Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Prepare 0.02% neutral red solution: Dilute 20 mL of 1% neutral red to 1000 mL with DI water.
2. Prepare 1% sodium bicarbonate solution: Dilute 100 mL of 10% sodium bicarbonate to 1000 mL with DI water.
3. Prepare 0.01 M solutions of the ammonium hydroxide, potassium hydroxide and sodium hydroxide. Add 90 mL of deionized water to each bottle. Cap and shake. The pH of each solutions should be 11–12.
4. Prepare the yeast suspension just prior to the lab. Add both packages of dry yeast to 500 mL of the 1% sodium bicarbonate solution. Mix thoroughly and swirl when dispensing. 
5. Prepare the boiling water baths:
   1. Fill a 250-mL beaker about one-third full with tap water.
   2. Add several boiling stones.
   3. Heat the water on a hot plate until the water just begins to boil.

Safety Precautions

Dilute hydrochloric acid, ammonium hydroxide, potassium hydroxide and sodium hydroxide solutions are skin and body tissue irritants and are slightly toxic by ingestion. Neutral red will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Boiling water is a burn hazard—use caution. 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. All of the waste solutions and excess sodium bicarbonate may be disposed of down the drain with an excess water according to Flinn Suggested Disposal Method #26b. Excess hydrochloric acid may be disposed of by neutralizing with base and then disposing of down the drain with plenty of excess water according to Flinn Suggested Disposal Method #24b. Excess sodium hydroxide, potassium hydroxide, and ammonium hydroxide may be disposed of by neutralizing with acid and then dispose of down the drain with an excess water according to Flinn Suggested Disposal Method #10.

Lab Hints

• Enough materials are provided in this kit for 30 students working in groups of two or for 15 groups of students. This laboratory activity can reasonably be completed in one 50-minute class period. The pre-laboratory assignment may be completed before coming to lab.
• Prelab Question 1 can be done as a demo prior to the lab. Do not shake the bottle vigorously or an emulsion may form.
• If the pH of the ammonium hydroxide is 9 or less, the neutral red will not change color.

Answers to Prelab Questions

1. All lipids have hydrophobic properties similar to those of the fatty acid tail of a phospholipid. One example of a lipid is cooking oil. Describe a property of oil that demonstrates the hydrophobic properties of lipids.

   A few milliliters of oil can be placed into a bottle of water. Shake the bottle and the oil will form droplets within the water. Eventually the oil droplets will rise to the surface of the water and form a separate layer on the surface of the water.

2. In this experiment the yeast cells are initially suspended in a sodium bicarbonate solution with a pH of about 8.5. When neutral red is added to the yeast suspension, what color would you expect the neutral red stain to be? What color would the neutral red stain be inside the yeast cell?

   The neutral red will appear yellow in the sodium bicarbonate solution and red inside the yeast cell.

Sample Data

Data Table 1. Neutral Red and Yeast

Data Table 2. Yeast–Neutral Red Suspension Data Table 3. Filtered Yeast–Neutral Red Suspension Data Table 4. Boiled Yeast–Neutral Red Suspension Data Table 5. Yeast–Neutral Red Suspension with Bases

Answers to Questions

1. Explain the color change observed in the yeast–neutral red solution in Data Table 2.

   The color changed as the neutral red was transported across the yeast cell membrane and encountered the acidic cytoplasm.

2. Explain the color change observed in the boiled yeast–neutral red solution in Data Table 4.

   Boiling altered the permeability of the yeast cell membrane, allowing the sodium bicarbonate to enter the cell and causing the cell to become basic. The neutral red stain turned yellow in the presence of the base.

3. Based upon the observations in Data Table 5, is there any evidence for transport of the bases across the yeast cell membrane? Explain.

   Transport of ammonium ion into the yeast cell caused the neutral red to turn yellow in the presence of the base.

Introduction

The cell membrane plays a vital role in regulating what goes into and out of the cell. Discover the characteristics of cell membranes that make this possible.

Concepts

• Cell membrane
• Active transport
• Diffusion
• Acid–base indicators

Background

Cell membranes are composed of lipids and proteins. The lipids form a double layer or bilayer (see Figure 1). Although there are many different types of lipids in a cell membrane, the major components in most cell membranes are phospholipids. Phospholipids are lipids that feature a phosphate group at one end of each molecule (see Figure 1). The phosphate end of the phospholipid is hydrophilic (“water-loving”) while the lipid tail is hydrophobic (“water-fearing”). As seen in Figure 1, in a bilayer, the hydrophobic lipid tails are oriented inwards and the hydrophilic phosphate groups are aligned so they face outwards, either toward the inside cytoplasm or the outside of the cell. Phospholipids will spontaneously form a bilayer when they are exposed to water. The lipid bilayer acts as a barrier to large polar molecules and macromolecules but allows small nonpolar molecules, such as oxygen, hydrocarbons, and fatty acids, as well as small uncharged polar molecules, such as water and carbon dioxide, to passively diffuse.

