Biology Laboratory Manual 11th Edition Vodopich Solutions Manual

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Exercise 9

DIFFUSION AND OSMOSIS: PASSIVE MOVEMENT OF MOLECULES IN BIOLOGICAL SYSTEMS

This exercise expands the definition of diffusion to include the concept of free energy. However, many students find this concept difficult to understand. They will need some help interpreting their results in terms of free energy rather than just concentration. Also important is the concept of a "model" in biological investigation. Specifically, the dialysis bag is used as a model of a cell.

If time permits, use this exercise to discuss aspects of experimentation such as hypotheses, quantification, sources of error, design of graphs, and individual versus class results.

Suggested Elements For An Introductory Lecture

• Heat causes molecules to move randomly and drives diffusion.

• Brownian movement is the random movements of small particles caused by collisions with other small particles, in this case water molecules.

• Diffusion is the net movement of molecules from an area of high free energy to an area of low free energy.

• Free energy is the energy available to do work.

• Osmosis is the diffusion of water across a differentially permeable membrane.

• A solvent is a fluid that dissolves substances. A solute is a substance dissolved in a solution.

• A hypotonic solution has a salt concentration lower than the concentration of a comparable solution.

• A hypertonic solution has a concentration of dissolved material higher than the concentration of a comparable solution.

• An isotonic solution has a concentration of dissolved material equal to the concentration of a comparable solution.

• The rate of osmosis or diffusion depends on the steepness of the free energy gradient. In biological systems that free energy gradient is primarily determined by concentration.

Activities

1. Observe Brownian movement of carmine red suspension particles viewed with high magnification.

2. Observe diffusion as affected by molecular weight. Potassium permanganate, malachite green, and methylene blue diffuse through an agar suspension to form three color halos of different sizes.

3. Observe diffusion across a differentially permeable membrane. One tied dialysis bag contains starch and is put in beaker with iodine that diffuses through the bag and makes the starch blue. Another bag contains phenolphthalein and is put in beaker with NaOH solution. Sodium diffuses into the bag and turns pink.

4. Observe osmosis along a concentration gradient. Water diffuses into the bags with various solutions of sugar. The higher the concentration, the faster water diffuses into the bag and the more the bag weighs.

5. Graph the rate of diffusion.

6. Determine the concentration of water in living plant cells.

7. Observe hemolysis of blood cells. Sheep's blood is dripped into distilled water and different salt solutions. The solution clears if cells are hemolyzed.

8. Observe plasmolysis of plant cells. Cells shrink from the cell wall when salt is applied to a wet mount preparation and viewed with microscope. Stalks of celery wilt when soaked in strong salt solution.

Vocabulary

agar

Brownian movement dependent variable dialysis dialysis tubing differentially permeable diffusion hemolysis hypertonic hypotonic independent variable iodine isotonic lysis molecular weight nonpolar molecule osmosis phenolphthalein plasmolysis polar molecule pressure potential random motion solute solute potential solution solvent

Materials For All Procedures

Number of lab sections

Work groups per section water potential

Total work groups

Students per work group

Time Line For Laboratory Preparation

Beginning of the semester:

Determine the number of sections, work groups, and students in the course. Inventory supplies and, if necessary, reorder supplies. After the supply of each material is verified, check off the supply in the spaces in the list(s) below.

Two weeks before lab:

Determine how many work groups you will have. Verify that the needed quantities of disposable supplies are available.

One–three days before lab: Prepare solutions. Purchase local items.

MATERIALS FOR PROCEDURE 9.1 OBSERVE BROWNIAN MOVEMENT

COMMENTS ON PROCEDURE 9.1

• This procedure is best set-up as a demonstration slide. However, you may want to designate only a few students to set up the Brownian movement demonstration.

• Viewing works best with the 100X/oil immersion.

• Unless otherwise noted, all catalog numbers are Ward’s Natural Science. Comparison shopping at the following scientific companies might save you money on some supplies: o Carolina Biological Supply Company, www.carolina.com o Fisher Scientific, www.fishersci.com

• Safety first: Be sure and cover any safety issues that may be specifically related to this lab procedure.

