Materials Included In Kit

Additional Materials Required

Prelab Preparation

Opportunities for Inquiry
Prepare one liter of 1.0 M sucrose solution.

1. Fill a 1-L volumetric flask approximately half-full with distilled or deionized water.
2. Add 342 g of sucrose to the flask. Invert several times until the sucrose has dissolved.
3. Fill the flask to the 1-L mark.
4. Repeat steps 1–4 to make 1 L of each of the specified concentrations of sucrose solutions listed in Table 1.
   {11119_Preparation_Table_1}

Safety Precautions

The chemicals used in this lab are considered nonhazardous. Once food grade items are brought into the lab they are considered chemicals and should not be consumed. Remind students to exercise caution when using sharp instruments to cut the tubers and onions. Food dyes will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The tubers and onions may be disposed of in the regular trash according to Flinn Suggested Disposal Method #26a. The solutions may be disposed of according to disposal method #26b.

Lab Hints

• Review the proper microscope techniques as needed before beginning this portion of the experiment.
• Disperse sodium chloride into separate containers so it is easily accessible for all laboratory groups.
• If time or materials are limited, it may be helpful to have different groups investigate different variables. For example, one group might use the same plant type in different sucrose concentrations. Another group might use three different types of tubers in the same sucrose concentration. Data may then be collaborated as a class to arrive at conclusions.
• A Density vs. Sucrose Concentration graph was included in this kit. If desired, students may measure the density of known standards and create their own curve. However, if time is limited students may simply measure the density of the unknowns and determine their concentration based on the reference graph.
  {11119_Hints_Figure_2}

Teacher Tips

• Before beginning this lab it is important that students understand the process of osmosis in living cells. A simple demonstration may be performed to clarify this process in students’ minds. Soak one celery stick in water and the other in 1 M sodium chloride solution. The sticks soaked in water have a high turgor pressure and break with a snap and those in sodium chloride are limp and difficult to break. Hence, why precut celery sticks in the grocery store are sold in plastic containers with water.
• The concept that molecules continued to move across the cell membrane even after equilibrium is obtained is a difficult concept for many students. Use Concentrating on Equilibrium, Flinn Catalog No. FB1638, to reinforce the dynamic state of equilibrium.

Further Extensions

Alignment to the Curriculum Framework of AP^® Biology 

Big Idea 2: Biological systems utilize free energy and molecular building blocks to grow, to reproduce, and to maintain dynamic homeostasis.

Enduring Understandings
2B1: Cell membranes are selectively permeable due to their structure.
2B2: Growth and dynamic homeostasis are maintained by the constant movement of molecules across membranes.

Learning Objectives
2A3: The student is able to use calculated surface area-to-volume ratios to predict which cell(s) might eliminate wastes or procure nutrients faster by diffusion.
2A3: the student is able to explain how cell size and shape affect the overall rate of nutrient intake and the rate of waste elimination.
2B1: The student is able to use representations and models to pose scientific questions about the properties of cell membranes and selective permeability based on molecular structure.

Answers to Prelab Questions

1. If a plant cell has a higher water potential than its surrounding environment will water flow into or out of the cell? Explain.

   If the water potential is higher inside the cell than the surrounding environment the water will exit the cell. This is because water moves from an area of higher potential to lower potential.

2. What would happen to plant tissue if saltwater were applied to the roots? Why?

   Water moves from an area of higher water potential to an area of lower water potential. The saltwater has a higher solute concentration than the cells inside the plant, hence a lower water potential. Therefore the water will travel from the area of higher water potential, inside the plant, to an area of lower potential, near the roots. This will dehydrate the plant and it will die.

Sample Data

Opportunities for Inquiry

Different student groups may choose to use different types of tubers to conduct this experiment. We tested two different types of tubers—white potato and Yukon Gold potatoes.

Answers to Questions

Baseline Activity

1. How does the cell membrane appear before and after being treated with sodium chloride solution?

   Both the vacuole and the cell membrane have condensed towards the center of the cell. The cell membrane pulls away from the cell wall.

2. Based on experimental results, when the 10% sodium chloride solution was added to the slide, was the water potential higher outside or inside the cells? Explain.