Proteins are an important component of the cell membrane. Carrier proteins and ion channel proteins are integral proteins that act as channels allowing ions, like chloride, and large polar molecules, like glucose, to completely cross the phospholipid bilayer. These channels may selectively close as necessary based upon the cell’s requirements.

Other proteins act as part of the active transport system to move specific molecules across the phospholipid bilayer. Additional proteins function as receptors which bind molecules, like hormones. Hormones provide the interior of the cell with information about the external environment of the cell. The position of a membrane protein depends upon its function. Receptor proteins are peripheral proteins—they adhere to the outer or inner surface of the phospholipid bilayer. Integral proteins that help transport molecules across the phospholipid bilayer cross through the bilayer with ends that often protrude into the cell or out from the cell (see Figure 2). Yeast cells are used in this experiment. Yeast cells are hardy, unicellular, eukaryotic organisms that divide quickly. Since yeast cells are eukaryotes they contain the same organelles as humans and their cell membranes function in a similar way. One difference between human cells and yeast cells is the pH of their cytoplasm. Human cytoplasm has an average pH of about 7.4. Yeast cytoplasm has a pH of about 5.8. In order to study the transport of molecules across the cell membrane, the cytoplasm must be stained with an indicator stain that does not immediately kill the cell. Stains that do not immediately kill living cells and unicellular organisms are called vital stains. Neutral red is a vital stain that also acts as an acid–base indicator. Acid–base indicators are chemicals that change color based on the pH of the solution to which they are added. Neutral red appears red in solutions with a pH less than 6.8. However, neutral red appears yellow in solutions with a pH greater than 8.0.

Experiment Overview

The purpose of this experiment is to visualize the permeability of a cell membrane to a variety of molecules.

Materials

Prelab Questions

1. All lipids have hydrophobic properties similar to those of the fatty acid tail of a phospholipid. One example of a lipid is cooking oil. Describe a property of oil that demonstrates the hydrophobic properties of lipids.
2. In this experiment the yeast cells are initially suspended in a sodium bicarbonate solution with a pH of about 8.5. When neutral red is added to the yeast suspension, what color would you expect the neutral red stain to be? What color would the neutral red stain be inside the yeast cell?

Safety Precautions

Dilute hydrochloric acid, ammonium hydroxide, potassium hydroxide and sodium hydroxide solutions are skin and body tissue irritants and slightly toxic by ingestion. Neutral red will stain skin and clothing. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles and chemical-resistant gloves and apron. Boiling water is a burn hazard—use caution. Wash hands thoroughly with soap and water before leaving the laboratory. Follow all laboratory safety guidelines.

Procedure

1. Place six test tubes in a test tube rack. Use a marker to label them with your group name and the numbers 1 through 6.
2. Using a graduated pipet, add 1 mL of neutral red solution into test tube 1.
3. Use a clean graduated pipet to add 1% sodium bicarbonate solution one drop at a time to test tube 1 until the color changes. Record the new color in Data Table 1 on the Permeability Worksheet.
4. Use a clean graduated pipet to add 0.01 M hydrochloric acid solution one drop at a time to test tube 1 until the color changes again. Record the color in Data Table 1 on the Permeability Worksheet.
5. Use a stirring rod to stir the yeast suspension. Using the graduated cylinder, transfer 25 mL of the yeast suspension to a 100-mL beaker. Record the initial color of the yeast suspension in Data Table 1 on the Permeability Worksheet.
6. Using the graduated cylinder, add 25 mL of the 0.02% neutral red solution to the yeast suspension in the beaker. Immediately record the color of the yeast–neutral red suspension in Data Table 2 on the Permeability Worksheet.
7. Observe and record the color of the suspension after 5 minutes in Data Table 2 on the Permeability Worksheet.
8. Place a funnel into test tube 2.
9. Fold a piece of filter paper in half and then in half again. Open the filter paper to form a filter paper cone (see Figure 3). Place the filter paper cone into the funnel.
   {10863_Procedure_Figure_3}
10. Filter 10 mL of the yeast–neutral red suspension from step 6 into test tube 2. Observe and record the color of the yeast cells in the filter paper and the liquid in the test tube in Data Table 3 on the Permeability Worksheet.
11. Pour 10 mL of the yeast–neutral red suspension from step 6 into test tube 3. Using a clamp, place the test tube into a boiling water bath for 5 minutes. Record the initial and final color of the yeast–neutral red suspension in the Data Table 4 on the Permeability Worksheet.
12. Pour 10 mL of the yeast–neutral red suspension from step 6 into test tubes 4–6.
13. Use a clean graduated pipet to add 1 mL of 0.01 M sodium hydroxide to test tube 4. Observe and record the color of the yeast cells in Data Table 5 on the Permeability Worksheet.
14. Use a clean graduated pipet to add 1 mL of 0.01 M potassium hydroxide to test tube 5. Observe and record the color of the yeast cells in Data Table 5 on the Permeability Worksheet.
15. Use a clean graduated pipet to add 1 mL of 0.01 M ammonium hydroxide to test tube 6. Observe and record the color of the yeast cells in Data Table 5 on the Permeability Worksheet.
16. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10863_Student1.pdf