MATERIALS FOR PROCEDURE 9.2

Observe

DIFFUSION AS A

Ffected By Molecular Weight

SOLUTION PREPARATION FOR PROCEDURE 9.2

• Prepare agar in petri plates by boiling 200 mL of water in a 500 mL flask and adding 4 g of granulated agar. Stir until the agar dissolves. Let the agar cool for 10 minutes and fill the petri plates (100 mm diameter or larger) to a depth of 5–7 mm.

• Prepare concentrated solutions of potassium permanganate (0.5–1.0 g), malachite green (0.5–1.0 g), and methylene blue (0.5–1.0 g) in dropper bottles with water (30 mL).

COMMENTS ON PROCEDURE 9.2

• We get better results using crystals rather than saturated solutions of the two pigments. Inoculate the agar with crystals of the two pigments applied about 5 cm apart with a spatula. Adequate diffusion will occur in 0.5–1.0 hours. Sometimes the potassium permanganate will oxidize in the agar and stop diffusing if the preparation ages too long. Consider using test tubes filled with agar and adding the drop or crystals to the top of the agar.

MATERIALS FOR PROCEDURE 9.3

SOLUTION PREPARATION FOR PROCEDURE 9.3

• Prepare phenolphthalein indicator solution by dissolving 1 g of crystalline phenolphthalein in 100 mL of 95% ethanol.

• Pouring solutions into the open end of dialysis bags can me messy. Try using pipets to dispense the solution.

• Prepare sodium hydroxide (1M) with 4.0 g of NaOH pellets dissolved in 100 mL of water. Potassium hydroxide can be substituted.

• Prepare the starch suspension (0.6%) by stirring 6 g of soluble starch powder into 100 mL of water. Bring 900 mL to a gentle boil and add the 100 mL of starch suspension. Boil and stir for 5 min, then cool. Filter the suspension through Whatman filter paper or a double coffee filter. The solution should be clear and have little or no sediment.

MATERIALS FOR PROCEDURE 9.4

SOLUTION PREPARATION FOR PROCEDURE 9.4

• Prepare solutions of sucrose (table sugar) in water at room temperature:

• 1% = 10 g/liter of water (or 20 mL of 50% sucrose in 980 mL water)

• 10% = 100 g/liter of water

• 25% = 250 g/liter of water

COMMENTS ON PROCEDURE 9.4

This procedure takes a long time to set-up so you may want to begin with this procedure.

• The 25% solution may require heat to dissolve quickly. Caution students not to waste the 25% solution.

• For 500 students we use 30 lbs sugar for solutions to prepare:

• 6 100-ft rolls of dialysis tubing

• Be aware that many students do not know what "gradient" means.

• Be sure and tell students to write in pencil, not ink when labeling each bag.

• You only need enough sucrose to “submerge” the bags consequently you might need 150 mL

• Sometimes the dialysis tubing will only contain 8 mL based on the size the dialysis tubing has been cut.

MATERIALS FOR PROCEDURE 9.5 GRAPH OSMOSIS

No materials needed

MATERIALS FOR PROCEDURE 9.6

DETERMINE THE CONCENTRATION OF SOLUTES IN LIVING PLANT CELLS

COMMENTS ON PROCEDURE 9.6

• A few hours before class the instructor should cut 10 6-cm cylinders of potato for each of five solution concentrations.

• Plastic rulers may be purchased locally at a discount store. Be sure and purchase rulers with the metric scale.

• If lab sections extend throughout a week, new potato cores may need to be prepared.

MATERIALS FOR PROCEDURE 9.7 OBSERVE HEMOLYSIS

SOLUTION PREPARATION FOR PROCEDURE 9.7

• Prepare 0.9% NaCl solution by mixing 9 g salts with enough water to make one liter.

• Prepare 10% NaCl solution by mixing 10 g salts with 90 mL of water to make 100 mL.

COMMENTS ON PROCEDURE 9.7

• Be aware that many students do not know that cells have "salts" in them. Generally "salt" refers to solutes.

