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8 Diffusion Across a Membrane and Osmosis

Background

Diffusion refers to the movement of molecules from an area of higher to one of lower concentration. It is the result of natural random movement of molecules and hence does not require energy. In the body of an organism, diffusion is a common means of transport. For instance, gases and other solutes diffuse into and out of cells of many organisms. For diffusion to occur, two conditions need to be met. First, there must be a difference in concentration of a substance. Second, the membrane must be permeable to the substance. For example, if the concentration of a substance is higher outside of cells than inside and the cell membrane is permeable to the diffusing molecules, then the substance diffuses into the cells. (If there is equal concentration then substances diffuse in and out).

Cells are able to keep molecules from leaking out or prevent unwanted molecules from coming in by having selectively permeable (or semi-permeable) membranes. Water, gases and other small uncharged molecules are generally able to easily diffuse across. The cell membrane, however, is impermeable to many others, particularly large organic molecules.

This process of water diffusing across a semi-permeable membrane is called osmosis. The net (overall) movement of water across the membrane has a tremendous impact on cells since water is the medium of transport in and out of cells. If the solution outside of cells has a lower solute concentration (hypotonic) and therefore a higher water concentration relative to the solution inside the cell, then net movement of water will diffuse into the cells. The result is an increase in the volume of cytosol that will cause the cell to swell. If the cell does not have a cell wall or some other means of protecting the membrane, it will burst! On the other hand, if the solution outside has a higher solute concentration (hypertonic) and therefore a lower water concentration than inside the cell, then the net movement of water will diffuse out of the cells causing the cell to lose water and shrink. Outside solutions with the same solute concentration as the inside the cell (isotonic) will have a zero net movement of water, and hence, minimal impact.

To review osmosis and learn about real life examples of osmosis, watch

Osmosis and Water Potential (Updated)

by Amoeba Sisters (9 min 49 sec, but watch only to 7 min 29 sec. Do not need to know water potential.)

Hypotheses and predictions

  • If the concentration of a substance is higher on one side of the membrane and the membrane is permeable to the substance, then substance will diffuse to the other side of the membrane.
  • If membranes are more permeable to small molecules, then small molecules will diffuse across the membrane more rapidly than large molecules.
  • If osmosis occurs, then cells will gain water and swell when placed in hypotonic environment and lose water and shrink when placed in hypertonic environments.

Purpose

  • Demonstrate diffusion of substances across a model cellulose membrane.
  • Determine the effect of molecular size on the permeability of a model cellulose membrane.
  • Demonstrate osmosis in both a model cell and living cell.

Diffusion of starch and iodine across a membrane

Materials (used in video)

  • Cellulose (dialysis) tubing (cut to 10cm)
  • Test tube
  • Rubber bands or tubing clips
  • Starch solution
  • Iodine (dropper)
  • One 250 ml beaker

Procedure ( demonstrated in video)

Watch “Diffusion of Through the Membrane Lab”

https://youtu.be/Kxsj37b0ruY

(4 min 29 sec) to view the set up and experiment with results. Focus on the starch solution and ignore the glucose.

  1. Obtain 1 piece of cellulose tubing approximately 10 cm in length. Open the tubing by wetting it and rubbing. Tie a knot at one end of the tubing.
  2. Pour the STARCH solution into the tubing almost to the top. Tie a knot, rubber band or clip the open end. Rinse the outside of the tube to remove any residual starch. Make sure the tubing does not leak. Record the initial color in the tubing in Table 1 below.
  3. Fill the beaker halfway with tap water. Put tubing into beaker. Then add enough iodine to turn the water a distinct yellow/amber color. Record initial color of water in beaker A in Table 1 below. Place beaker aside for the rest of the lab period.

Osmosis in potato (DIY experiment)

Materials

  • 4 slices of potato (~ 1 inch or 2 cm thick)
  • salt (~2 Tablespoons)
  • tap water
  • two bowls

Procedure

Watch ”DIY Science Experiment on the Osmosis of a Potato”

(1 min 9 sec) which demonstrates the experiment.

  1. Obtain two bowls. Pour water into the two bowls. Add approximately 2 tablespoons of salt to one bowl & label salt. Label the other bowl
  2. Obtain four slices of potato. Determine initial turgidity (how hard or soft) by gently giving the potato slices a squeeze. Turgid potato will be firm or hard whereas flaccid potato will feel soft or soggy. This is the “control” condition with the potato out of water. Record observations in Table 2.
  3. Place two potato slices in each bowl.
  4. Allow the potato to stay in the bowls for at least 20 min.
  5. Remove the potato slices and determine final turgidity. Record observation in Table 2.

