibodies (called anti-A antibodies) against the foreign red cells. These antibodies will cause the blood cells that have the “A” antigen to clump together and block blood vessels, thus posing a threat to the well being of the person who receives the transfusion. This is called an aggultination reaction. Ultimately, it is the genes carried on the chromosomes that determine whether or not a given antigen will be produced by an individual.

In today’s laboratory, we will be investigating two types of antigens. The first type determines the so-called “A-B-O” blood group. The second antigen determines the “Rh” type of the red cell. In humans, the “A” antigen and/or the “B” antigen can be present; or, alternatively, no antigen is present. If there is no antigen, the person’s blood type is said to be type “O”. In terms of the genes present, there is a dominant allele that leads to the production of the “A” antigen and another dominant allele that leads to the production of the “B” antigen. A person may have either one or both of these alleles (remember that genes always come in pairs). However, if a person has neither of these alleles, he/she is said to have a pair of recessive alleles that result in no antigen production for the “A-B-O” blood group (i.e., individuals with type “O” blood). With respect to the Rh antigen, individuals are said to be either “Rh positive” (i.e., they have the antigen) or “Rh negative” (i.e., they do not have the antigen). In terms of genes, this means that individuals who are “Rh +” have at least one dominant allele that caused the production of the Rh antigen whereas individuals who are “Rh –“ have two recessive alleles and do not make the antigen. Thus, any given individual will have 4 alleles for these two blood types. One pair of alleles will determine the “A-B-O” blood type while the second pair will determine the “Rh” type. Furthermore, six combinations of “A-B-O” alleles are possible:

1. A person may have two dominant alleles for type “A” antigens (usually designated as IAIA) and will have type “A” blood;

2. A person may have one dominant allele for type “A” antigens and a recessive allele (designated as IAi) and will still have type “A” blood;

3. A person may have two dominant alleles for type “B” antigens (designated as IBIB) and will have type “B” blood;

4. A person may have one dominant allele for type “B” antigens and a recessive allele (designated as IBi) and will still have type “B” blood;

5. A person may have one dominant allele for type “A” antigens and another dominant allele for type “B” antigens (designated as IAIB) and will therefore have type “AB” blood; or

6. A person may have two recessive alleles and will therefore make no antigens for this blood group (designated as ii ) and will have type “O” blood.

For the “Rh” antigen, there are only three combinations of allelespossible:

1. A person may have two dominant alleles for production of the “Rh” antigen (designated as +/+) and will be “Rh positive”;

2. A person may have one dominant allele and one recessive allele (designated as +/-) and will still be “Rh positive”; or

3. A person may have two recessive alleles, make no Rh antigen (designated as -/-) and will be “Rh negative”.

The pairs of allele for these two traits (i.e., “A-B-O” blood type and “Rh”type) are inherited independently (i.e., they are on different pairs of chromosomes). Therefore, for each of the six allele combinations of the “A-B-O” blood group, there are two combinations of Rh alleles (i.e., + or -) . This means that there are 12 possible genotypes and 8 possible phenotypes. Be sure you understand this last statement before you proceed. You will need to use all of this information in today’s laboratory.

Determining Paternity

It sometimes happens that a woman gives birth to a child whose father is not known with certainty. In such cases, the woman is usually able to limit the father to several possible males. Evidence from blood typing can often be useful in resolving paternity among the possible fathers. In today’s laboratory, you will collect data on the blood types of a mother, her child, and three potential fathers. Using genetic analysis, you will attempt to determine which of the three men might be the father. You will be required to report on your results using the data you collect to justify your tentative conclusions. Your instructor will show you how to determine blood types for the “A-B-O” blood groups and for the “Rh” factor. Using this method, you should test all 5 samples and then analyze the results using your knowledge of the results and of the rules of inheritance for human blood groups that were previously discussed. Once you have finished your data collection and analysis, write answers to the following questions:

1. What is the blood type of the mother? Her child? The three potential fathers?

2. Given this information, which genotypes can you determine with certainty? Justify your answer.

3. Assuming that the three males who provided the blood samples in this case are the only possible fathers, is it possible to say with certainty which one was the father? Justify your answer.

4. Assuming that these are not the only three males who could have fathered this child, can you still determine paternity with certainty? Explain why or why not.

5. Suppose that this same mother marries the suspected father (based upon the results). If they have another child who is blood type “O negative”, would this cast doubt on the paternity of the first child? Explain why or why not.

Molecular Genetics: Investigating theStructure and Function of DNA

Most of you have probably heard about a molecule called DNA. You may even recall that DNA has something to do with how you get your characteristics from your parents (e.g., if your parents both have red hair, you are likely to have red hair, too). DNA stands for deoxyribonucleic acid, so you can see why they call it DNA! But what does this stuff really look like? Today, we will try to find out by extracting DNA from plant material. The plant material we will use is called wheat germ (wheat germ consists mainly of the embryos of wheat plants).