MATERIALS FOR PROCEDURE 9.8 OBSERVE PLASMOLYSIS

stalks with leaves attached, soaked in water overnight celery stalks with leaves attached, soaked in 30% NaCl overnight dropper bottles

Solutions

NaCl solution 30%, in dropper bottles

SOLUTION PREPARATION FOR 9.8

W 9706

• Prepare 30% NaCl by adding 150 g of table salt to a 500 mL flask, and then add enough water to make 500 mL of solution.

COMMENTS ON PROCEDURE 9.8

• Put the bottom of the celery stalks into two separate beakers: one with tap water and the other with 30% salt or greater. Soak them for 12 hours or more.

• This plasmolysis procedure may require an extended discussion of hypo-, hyper-, and isotonic solutions.

Comments On All Procedures

• All experiments can be conducted in groups of 2–4 students as your circumstances dictate. Demonstrations of Brownian movement might be set up by a few students for the observation of the rest of the class. Fine focus for Brownian movement is the key to success. Encourage students to show and describe the movement of the India ink particles to each other.

• The procedure using dialysis bags requires that the bags be tied tightly. The bag containing phenolphthalein must be very carefully rinsed especially and tied tightly. The seal is best if the end of the bag is folded transversely, then again longitudinally before tying. A leaking bag of sucrose during the treatment can be detected if you see density lines of sugar solution at the bottom of the beaker when it is held to the light and gently swirled.

• The bags and their contents for the osmosis procedure can easily be labeled with small circles of paper cut with a hole-punch and labeled in pencil.

• The procedure with sheep's blood requires repetition by students to ensure accuracy. The blood may be diluted 1 part blood to 2 parts 0.9% saline and refrigerated before use. Samples of human blood may also be available at the Red Cross for educational use. Ward's offers an osmosis diffusion lab activity (36 W 5405) and a diffusion and cell size lab activity (36 W 1241).

Safety

❖ Avoid creating dust conditions when handling any solid biological stain.

❖ Potassium permanganate is a REACTIVE CHEMICAL.

❖ Wear PERSONAL PROTECTIVE EQUIPMENT, including face shield.

❖ Avoid contact with: powerful oxidizing materials, alcohols, arsenites, iodides, charcoals, hydrochloric acid, bromides, organic materials, ferrous or mercurial salts, hyposulfites, sulfites, peroxides.

❖ Sodium hydroxide is a CORROSIVE SOLID.

❖ Wear PERSONAL PROTECTIVE EQUIPMENT, including face shield.

❖ Avoid dusting conditions.

❖ Avoid contact with: water, acids, most common metals (zinc, aluminum, lead, etc.) liberating flammable hydrogen.

❖ Potassium hydroxide is a CORROSIVE SOLID.

❖ Wear PERSONAL PROTECTIVE EQUIPMENT.

❖ Avoid contact with: gaseous ammonia or its solutions with free iodine to prevent formation of explosive “nitrogen iodide”; sodium azide, acetaldehyde, sodium hydride.

❖ Sheep blood - defibrogenated is a potential HEALTH HAZARD.

❖ Use Universal Protective Measures: Gloves, goggles, mask.

Investigative Procedure

• Inventory/survey class on what supplies are needed for this procedure.

Answers To Questions

1. a. Briefly describe your observation of the moving pigment particles. Thousands of small black particles vibrating rapidly and making the entire field appear gray b. Does the movement of particles change visibly with heat? If so, how?

Increase the temperature; heat causes faster motion of molecules and increases frequency of collisions

2. Which would you predict would diffuse faster: a substance having a high molecular weight or a substance having a low molecular weight? Why? Low molecular weight, can diffuse faster b. Do molecules stop moving when diffusion stops? Explain your answer. No, molecules are always in motion if heat is present

3. a. Considering the different molecular weights of potassium permanganate, malachite green, and methylene blue, which should have the larger halo after the same amount of time? Why?

Potassium permanganate should have the largest halo because it has the smaller molecular weight and diffuses faster.

