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LESSON 1

SIMPLE DIFFUSION AND OSMOSIS: EFFECT OF DIFFERENT MOLECULES ACROSS A SEMI-PERMEABLE MEMBRANE

PURPOSE

1. Study the principles of simple diffusion and osmosis and how they relate to physiology.

2. Learn what factors affect the rate of simple diffusion across a semi-permeable membrane.

3. Compare the effect of different solute concentrations on osmosis.

4. Understand the correlation between osmolarity and tonicity of a solution.

5. Study the effect of permeant and impermeant molecules on red blood cell volume.

INTRODUCTION

The uptake and release of substances by cells and the exchange of molecules between cells and with their immediate environment are essential for maintaining homeostasis within a living organism. Many factors are involved in determining whether or not a given substance will cross the cell membrane spontaneously and how rapidly it moves. In this exercise, we will study the transport mechanisms of simple diffusion and osmosis.

A. SIMPLE DIFFUSION

Diffusion is an important transport mechanism by which certain molecules travel across living cells. It is a passive movement of molecules from an area of its higher concentration to an area of its lower concentration due to random motion caused by their own thermal energy. Random motion or Brownian motion is a spontaneous movement of molecules bouncing in all directions. Simple diffusion is one of the examples of a passive transport mechanism. Passive transport is when molecules do not require metabolic energy (ATP) to cross a semi-permeable membrane, a membrane which is selectively permeable to some molecules but not to others. In simple diffusion, molecules do not require mediated transporter. Over time, the net diffusion of the molecules across will reach equilibrium. Simple diffusion is used by gases such as oxygen and carbon dioxide, and other hydrophobic substances. Can you name other hydrophobic or lipid soluble substances (lipophilic)?

Rate of simple diffusion is based on several factors as described in Ficks Law of Diffusion. Concentration gradient (or difference) of substances across the membrane, surface area of the diffusion pathway, and the membrane permeability which is influenced by temperature, lipid solubility of the diffusing substance, membrane thickness, molecular

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weight, and size and shape of the substance (colloids vs. crystalloids) will affect the rate of diffusion of the molecules over time.

Experiment 1 will examine the net diffusion of three different substances through a colloidal aqueous medium consisting of an agar gel set at two different temperatures. Agar gels reduce the rate of net diffusion by restricting the cross sectional area of the channels or interstices through which a given substance can cross. For example, a 4% agar gel contains aqueous interstices of 1 to 2 nanometers in diameter whereas an agar gel of 2% has interstices of 5 to 6 nanometers in diameter. The area for diffusion will be greater in the 2% than in the 4% gel. In fact, a purified form of agar, agarose, is used extensively in research to separate DNA fragments by size using an electrical current.

PROCEDURE

EXPERIMENT 1: COMPARING THE RATE OF NET DIFFUSION OF THREE DIFFERENT SUBSTANCES ACROSS A 2% AGAR GEL

We will observe and compare the rate of net diffusion of the following substances.

Substances Molecular Weight Color 1.0% Congo Red 697 g/mol Dark Red 1.0% Eosin 580 g/mol Orange 0.5% Methylene Blue 320 g/mol Dark Blue

Two petri dishes containing 2% agar gel will be provided to each group. Turn both dishes up-side-down so the cover is on the top. On each dish, remove the cover and make 3 small wells at equal distance using a cork borer. It is important to use the same cork borer for both dishes to keep the wells at the same radius. Carefully instill 4 drops of the substances provided in separate well as shown in Figure 1. Note the time when the substance is added and place the cover back on top. Write your name on the cover using a marking pen so you can identify your dishes later. Place one of the 2% agar dishes in an incubator set at 42C or higher. Keep the remaining one at room temperature. Since the membrane is permeable to the substances, diffusion will begin starting from the well into the agar gel. As the substances diffuse farther, the color will be more transparent. At exactly 2 hours (or as noted by your lab instructor), take both dishes and place them on the table. Observe and mark the farthest point of the diffusion for each substance using a toothpick. Place the dish on top of a photocopied metric ruler provided on page 3 and measure the distance starting from the edge of the well to the farthest point of the diffusion.