Results

Screen Shot 2020-06-21 at 4.53.02 PM.png

Discussion/Conclusion

Diffusion of starch and iodine across membrane

  1. Did the iodine molecules diffuse across the membrane? Yes or No? _____
    1. What results in Table 1 support your conclusion?
    2. Explain why iodine diffused or did not diffuse based on difference in concentration of iodine, size of iodine molecules and permeability of tubing membrane.
  2. Did the starch molecules diffuse across the membrane? Yes or No? _____
    1. What results in table 1 support your conclusion?
    2. Explain why starch diffused or did not diffuse based on difference in concentration of starch, size of starch molecules and permeability of tubing membrane.

Osmosis

  1. Was the potato in the “water” beaker placed in a hypertonic, isotonic or hypotonic environment? ____________________
    1. What direction did the water move? _________________________________________
    2. What results in table 2 support your conclusion?
    3. Why did the water move in that direction?
  2. Was the potato in the “salt water” beaker was placed in a hypertonic or hypotonic environment? ____________________
    1. What direction did the water move? ______________________________
    2. What results in table 2 support your conclusion?
    3. Why did the water move in that direction?
  3. Even though the potato cells have lost or gained water in each of the experiments, the overall size of the potato columns did not change. Why not?
  4. If you don’t want a soggy salad, why should you wait to put the dressing on just before eating it? Explain using the concept of osmosis.

9 Enzymes in Living Tissues

Background

Enzymes are present in all living tissues because they are critical for chemical reactions. Without enzymes, reactions such as breaking down a sugar molecule would occur too slowly to be useful, perhaps not even in your lifetime. Enzymes are catalysts, which are molecules that speed up chemical reactions. They typically speed up chemical reactions by a very large factor, from 10,000 to 1,000,000 times.

Since enzymes are proteins, they consist of chains of amino acids folded together in a specific 3D shape. If enzymes lose their shape, they lose their ability to bind to their substrates, and their ability to function as a catalyst. This change in the specific 3D shape is called denaturation.

For a review of enzymes, watch“Enzymes” by Amoeba Sisters (5 min 46 sec).

This laboratory will demonstrate the presence of catalase, an enzyme located inside both animal and plant cells, and study its properties. Catalase breaks down toxic hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2). The H2O2 is the substrate for catalase. Water (H2O) and oxygen (O2) are the products. Oxygen (O2) is a gas that creates bubbles that are detectable and an indication that the reaction occurred.

Screen Shot 2020-06-21 at 8.55.40 PM.png

Hypothesis and prediction

If catalase is present in the cells and functioning, then catalase will break down H2O2 and oxygen gas (O2) will be released.

Purpose

  • To determine the presence of the enzyme, catalase, in plant and animal tissues inside the cell.
  • To determine the effect of temperature on enzyme activity.

Materials

  • Ground potato (raw and boiled)
  • Ground liver (raw and boiled)
  • Hydrogen peroxide (3%)
  • Glass stir rods
  • Test tube racks
  • Test Tubes
  • Forceps
  • Disposable pipettes
  • Ruler with mm markings

Procedure

Test for control condition, catalase enzyme in raw and boiled liver.

Watch “Liver and catalase make-up lab” and follow along with the procedure (only up to 9 min and 30 sec).

  1. Observe whether O2 gas bubbles form or not in the graduated cylinder with only H2O2. If O2 gas bubbles form, record the maximum height (in mm) that the bubbles reach within 10 seconds in Table 1. Select one of these options as to how many mm of O2 gas bubbles form: 0, 2, 50, 100 or 200 mm. Record in Table 1. Note: Graduated cylinder in the video is ~110 mm in height.
  2. Test tube 1: Place a piece of raw liver into test tube. Push it all the way down then add 2 ml of H2O2. Select one of these options as to how many mm of O2 gas bubbles form: 0, 2, 50, 100 or 200 mm. Record in Table 1. Note: Test tube in the video are ~110 mm in height.

*Note: Do not record data for test tube #2 with reused liquid.

  1. Test tube 1 retest: After draining previously used liquid in test tube 1, add 2 ml more of new H2O2 to the previously used liver in test tube #1. If O2 gas bubbles form, record the maximum height (in mm) that the bubbles reach within 10 seconds. Select one of these options as to how many mm of O2 gas bubbles form: 0, 2, 50, 100 or 200 mm. Record in Table 1.
  2. Test tube 3: Boil liver with water for 5 min. Let it cool & drain water from test tube. Add 2 ml of H2O2. If O2 gas bubbles form, record the maximum height (in mm) that the bubbles reach within 10 seconds. Select one of these options as to how many mm of O2 gas bubbles form: 0, 2, 50, 100 or 200 mm. Record in Table 1.

