Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Prepare an unknown concentration of ethyl alcohol solution for students to test. The sample data was obtained using a 23% ethyl alcohol solution. To make 50 mL of 23% ethyl alcohol solution measure 29 mL of 40% ethyl alcohol and dilute to 50 mL with distilled water.
2. Prepare 50 mL of 20% n-propyl alcohol by measuring 10 mL of anhydrous alcohol into a 50-mL graduated cylinder. Dilute to 50 mL using distilled water.
3. Place the required alcohol solutions around the room so they will be accessible to all lab groups.

Safety Precautions

Ethyl alcohol is a flammable solvent and a dangerous fire risk. Addition of a denaturant makes the product poisonous—it cannot be made nonpoisonous. Scalpels are sharp objects, always cut away from yourself and others. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines. 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. Resulting solutions may be flushed down the drain according to Flinn Suggested Disposal Method #26b.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs, or for 15 groups of students. This laboratory activity can reasonably be completed in one 50-minute class period. The prelaboratory assignment may be completed before coming to lab, and the data compilation and calculations may be completed the day after the lab.

Teacher Tips

• If time permits, allow students to test their self-designed experiments.
• The Permeability of Yeast Membranes Kit, (Catalog No. FB1907), which also studies cell membrane properties, is available from Flinn Scientific.

Answers to Prelab Questions

1. Why is it important to place beets of uniform size in each solution?

   The size of the beets and the volume of solution must remain constant so that the cell damage can accurately be assessed. If one solution has a larger beet it will appear that more damage has occurred than has actually occurred. Conversely, if the volume of solution is greater in one sample it will appear that less damage occurred than actually has occurred.

2. Explain why it is important to stir the solution gently while the beets are soaking.

   The beets should be stirred gently because if they are stirred too vigorously physical damage can occur causing higher than expected absorbance readings.

Sample Data

*Measurements were taken at 565 nm. Regardless of the setting the same trends should be visible.

Answers to Questions

1. Describe the qualitative and quantitative evidence that the beet membrane has been damaged by alcohol.

   The qualitative evidence was shown by the intensified red color of solution. The quantitative evidence is the increased absorbance with more concentrated alcohol solutions.

2. Review the data table. At what concentration did the ethyl alcohol begin to damage the membrane?

   Pigments began to leak into the alcohol solution even at low concentrations, as evidenced by the gradual increase in absorbance. However, the intensity and absorbance began to increase much more significantly at an alcohol concentration of 30%.

3. Create a graph comparing the absorbance versus concentration of ethyl alcohol solution.
   {11038_Answers_Figure_2}
4. Based upon the curve created in the graph and the absorbance of the unknown predict the concentration of the unknown solution of ethyl alcohol.

   Student answers will slightly vary. Students should see that the absorbance 0.098 intersects the curve somewhere between 23–27%.

5. Compare the absorbance of the 20% ethyl alcohol (a two-carbon molecule) to the 20% n-propyl alcohol (a three-carbon molecule). Explain why the different absorbance might occur and what tests could be done to confirm this hypothesis.

   Based on these two samples it appears that the molecule size affects the amount of membrane damage. The larger the molecule the more damage is caused. This could be tested by exposing the membranes to a one carbon alcohol (methyl alcohol) to see if less damage occurs.

6. Design an experiment testing cell membrane properties using another variable.

   Student answers will vary. The background suggests that osmotic balance and pH changes can compromise the membrane. Students may choose to alter the osmotic balance using sodium chloride or change the pH by adding an acid or base.

References

Campbell, N.A. Biology, 6th Ed.; Benjamin Cummings; San Francisco; 2002; pp 138–143.

Introduction

Cells need to be able to withstand a variety of environmental conditions in order to survive. The plasma membrane protects the cell from the external environment. Discover the effect of alcohol on the ability of the plasma membrane to function properly.

Concepts

• Cells
• Membrane properties

Background

Cells must be able to interact with their environment in order to survive. The plasma membrane is the physical boundary that separates the living cell from its surroundings. Its job is to control what comes into and goes out of the cell. The plasma membrane is said to be selectively permeable, which means that it allows some substances to cross through but not others. The plasma membrane primarily consists of a phospholipid bilayer with interspersed proteins. Phospholipid molecules have both a hydrophilic (water-loving) and hydrophobic (water-fearing) chain or “tail.” See Figure 1 for general schematic of the structure of a cell membrane. Membranes are held together by hydrophobic interactions.