   Water moves from an area of higher water potential to an area of lower water potential. Since the vacuole and cell membrane shrunk due to water loss, the water potential was higher inside the cell than in the 10% sodium chloride solution.

3. How can the concentration of the sucrose solutions be identified?

   The concentration of each solution can be determined by the density. Density is a measurement of mass divided by volume. Either known solutions can be measured as references or the unknown densities can be located on a reference curve to find their corresponding concentrations. The unknown concentrations of sucrose solutions will be determined by their density. The density of each solution will be measured and referenced on the calibration curve to find its corresponding concentration. Tuber plant cells of the same size cell will be soaked in different concentrations of sucrose solutions to determine if the water potential of the cell is less than, equal to, or greater than the surrounding solution. The cubes will be massed before entering the solution, soaked for 40 minutes and then massed again to determine the change.

Opportunities for Inquiry

1. How could the plant samples be measured to determine the rate of osmosis?

   Osmosis rate can be measured by a change in mass, volume or length of the sample.

2. How is water potential calculated in the sample cells?

   –(1)(0.4)(0.0831)(295) = –9.81 bars

References

Campbell, N. A. Biology; Benjamin Cummings: San Francisco, CA; 2004; 6th Edition.

AP Biology Investigative Labs: An Inquiry-Based Approach. College Entrance Examination Board: New York; 2012.

Introduction

The way in which selectively permeable membranes, water, and solutes interact is critical to homeostasis. In plants, water and nutrients move from the roots to the stem and leaves due to differences in water potentials.

Concepts

• Water potential
• Osmosis
• Diffusion
• Turgor pressure

Background

Plants must be able to balance water uptake and loss to survive. In animal cells the direction of osmosis is easily predicted based on solute concentration. Water will flow from the hypotonic (high concentration) region to the hypertonic (low concentration) region in the cellular environment. In plant cells, this is only half the story. Plants have rigid cell walls that affect the cells’ physical pressure. The effects of both solute concentration and physical pressure are incorporated into a single measurement called water potential, represented by the Greek letter psi (ψ). Water potential is defined in terms of the free energy per mole of water and is measured in bars. The total water potential (ψ) may be determined by adding the water potential due to pressure (ψP) and the water potential due to solute concentration (ψS) (Equation 1).

The key to understanding water potential is that water spontaneously moves from an area of higher potential (higher free energy, more water molecules) to an area of lower water potential (lower free energy, fewer water molecules). In basic terms water potential measures the tendency of water to diffuse from one area to another.

Solute potential, ψ_S, is dependent on solute concentration. The solute concentration in the area surrounding the cell influences the properties of the cell. Solutes decrease the water potential in a solution, thus causing water to diffuse into an area with a higher solute concentration. If the solute concentration is higher outside the cell, water will leave the cell and enter the surrounding solution. The solute potential is calculated using Equation 2. where “i” is the ionization constant (for sucrose this is 1.0 because it does not ionize in water)

C is the molar concentration
R is the pressure constant (R = 0.0831 liter•bar/mole•K)
T is the absolute temperature of solution in Kelvin (K = 273 + °C)

The units of measure will cancel as in the following example:

For a 1.0 M sucrose solution at 22 °C at standard atmospheric pressure:

ψ_S is the –iCRT
ψ_S is the –(1)(1.0 mole/liter)(0.0831 liter•bar/mole•K)(295 K)
ψ_S is –24.51 bars

The pressure potential (ψ_P) results from an exertion of pressure, positive or negative, on a solution. The pressure potential affects a cell because an increase in pressure causes water to leave the cell and enter the lower pressure area. If the pressure potential is known to be zero (ψ_P = 0) then the water potential equals the solute potential (Equation 3). The normal turgid (rigid) state of plant cells is the result of water potential. It is this turgid state that makes the green portions of plants “stand” upright. This phenomenon involves the movement of water by osmosis into each plant cell from a region of higher water potential outside the cell to the vacuole inside the cell, which has a lower water potential. The increasing volume of water in the vacuole causes it to enlarge and press the cell contents against the cell wall. Eventually a point is reached when the cell wall cannot stretch anymore. At this point there will be no further net uptake of water by osmosis—the water potential inside the cell equals the water pressure outside the cell. A wilted plant is usually the result of a loss of turgidity of the tissues as a consequence of excessive water loss.

