Lab Topic 10

Lab Topic 5

Diffusion and Osmosis

Before coming to lab, you should read through all of Lab Topic 5

Written by Jacqueline A. Jordan and J. Yvette Gardner-Johnson, Clayton State University Morrow, GA

Objectives

1. Understand and be able to use all the bold terms in this lab.

2. Define what is meant by a semi-permeable or selectively-permeable membrane.

3. Know the factors that affect diffusion.

4. Understand osmosis and what happens to a cell placed in a hypertonic, hypotonic, or isotonic solution.

Introduction

Cells are the structural and functional units of life. The human body is composed of over 75 trillion cells that contain between 75 90% water. In order for cells to survive, they must exchange materials like water, nutrients, and gases (CO2 and O2). These molecules move in and out of cells through a plasma membrane that is composed of a mosaic of protein molecules moving around in a fluid bilayer of phospholipids (fluid mosaic model). This composition creates an environment that regulates the movement of materials into and out of the cell. There are two primary ways molecules move through the cell membrane: passive and active transport. Active transport requires a cell to use energy (usually in the form of ATP) to move a molecule against a concentration gradient. During passive transport, the cell does not expend energy to move materials across the cell membrane. Diffusion is defined as the movement of molecules from a region of high concentration to a low concentration without the assistance of a transport protein. Molecules are propelled by kinetic energy (the energy of motion). The botanist, Robert Brown, was the first person to observe the random movement of small particles which is now defined as the Brownian movement. Several factors can influence the rate of diffusion. These include the steepness of the concentration gradient, temperature, and size of the molecule. Molecules will move by diffusion until they reach a state of dynamic equilibrium, equal movement of molecules in both directions.

In this lab, you will examine several principles of diffusion. In exercise 5.1, you will examine one of the factors that affect the rate of diffusion: size (molecular weight) of the molecule. The size of the molecules is inversely related to its rate of diffusion; therefore the larger the molecule the slower the rate of diffusion. In exercise 5.2 and 5.3, you will observe the diffusion of water through a cell membrane, termed osmosis. First, you will investigate the movement of molecules through an osmometer, a simulated plasma membrane constructed with dialysis tubing. This experiment demonstrates the same properties of semi-permeability that is observed in living cell membranes. The membrane will allow the passage of some molecules but not others. The process of the selective movement of chemicals across a membrane is known as dialysis. Lastly, changes in red blood cells following various osmotic environments will be demonstrated by your instructor. You will observe the importance of the regulation of the movement of water across cell membranes is critical to the survival of the cell.Exercise 5.1

Diffusion of Dye through Agar Gel

Agar is a carbohydrate derived from seaweed that will form a gel-like solid when mixed with water, heated and cooled. Materials can diffuse through agar easily because there are water channels with the matrix of carbohydrates. In this experiment you will determine the effect of size (molecular weight) and temperature on the diffusion rate through this semisolid material. You will use two dyes of different molecular weights, methyl orange (MW=327) and potassium permanganate (MW= 158), and measure the distance they diffuse through the semisolid medium. State your hypothesis and make a prediction related to the diffusion rate of the two dyes on your lab report sheet.

Materials per group for Exercise 5.1

1 pre-corked agar plate

dropper bottle of potassium permanganate

dropper bottle of methyl orange

Ruler (mm)

Procedure Exercise 5.1

1. Obtain a pre-corked agar plate and locate the solutions of potassium permanganate and methyl orange.2. Fill one well with 2-5 drops of potassium permanganate (depending on the depth of the well). Fill the second well with 2-5 drops of methyl orange. Make sure you add the same amount of drops to each well. Be careful not to overflow the wells. 3. Record the initial diameter of the colored wells in millimeter (mm) in Table 1 on your report sheet. This is time 0.