Test for catalase enzyme in raw and boiled potato

Watch Catalase Enzymeand following along with procedure (view up to 33 sec. only).

  1. Place a piece of potato into test tube. Add 2 ml of H2O2. If O2 gas bubbles form, record the maximum height (in mm) that the bubbles reach within 10 seconds. Select one of these options as to how many mm of O2 gas bubbles form: 0, 2, 50, 100 or 200 mm. Record in Table 1.
  2. Place boiled potato into test tube. Add 2 ml of H2O2. If O2 gas bubbles form, record the maximum height (in mm) that the bubbles reach within 10 seconds. (No video available; predict the results.)

Results

enzyme table.png

enzyme graph.png

Discussion/Conclusion

  1. What is the reaction catalyzed by catalase? ______________________________________
    1. What is the substrate: __________
    2. What are the products? __________ and __________
  2. Recall that the 4 main organic substances are carbohydrates, lipids, proteins and nucleic acids. Which organic substance are enzymes such as catalase made of? _______________
  3. Let’s look at the presence of functional catalase in living tissues.
    1. What is the control condition and why do we need it?
    2. Was catalase present in animal cells? _____ Which result(s) supports your answer.
    3. Was catalase in animal cells reusable? _____ Which result(s) supports your answer?
    4. Was catalase present in plant cells? _____ Which result(s) supports your answer.
    5. Was catalase functional in raw tissues? ______ Which result(s) supports your answer.
    6. Was catalase functional in boiled tissues? _______ Which result(s) supports your answer.
    7. Explain why the results were the same or different in raw and boiled tissues. Discuss your predicted result for boiled potato.
  4. Do your results support your hypothesis? Explain.

10 Function of Chlorophyll

Background

Various pigments are found in leaves. We see these in autumn leaves when the green starts to fade. In some plants like Coleus, the colors can be seen all the time. Pigment colors may be bright orange, yellow, red, purple or green. The green pigment is chlorophyll, a molecule that strongly absorbs light energy and stores it in its chemical form.

Chlorophyll plays a role in photosynthesis, the process that plants use to convert the energy of sunlight into chemical energy in carbohydrates. They do this by using CO2 and H20 as raw materials to manufacture glucose in the presence of light. The glucose can then be transported as sucrose (table sugar) or be stored as starch.

To find out the function of chlorophyll in leaves, the green areas of a leaf will be matched with the location of starch after all the pigments have been extracted. Iodine solution will be used to locate starch in the leaf.

For a review of photosynthesis, watch “Photosynthesis and the Teeny Tiny Pigment Pancakes” by Amoeba Sisters (7 min 45 sec).

Hypothesis and prediction

If chlorophyll is required in photosynthesis, then we predict that starch (which are products of photosynthesis) will be found in parts of the leaf that contain chlorophyll.

Purpose

Demonstrate the role of chlorophyll in photosynthesis.

Materials (used in the video)

  • Ethanol alcohol
  • Variegated leaf
  • Forceps
  • Bunsen burner
  • Test tube
  • Water bath
  • Iodine
  • Beakers
  • Tripod

Procedure

Testing a leaf for starch

Watch Testing a leaf for starch (6 min 25 sec) and follow the procedure below to collect your data.

  1. Obtain a fresh variegated leaf and draw its color patterns. Note particularly where the green color is located.

Figure 1. Fresh Coleus Leaf with oval to draw leaf specimen.

  1. Place the leaf into a boiling water bath for 20 seconds to stop photosynthesis & denature enzymes.
  2. Place the leaf into a test tube with alcohol. Place the test tube with alcohol & leaf in the boiling water. Wait until the green color is removed and the leaf is completely clear.
  3. Rinse the leaf in water bath and spread the leaf in Petri dish.
  4. Saturate the leaf with iodine solution, followed by a rinse in cold water. Diagram the leaf and note location of color patterns.

Figure 2. Leaf after iodine solution with oval to draw leaf specimen.