Materials:

Raw wheat germ Cheesecloth Sample cups for weighing wheat germGlass stirring rodsIce-cold nuclear buffer 10 mM Tris/1 mM EDTA15-mL centrifuge tubes 10% SDSMotars and pestles 7.5 M ammonium acetateGlass beakers Ice-cold 95% ethanolCentrifuges Spooling rods700 C water baths Glass test tubesIce baths DNA model kits

Procedure:

1. Put 2-3 grams of wheat germ into a cold mortar. Pour in 30 mL of ice-cold nuclear buffer.

2. Grind the wheat germ and the nuclear buffer with the pestle until a smooth mixture is formed.

3. Pour the mixture through two layers of cheesecloth into a glass beaker, squeeze the cloth to get out as much of liquid as you can, and then pour the filtered liquid into two 15-mL centrifuge tubes. Label one of your tubes for identification and put the other tube aside for possible future use.

4. Take your tube to one of the centrifuges in the lab. Your instructor will make sure all the tubes are balanced and will put them into the centrifuge and turn it on. The centrifuge will spin the tubes at low speed for 5 minutes.

5. Get your tube from the centrifuge. Notice that there is a solid pellet at the bottom of the tube and liquid on top. Pour off the liquid into your glass beaker (to be discarded later).

6. Use one of the 5 mL pipettes at your station to transfer 2.5 mL of the TRIS/EDTA solution to your tube. Then transfer 1.0 mL of 10% SDS to the same tube.

7. Now use a glass stirring rod to mix the pellet and the liquid until most of the pellet is suspended in the liquid.

8. Put your tube into the 70o C water bath. Leave the tube in the water bath for 10 minutes and shake it every 2 or 3 minutes during this time.

9. Transfer your tube from the hot water bath to the ice bath closest to your station; using a clean, 5.0 mL pipette, add 1.8 ml of 7.5 M ammonium acetate. Mix the contents of the tube completely and leave the tube in the ice for 10 minutes. During this time, you will discuss what you have been doing.

10. Take your tube to one of the centrifuges again. Your teacher will match your tube with a tube belonging to another group and will spin the

tubes for 15 minutes at high speed. During this time, the class will discuss some information about DNA.

11. Remove the tube from the centrifuge and CAREFULLY pour the liquid into the clean, glass tube at your station. BE SURE NOT TO TRANSFER ANY OF THE PELLET. Now observe the amount of liquid in the glass tube and SLOWLY pour in about twice as much ice-cold 95% Ethyl Alcohol from the labeled tube down the side onto the top of the liquid in your tube. The alcohol should remain on top of the liquid.

12. Now, get a glass spooling rod from your instructor. Take the rod and slowly move it up and down between the liquid in the bottom of the tube and the alcohol, turning the rod as you move it up and down through the interface between the two layers. As you do this, fine white fibers should appear and stick to the glass rod. These fibers should be DNA molecules.

_______________________________________________________________Part II:

Using a Model To Understand DNA

Structure and Function

You have isolated DNA from wheat germ. However, all you can see is a white glob of fibers. Scientists claim these fibers are made up of DNA molecules. As with all molecules, individual molecules DNA cannot be seen with the naked eye. One way to try to understand what DNA molecules might be like is study a scale model of the molecule. You will try to learn something about DNA using a simple scale model. As you study this model and answer questions about it, try to remember that it is a model, not the real thing. Nevertheless, the model is based upon what scientists believe that they know about real DNA molecules.

Compared to everyday objects in our world, DNA molecules are very small. However, compared to many other molecules, DNA molecules are large. In fact, scientists believe that DNA molecules are put together by

living things using a number of smaller molecules. The different parts of our model represent these smaller molecules that make up the DNA molecule. Here is a list of the parts used to build the model along with the names that scientists give to these molecules that make up DNA:

Quantity Name of Part Molecule Represented

by the Part

0-6 Blue straw Adenine0-6 Red straw Thymine0-6 Green straw Guanine0-6 Grey straw Cytosine

12 Black, 3-pronged Deoxyribose connector (a sugar)

12 Red, 2-pronged Phosphate connector

6 White, 2-pronged Hydrogen bondconnector

24 Yellow straw Chemical bond

joining deoxy-ribose

and phosphate 1 Long straw Support stand*

1 Triangular base Support stand*

*These two parts hold the model together, but do not represent parts of the real DNA molecule.

The finished model should look something like a twisted ladder (called a double helix). The rungs of the ladder are the red-blue and/or green-gray pairs of straws. The sides of the ladder represent the two "backbones" of the DNA molecule. Notice that each backbone consists of repeating units. A black connector (representing a deoxyribose sugar molecule) is joined by a yellow straw to a red connector (representing a phosphate molecule) which is joined by a yellow straw to another black connector which is joined by a yellow straw to a red connector and so on all the way up the length of the backbone. The yellow straws represent

chemical bonds (i.e., sugar-phosphate bonds). The straws (i.e., representing bases) sticking out from two sides of the ladder are held together by hydrogen bonds (represented by white connectors). NOTICE THAT RED STRAWS ARE ALWAYS PAIRED WITH BLUE STRAWS AND GREEN STRAWS ARE ALWAYS PAIRED WITH GRAY STRAWS. This is a basic “rule” of DNA structure determined by the chemical structure of the actual molecules represented by these straws.