4. a. Describe color changes in the two bags and their surrounding solutions. Contents of starch bag appears blue, surrounding solution appears clear; phenolphthalein bag appears red, surrounding solution appears clear b. For which molecules and ions (phenolphthalein, iodine, starch, Na+, OH-) does your experiment give evidence for passage through the semipermeable membrane?

Sodium ion, hydroxyl ion, and iodine pass through c. What characteristic distinguishes those molecules and ions passing through the membrane from those that do not pass through the membrane? Size of the molecule

5. a. Did water move across the membrane in all bags containing solutions of sugar? Yes b. In which bags did osmosis occur?

Bags a, c, d c. A concentration gradient for water must be present in cells for osmosis to occur. Which bag represented the steepest concentration gradient relative to its surrounding environment?

Bags a and d both had high, steep gradients d. The steepest gradient should result in the highest rate of diffusion. Examine the data in Table 9.1 for Change in Weight during the 15- and 30-minute intervals. Did the greatest changes in weight occur in cells with the steepest concentration gradients? Why or why not? b. What influence on diffusion (i.e., temperature, pressure, concentration) causes the curves for bags C and D eventually to become horizontal (i.e., have a slope = 0)? Increase in pressure in the bag

Yes, if there was no leakage, major temperature change, or weighing error.

6. a. Refer to your graph. How does the slope of a segment of a curve relate to the rate of diffusion?

The higher the slope, the greater the rate of diffusion.

7. a. Which potato cylinders increased in size or weight? Why?

Those in 0% salt solution, because water diffused into the cells and increased their size b. Which solution(s) contained a higher concentration of solutes and therefore a lower water potential than in the potato cells? Explain your answer.

5, 10, and 15% solution were probably all demonstrated to have higher concentrations of solutes c. Which salt solution best approximated the water potential in the potato cells? How do you know this?

0.9% salt concentration d. For a growing potato plant what would you predict as the water potential of the potato relative to the soil? Relative to the leaves?

The potential is most likely lower than the potential of the soil, and higher than the potential of the leaves e. What might be some sources of error in this experiment? Inaccurate cutting of the cylinders, stressed cells f. How could a graph of your data help you estimate the solute concentration of potato cells?

The solution causing no change in the length of the potato cylinder would be the same solute concentration as the potato cells.

8. a. Through which test tubes could you read the printed page? Why?

Tube 3 becomes clear and allows for the quickest reading because the cells were quickly lysed. Tube 1 may clarify somewhat, but the crenated cells keep the solution cloudy b. Which concentration of NaCl lysed the cells?

Tube 3 containing 0% chloride c. Which of the three solutions most closely approximates the solute concentration in a red blood cell? How do you know?

Tube 2 containing 0.9% sodium chloride; the red blood cells did not lyse

9. a. Why did the plant cells plasmolyze when immersed in a hypertonic solution?

Loss of water from cell b. What can you conclude about the permeability of the cell membrane (i.e., the membrane surrounding the cytoplasm) and vacuolar membrane (i.e., the membrane surrounding the vacuole) to water?

Very permeable to water

10. What causes crispness (i.e., firmness, crunchiness) in celery? Turgor due to adequate water in cells

Questions for Further Thought and Study

1. Why must particles be extremely small to demonstrate Brownian movement? They must be small enough to move in response to collisions with water molecules. Smaller the particle, smaller mass, greater influence by collision.

2. What is the difference between molecular motion and diffusion? Molecular motion is random. Diffusion is directional. Diffusion is movement of particles from a high concentration (high free energy) to an area of low concentration (low free energy).

3. If you immerse your hand in distilled water for 15 min, will your cells lyse? Why or why not?

No, protected keratinized cells of epidermis and lipids produced by this layer. Keratin and lipids help prevent water from entering a cell.

4. Your data for diffusion of water across a differentially permeable membrane in response to a sucrose gradient could be graphed with Change in Weight on the vertical axis rather than Total Weight. How would you interpret the slope of the curves produced when you do this?