Discussion/Conclusion

  1. The green areas in the leaf contained what pigment? ___________________
  2. Why do you use alcohol to extract chlorophyll?
  3. What is the purpose of iodine in this experiment?
  4. Blue/purple areas in the leaf after iodine was added contained what molecule? ____________
  5. Are the blue/purple areas found where the green sites used to be? ________________. What does this result imply regarding chlorophyll function in a plant leaf?
  6. Light is a critical component of photosynthesis. In the following experiment, one leaf is exposed to light and another leaf is covered with aluminum foil. Then both leaves are tested for the presence of starch using iodine.
    1. Complete this hypothesis with a prediction. If a leaf is not exposed to light, then ____________________________________________________________________________.
    2. Watch

    Starch Detection in Leaves(2 min 34 sec) which follows a similar procedure as listed above. What are the results of starch detection in this experiment?

  7. Do the results support your hypothesis in 6? Explain using the obtained data.

11 Anaerobic Respiration in Fungi: From Sugar to Alcohol

Background

How do we get our energy? Like fungi, we are heterotrophs, which means we cannot make our own food. Instead, we must get our food from the environment. Fungi do the same. They absorb their food in the form of sugar. Fungi must then convert the stored energy in sugar (ie. glucose) into the usable form of energy in ATP. This process can be carried out with (aerobic) or without (anaerobic) oxygen gas. Fermentation is the process of an organism converting chemical energy from sugar into ATP without oxygen gas. Depending on the type of organism, specific fermentation products are produced. The fungi, Baker’s yeast (Saccharomyces cerevisiae), famously ferments sugars to produce ethanol alcohol in bread, beers, wines and other alcoholic drinks.

The fermentation process is useful in the field of biotechnology. Recombinant DNA technology allows us to insert genes of interest into the yeast plasmid DNA. When the yeast goes through fermentation, it will produce our desired product of interest from the inserted gene. Many products can be manufactured using this method such as foods (ie. cheese, milk, yogurt), pharmaceuticals (drugs), hormones, proteins, vaccines and other products.

In yeast, alcohol is the natural fermentation product and occurs according to this metabolic pathway:

Screen Shot 2020-06-22 at 11.12.34 AM.png

In this exercise, yeast will be allowed to consume sugar containing protein under anaerobic conditions to demonstrate fermentation. We will monitor the release of the endproducts, ethanol alcohol and carbon dioxide (CO2).

To review fermentation, watch Fermentation by Amoeba Sisters (8 min 34 sec).

Hypothesis and prediction

If yeast is given sugar in the absence of oxygen gas, then they will go through the process of fermentation to produce ethanol, carbon dioxide and ATP.

Materials (used in video)

  • Baker’s yeast
  • Sugar
  • 100 mL water
  • Balloon
  • Timer
  • Bottles

Procedure

Watch Fermentation of Yeast & Sugar – The Sci Guys: Science at Home (4 min 17 sec) and follow along with the procedure below.

Bottle Preparation

  1. Prepare 3 bottles. Add 2.25 teaspoons of yeast to each bottle.
  2. In bottle 1, add 1 teaspoon of sugar. In bottle 2, add 2 teaspoons of sugar. In bottle 3, add 3 teaspoons of sugar.
  3. Add 1 cup (237 mL) of warm water to each bottle.
  4. Cover each bottle with a balloon and let sit for 1 hour.

Monitor Fermentation

  1. On the Results sheet attached, the starting CO2volume in the balloon in the Table 1 is recorded. This is run time 0 and begins when you put the balloon on the bottle.
  2. The total CO2 volume in the balloon for bottle #1 (with 1 teaspoon of sugar) is recorded every 2 minutes for 30 minutes.
  3. Note changes occurring inside the flask (ie. color, movement, volume) during fermentation. Look at the video time 1 min 43 sec versus 3 min 22 seconds. Record this data.

Note: Human consumption of this experiment’s product is NOT ALLOWED!

Results

Screen Shot 2020-06-22 at 11.07.47 AM.png

Compare the initial reactants in the bottle to the final product (color, transparency):

Record your data from Table 1 for bottle #1 on the graph below. Put time on the X-axis and CO2 released on the Y-axis. Be sure to label your axes.

Next, draw on figure 1 what you predict the data to be for bottle #2 and bottle #3.

fermentation graph.png

Discussion/Conclusion

  1. Describe all observations that demonstrate that fermentation occurred in this experiment.
  1. What were the roles of the yeast, sugar and balloon in the fermentation process?

Yeast:

Sugar:

Balloon :

  1. Why does carbon dioxide production increase over time?
  1. Explain the basis of your predicted hypothetical data drawn on Figure 1 for bottle #2 and #3.
  1. How can yeast fermentation be used in biotechnology to make useful products for humans?

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