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4 drops of Methylene Blue

4 drops of Eosin

4 drops of Congo Red well

Figure 1. Set-up of the agar dish

(CAUTION: Make sure the number of drops placed in each well is equal and no cracks are visible on the gel.)

RESULTS Use the photocopied ruler below to measure the distance in millimeter (mm). (1 cm = 10 mm)

each line is equivalent to 1 mm 1 cm

Table 1. Distances of net diffusion of the three substances in 2% agar gel at different temperature recorded after two hours

Substances Distance (mm) at room temperature

Distance (mm) at 42C or higher

1% Congo Red

1% Eosin

0.5% Methylene Blue

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POSSIBLE QUESTIONS TO THINK ABOUT

1. Differentiate between the mechanisms of simple diffusion, facilitated diffusion, and active transport? What type of molecules will only travel by simple diffusion?

2. What factors according to Fick's Law had affected the data collected? What factors did not affect the data collected? Why?

3. What are colloids and crystalloids? Give examples.

4. Given the molecular weight, what is the relative size of each of the substances based on their rate of net diffusion?

B. OSMOSIS

Osmosis is an essential process in physiology. The movement of fluid into and out of the circulatory system, as well as the function of our kidneys, is dependent on osmosis. It is the process by which water diffuses into and out of the cell in the body. When a semi-permeable membrane separates two different concentration of solutions (a solution is a solvent containing dissolved solute) and allows water (solvent) to pass through it passively but not the solute (substance dissolved in solvent), water will diffuse from an area of lower solute concentration (higher water concentration) to one of higher solute concentration (lower water concentration).

Concentration is the amount of solute per unit volume of a solution and it is expressed in molarity (moles per liter of solution, or abbreviated as M). Since certain solutes, like NaCl, dissociate into particles when place in a solution, the total solute particle concentration of a solution is expressed in terms of osmolarity (osmole per liter of solution, or abbreviated as Osm).

The higher the osmolarity of a solution in comparison to another solution means the higher its solute concentration and the lower its water concentration. Solution with the higher osmolarity relative to another is said to be hyperosmotic solution. Solution with the lower osmolarity relative to another is said to be hypoosmotic. Two solutions with the same osmolarity are said to be isoosmotic to each other. Therefore, water will always diffuses from hypoosmotic solution to hyperosmotic solution. What is the direction of diffusible solutes compared to non-diffusible solutes?

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A B

WATER

(hypoosmotic solution) (hyperosmotic solution)

Figure 2. Compartments A and B are separated by a membrane which is permeable to both water and solute X. Solute X is found in both compartments but in different concentrations. Is there a concentration gradient between compartments A and B? If yes, what is the direction of the diffusion of water and solute X?

Tonicity describes the effect of a solution (ex. internal environment) to the cell volume or on the osmotic movement of water. It is depended on the solute concentration as well as the permeability of the membrane to the solute. Red blood cells (RBCs) when added to an isoosmotic solution of non-diffusible solutes, which cannot permeate across the RBC membrane, the cells will neither swell nor crenate (shrink) because the concentration of water and solute across is at equilibrium. The osmolarity of the solution and the cell volume will remain the same. The solution is said to be isotonic relative to the red blood cells. When red blood cells are placed in hypoosmotic solution of non-diffusible solutes, water will enter the cells and cause the cells to swell and hemolyse (burst). The solution is said to be hypotonic relative to the red blood cells. When red blood cells are placed in hyperosmotic solution of non-diffusible solutes, water will leave the cells and the cells will crenate. The solution is said to be hypertonic relative to the red blood cells.

PROCEDURE

EXPERIMENT 2: EFFECT OF DIFFERENT CONCENTRATIONS OF SUCROSE SOLUTION ON THE RATE OF OSMOSIS ACROSS A SEMI- PERMEABLE MEMBRANE

Experiment 2 will demonstrate the influence of 0.5M Sucrose and 1.0M Sucrose on the rate of net osmosis across a semi-permeable membrane. The instructor will set up two osmometers before the lab as shown in Figure 3. Osmometer is made by securely fastening a piece of semi-permeable dialysis membrane held by a rubber band to the open bottom of a thistle tube and filled with solute solution. The tube is then immersed in a beaker filled with distilled water at time zero. Water will diffuse by osmosis across the dialysis membrane into the thistle tube raising the fluid level in the tube. The fluid in the thistle tube will rise until the hydrostatic pressure equals the osmotic pressure of the solution.