4. Continue to measure the diameter of the colored wells every 10 minutes. 5. At the end of 30 minutes, prepare a graph (using the grid on your report sheet) showing the changes in the diameter of the colored wells of each dye. REVISION TO EXERCISE 5.2Exercise 5.2

Movement of Water through an Osmometer (Dialysis Tubing)

In the previous exercise you examined factors that affect the rate of diffusion. In this exercise you will explore the semi-permeability of cellular membranes using an osmometer or dialysis tubing. Recall diffusion is the movement of molecules from a higher concentrated area to a less concentrated area. Osmosis is the diffusion of water through a permeable membrane from a region containing a high concentration of water to a region of low concentration of water. Solutes are dissolved molecules that affect osmosis. If a region has a higher solute particle concentration, it must have a lower water concentration when compared to another region that has lower solute particle concentration and a higher water concentration. Therefore, the concentration of water to solute particle concentration is inversely related. The terms hypertonic and hypotonic are only in reference to the solute particle concentration, not the water concentration. In comparing two solutions, the solution with a higher concentration of solutes is considered hypertonic, the solution with the lower solute concentration is considered hypotonic. Solutions of equal solute concentration are considered isotonic. In a hypertonic solution, you would expect the net flux of water to move out of the bag. In a hypotonic solution, you would expect the net flux of water to move into the bag. Equal movement of water inside and outside of the bag is considered isotonic. The dialysis tubing used in the experiment has microscopic pores of a certain size; only molecules smaller than the pore size can pass through the membrane (semipermeable). The weight of the dialysis tubing is measured to determine the net movement of water. You will prepare one dialysis bag containing 10M glucose. State your hypothesis and make prediction related to the amount of water that will move into this dialysis bag.

Materials per group for Exercise 5.2

1 (10cm) pieces of dialysis tubing

1 250ml beakers

2 pieces of string

1 pipette

balance

distilled water

3 M glucose/starch solution

Procedure

1. Using a wax pencil, label one beaker distilled water.

2. Fill the beaker with 100ml of distilled water.3. Set this beaker to the side.

4. Obtain one piece of pre-wetted dialysis tubing.

5. Tie one end of the dialysis tubing with string. Fill the dialysis tubing with 4 ml of 3M glucose/starch solution. Tie the other end with string. This is a 3M glucose/starch osmometer. (See picture below).

6. Gently remove excess liquid from the outside of the osmometers with a paper towel.

7. Weigh the osmometer and record the starting weight in Table 5.2 in your report sheet.

8. Place the osmometer in the beaker.9. After 30 minute incubation, remove the osmometer from each beaker, dry off with a paper towel, and weigh.

10. Record the ending weight in Table 5.2 for each osmometer.

11. Remove 3 ml of water from the beaker. Place contents in a test tube. Add 10 drops of Benedicts reagent to each tube to test for reducing sugar. Incubate in hot water bath for 3-5 minutes.

12. Remove 3 ml of water from the beaker. Place contents in a test tube. Add 10 drops of Lugols starch solution to test for starch

13. Record your results of both tests in Table 2 on your report sheet.

Figure 1 OsmometerExercise 5.3

Osmosis and its affects on Red Blood Cells

In the previous experiment we learned about the principles of osmosis and the importance in regulating the movement of water in and out of the cell. This experiment is designed to demonstrate the movement of water (osmosis) through a living cell when placed in a hypertonic, hypotonic, or isotonic solution. Red blood cells are commonly used to study the semi-permeability of living cell membranes. The plasma membrane of these cells is permeable to water, but relatively impermeable to salts. Normal red blood cells have a physiological concentration of 0.9% NaCl. This is important in maintaining the normal tonicity (shape and function) of the cells. If a normal red blood cell is placed in an environment that is isotonic to the cell, there will be no net movement of water across the plasma membrane. Cells will have a healthy normal biconcave appearance. If a normal red blood cell is placed in a solution that is more than or less than 0.9% NaCl the cells will either swell (even perhaps burst) or crenate (shrink). State your hypothesis and predict the outcome of your experiment. Your instructor will demonstrate the effects of red blood cells in each environment.DEMO BY INSTRUCTOR1. The instructor will prepare 4 test tubes containing the following solutions: 0.05%, 0.9%, and 10% NaCL solutions.2. The instructor will add one drop of ox blood to each tube and mix gently but thoroughly. Record the observations of each test tube on your report sheet.

3. Complete the Table 3 on your Report Sheet.

5-6 Lab Topic 5: Diffusion and Osmosis