Notice also that the two ends of the backbones look different (i.e., one end will have only a yellow straw sticking out while the other end will have a yellow straw with a red connector sticking out). Furthermore, one backbone "points" in one direction, while the other backbone "points" in the opposite direction. This is another feature of the DNA molecule that is important. The two strands are described as being antiparallel.

Now that you have a completed model of a DNA molecule, you are ready to try to learn something about DNA and how it works. DNA is found inside each one of the trillions of cells inside your body. It is believed to carry the information that makes you what you are and makes you different from other people. It also makes humans different from other kinds of living things. How does this happen?

Each pair of students in the class should have a model of a DNA molecule. All the models should look about the same. But are they exactly the same? Carefully compare your model to the models of other pairs of students. Are there any differences? If so, explain what the differences are.

Now, look at your model. Starting at the top, write down thecolor sequence (i.e., the order of blue, red, green, or gray straws) on the side of the model that has the yellow straw with the red connector sticking UP.

(top) ______ ______ ______ ______ ______ ______ (bottom)

Compare your color sequence with the color sequences of other groups. How many different color sequences can you find in the whole class?

Now, trade your written color sequence with the sequence of another group. Recall that there is a rule for making colored PAIRS in our models of DNA molecules. Using this rule, can you figure out the top-to-bottom color sequence that is attached to the other backbone of the other group's DNA model without looking at their model? If you think so, write the sequence below and then look at their model to see if you were correct.

(top) ______ ______ ______ ______ ______ ______ (bottom)

Now, suppose that your teacher were to give you half of a DNA model (i.e., one backbone with only one set of blue, red, green, and gray straws attached to the black connectors). Could you build the other half of the model without any instructions? Explain how you think it could be done.

Each time a living cell divides, scientists believe that real DNA molecules inside of that cell make copies of themselves. Does what we just did with our models give you an idea about how this could happen? Explain.

Now let's think about a different problem using our DNA models.Every model in the class has 6 colored pairs of straws. Assuming that you could have all the blue, red, green, and gray straws you wanted, how many DIFFERENT models with six colored straw pairs could you make?

In real DNA molecules, scientists call the things that make the "rungs" of the ladder BASES. There are four bases, called ADENINE, THYMINE, GUANINE, and CYTOSINE. In our model we represented these bases with red, blue, green, and gray straws. As we stated earlier, there is a rule in real DNA molecules that determines which bases can form pairs. Recall this rule. In molecular language, adenine must pair with thymine and guanine must pair with cytosine. Each model we built would represent a DNA molecule containing 6 correctly matched base pairs.

However, real DNA molecules have many more than 6 base pairs. A single set of 23 human DNA molecules contains about 3,000,000,000 base pairs and each of your cells has 2 such sets (one from your father and one from your mother). To get some idea of how complicated this gets, do the following exercise. Have everyone in the class join their DNA models end-to-end. Before you connect your model to another group’s model, both groups should remove the models from the support stands. Remember that the position of the yellow straws and the yellow straws with the red connectors will determine which end fits with another group's model. HOWEVER, THE ORDER IN WHICH DIFFERENT GROUPS JOIN WILL DETERMINE THE COLOR SEQUENCE OF THE FINISHED MOLECULE. Once all the molecules are joined, your teacher will help you to write down the color sequence of the entire model.

How many base pairs are represented by this "big" model?

__________________________________

If everyone had as many blue, red, green, and gray straws as he/she wanted, how many different color sequences could this "big" model have?

_________________________________

How many models with 6 pairs of colored straws each would you need in order to represent ONE set of human DNA molecules?

_________________________________

Now measure the length (in centimeters) of one of the models (i.e., a model with 6 pairs of colored straws). How many meters long would a scale model of ONE set of human DNA molecules have to be? (Hint: Recall how many bases pairs are in one set of human DNA molecules.)

_________________________________

A single set of "real" human DNA molecules has an actual length of about 1 meter (i.e., this is the actual length of the DNA molecules in a set of 23 chromosomes stretched out end-to-end). How many times the size of real DNA is our DNA model? This number represents the scale of the model.

_________________________________

The number of possible sequences of colored straws that a scale model of a single set of human DNA molecules could have is:

43,000,000,000

In other words, this number is 4 multiplied by itself 3,000,000,000 times! The number is much larger that the total number of atoms scientists believe to be present in the known universe. Even supercomputers cannot handle calculations using this number. Don't try to write this number down because you will be dead long before you could finish.

Now, why don't you try to solve this additional problem? Suppose that you want to send secret messages to other students in the class. Also suppose that you have to use the DNA models to send these messages and the models have to be made using the rules you learned about. How could you do this? (Hint: if any single letter in the alphabet can be represented by more that one base at a time, then what is the minimum number of bases required to represent each one of the 26 letters in the alphabet?).