Animals must also compensate for the effects of water potential on their cells. In very dilute solutions, animal cells will swell and burst. In concentrated solutions, water will exit the animal cell by osmosis and the cell will shrivel. Consequently, animal cells must always be bathed in a solution having the same water potential as their cytoplasm, or the animals must have methods to regulate the water potential. The regulation of water and ion concentrations in the body is called osmoregulation. In humans, the kidneys regulate the amount of water and mineral salts in the blood under the direction of the hypothalamus. Other animals have methods of conserving water on dry land or in seawater or of ridding their bodies of excess water if they reside in freshwater habitats.

Experiment Overview

In the Baseline Activity an onion epidermis sample is placed on a wet mount. The sample cells are then observed with and without treatment of sodium chloride solution. The results of the Baseline Activity will be considered and used as the foundation for the development of a procedure to determine the water potential of various tuber plants.

Materials

Prelab Questions

1. If a plant cell has a higher water potential than its surrounding environment, will water flow into or out of the cell? Explain.
2. What would happen to plant tissue if saltwater was applied to the roots? Why?
3. What is a tuber? How does the function of the plant tuber likely affect its water potential?

Safety Precautions

Exercise caution when working with scalpels and knives as they are sharp instruments. Never cut toward yourself or others. Once food items are brought into the lab they are considered chemicals and should never be consumed. Sodium chloride is irritating to eyes. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Baseline Activity

1. Place a drop of water on a clean microscope slide.
2. Using forceps, peel the thin purple epidermis off of the concave side of the onion bulb’s leaf scale.
   {11119_Procedure_Figure_1}
3. Place the epidermis sample on the wet mount slide.
4. Place a cover slip on the sample.
5. Place the slide on the microscope stage and focus the specimen.
6. Observe the purple epidermal cells and the cell wall.
7. Observe the cell membrane and record observations.
8. Add a drop of the sodium chloride solution on the microscope slide next to the edge of the cover slip.
9. Place the edge of a paper towel on the opposite side of the cover slip as the sodium chloride drop to draw the solution across the slide.
10. Observe the cells’ organelles. How are they the same and how are they different than when treated with water?
11. Based on experimental results, when the 10% sodium chloride solution was added to the slide, was the water potential higher outside or inside the epidermal cells?

Based on the information in the Background section and your results from the previous activity design an experiment to identify the concentrations of unknown sucrose solutions and use the solutions. This method will be used to determine the concentration of the sucrose solutions used in the Opportunities for Inquiry activity.

Opportunities for Inquiry

A series containing five different concentrations of sucrose will be provided to each lab group as the solute solution to be used in the sucrose inquiry portion of this lab. Develop a procedure to determine the concentration of each solution.

1. A small sample of each solution may be consumed during this testing. The solutions may not be tasted.
2. Each group should develop at least one method to determine the sucrose concentration.
3. Decide, as a class, the procedure that will be used.
4. The sucrose concentrations will need to be included in the data and summary sections of the inquiry lab.

1. Consider the following questions while reflecting upon your knowledge of water potential.
   1. How can water potential be determined for living plant tissues?
   2. Is the water potential of different plant cells the same?
   3. How would cooking affect water potential?
   4. Do different plant tissues on the same plant all have the same water potential?

2. Plan, discuss, evaluate, execute and justify an experiment to determine the water potential of different tuber plants, different plant tissues, or other changes to the tubers. Note: Two experiments may be conducted to answer the preceding questions.
1. Develop a testable hypothesis.
2. Discuss and design a controlled experiment to test the hypothesis.
3. List any safety concerns or precautions that will be taken to protect yourself, your classmates and your instructor during the experiment.
4. Determine how you will collect and record raw data.
5. How will you analyze raw data to test your hypothesis?
6. Review your hypothesis, safety precautions, procedure, data tables, and proposed analysis with your instructor prior to beginning your experiment.
7. Once the experiment and analysis are complete, evaluate your hypothesis and justify why or why not the hypothesis was supported by your data.
8. Present and defend your findings to the class.
9. Make suggestions for a new or revised experiment to modify or retest your hypothesis.