The plasma membrane has a diverse collection of proteins embedded in the space between the phospholipid bilayer. Two main types of proteins are found in the plasma membrane. Integral proteins are found in the hydrophobic core of the lipid bilayer. Many integral proteins are also transmembrane proteins meaning they span the entire membrane. Therefore the hydrophilic portion of the protein is found on the outside of the membrane and the hydrophobic portion is found within the membrane. Peripheral proteins are loosely attached to the surface of the membrane. Peripheral proteins are hydrophilic proteins as they are found in the aqueous fluid outside the cell. Membranes have a variety of properties and functions that are largely determined by the proteins within the membrane. Different stresses such as detergents, pH, and altering the osmotic balance can affect the cell membrane’s permeability.

Plant cells contain vacuoles that store water and other molecules. Vacuoles are enclosed by a membrane known as the tonoplast. Like other cellular membranes, the tonoplast is selective regarding what can pass through the membrane. Vacuoles are diverse organelles that store a wide variety of organic compounds and inorganic ions. They may also serve as disposal sites for metabolic byproducts that could harm the cell. Some plant vacuoles contain pigments that color the cell. The vacuole of a beet cell contains a water-soluble pigment known as betacyanin. This pigment gives the beet its characteristic deep reddish-purple color. When cells are healthy this water-soluble pigment remains in the vacuole. If the tonoplast becomes damaged the contents of the vacuole will be released into the cell. Damage to the plasma membrane allows the betacyanin to spill out of the plant cell into the surrounding environment. If the beet cell is surrounded by a fluid, the intensity of the color in the fluid correlates with the amount of damage that has been done to the cellular membrane. Therefore, membrane damage can be quantitatively measured using a colorimeter or spectrophotometer to determine the amount of pigment released from the vacuoles.

The colorimeter is used to measure the pigment intensity of the solution. The light source of the colorimeter passes through the solution and strikes a photocell. If the solution is colorless then the cell membranes are completely intact. If the membranes are damaged the beet pigment will leak into the cell causing it to turn red. A solution with less pigment will absorb less light and transmit more light indicating minimal membrane damage. A solution with more pigment will absorb more light and transmit less light indicating greater membrane damage. The amount of light transmitted (or absorbed) can be plotted on a graph. If known concentrations are first prepared and analyzed, unknown concentrations can then be determined.

Experiment Overview

The purpose of this activity is to analyze the effect of different concentrations and types of alcohol on cell membranes.

Materials

Prelab Questions

Read through the Background and Procedure sections before completing Prelab Questions.

1. Why is it important to place beets of uniform size in each solution?
2. Explain why it is important to stir the solution gently while the beets are soaking.

Safety Precautions

Ethyl alcohol is a flammable solvent and a dangerous fire risk—keep away from flames, sparks and other sources of ignition. Addition of a denaturant makes the product poisonous; it cannot be made nonpoisonous. Scalpels are sharp instruments; use caution when cutting. Always cut away from your body and away from others. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Alcohol Test

1. Obtain seven test tubes. Using a marker, label them as follows—0%, 10%, 20%, 30% and 40%, 20% n and U (unknown).
2. Using a clean pipet, add 3 mL of distilled water to the 0% test tube.
3. Using a clean pipet for each solution, place 3 mL of each solution listed in the table below into the appropriately labeled test tube. Hint: It may be helpful to label the pipets with a permanent marker to ensure their identities later.
   {11038_Procedure_Table_1}
4. Obtain a piece of beet. Cut the beet into seven equally sized cubes, 0.25 g each (approximately 5 mm along each side). Make sure each cube does not contain any skin or rough edges.
5. Lightly rinse the beet cubes in distilled water. Do not over-rinse so as to wash away excess pigment.
6. Gently place one beet cube into each of the seven test tubes.
7. Soak the beets in solution for 10 minutes, gently irrigating the beet pieces every minute with the corresponding pipet. Draw up the solution into the pipet and gently eject it back into the test tube. Note: Exercise caution so as not to damage or puncture the beet while irrigating. Doing so will expose additional cells to the solution and bias the results.
8. After 10 minutes, gently remove the beet from each solution using forceps.
9. Prepare a blank solution by filling a cuvet with distilled or deionized water.
10. Wipe the outside of the cuvet with lens paper to remove any residue.
11. Insert the blank into the colorimeter and zero the instrument to calibrate.
12. Remove the cuvet and discard the water.
13. Fill the cuvet with solution from the distilled water/beet solution (0% tube).
14. Wipe the cuvet with lens paper to remove any fingerprints.
15. Place the cuvet in the colorimeter and measure the absorbance. Record results on the Membrane Properties Worksheet.
16. Discard the solution into the waste collection beaker.
17. Repeat steps 13–16 six more times to measure the absorbance of each beet solution.
18. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

11038_Student1.pdf