Before the end of the lab (or as noted by your lab instructor), measure the level of the fluid of each osmometer using a ruler and compare the rate of net osmosis. Input results in Table 2.

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Figure 3. Osmometer apparatus

RESULTS

Table 2. Distances of net osmosis across a dialysis membrane of 0.5M Sucrose and 1.0 M Sucrose solution over time

Time (T)

Distance (mm) (0.5M Sucrose)

Distance (mm) 1.0 M Sucrose

At T0 (initial) 0mm 0mm At Tfinal

POSSIBLE QUESTIONS TO THINK ABOUT

1. Define osmosis.

2. Define osmolarity. What is the osmolarity of 0.5M Sucrose and 1.0 M Sucrose?

3. From the experiment, which of the osmometers contained a greater amount of fluid overtime? Why?

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EXPERIMENT 3: TONICITY OF 0.9% NaCl, 20% NaCl, AND DISTILLED WATER RELATIVE TO THE RED BLOOD CELLS

Add approximately 20 drops of the following solutions into separate individual test tubes.

0.9% NaCl 20% NaCl Distilled Water

Add one drop of sheep blood to each solution one tube at a time. Gently mix the solution by tapping the tube with your finger. Carefully identify what solution is cloudy, semi-cloudy, and clear. Input your observation in Table 3.

Then pipette a small aliquot of each solution and carefully place a drop on top of a microscope slide. Cover it with a cover slip. Use the 10x objective lens to find the cells. Then carefully swing the 40x objective lens (400x magnification). Slightly lower the stage until a clear view of individual cells is visible. Examine the structure of the cells.

(10x ocular lens and 40x objection lens: 10 x 40 = 400x magnification)

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RESULTS

Table 3. Results of 0.9% NaCl, 20% NaCl, and distilled water relative to red blood cells

Solution

Osmolarity of the solution relative to the RBCs before blood is added. Select one: isoosmotic, hypoosmotic, or hyperosmotic

Appearance of the solution after blood is added. Select one: cloudy, semi-cloudy, or clear

Appearance of the RBCs under the microscope. Select one: crenated, hemolysed, or round

Osmolarity of the solution relative to the RBCs after blood is added. Select one: isoosmotic, hypoosmotic, or hyperosmotic

Tonicity of the solution relative to the RBCs. Select one: isotonic, hypotonic, or hypertonic

0.9% NaCl

20% NaCl

Distilled Water

POSSIBLE QUESTIONS TO THINK ABOUT

1. Explain the difference between osmolarity and tonicity of a solution?

2. What is the osmolarity of blood plasma, 0.9% NaCl, and 20% NaCl? Molecular weight of NaCl = 58g/mole.

3. Explain how RBCs crenate in a hyperosmotic solution, hemolyse in a hypoosmotic solution, and remained round in an iso-osmotic solution given that the cell membrane is impermeable to the solutes. What is the tonicity of these solutions?

4. According to the experiment, did the osmolarity of each solution change after the blood was added? Why or why not?

5. Can you determine the tonicity of a solution relative to the red blood cells without examining the appearance of the cells under a microscope? If yes, how?

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Figure 4. Compound Microscope

1. Base 8. Objective lenses (low, high power, and oil immersion) 2. Condenser 9. Coarse adjustment knob 3. Stage (mechanical) 10. Fine adjustment knob 4. Condenser height adjustment 11. Ocular (eyepiece) 5. Iris diaphragm 12. Arm 6. Nose piece 13. Light source 7. Body tube 14. Filter

Note: The iris diaphragm controls the amount of light coming from the light source to the stage.

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EXPERIMENT 4: TONICITY OF PERMEANT AND IMPERMEANT SOLUTE SOLUTIONS RELATIVE TO THE RED BLOOD CELLS

Place approximately 20 drops of each of the following solutions in separate individual test tubes. Add one drop of sheep blood to each tube one tube at a time and gently mix quickly and thoroughly. Observe the appearance of each solution. Use the results obtained from experiment 3 as your reference. Input the results in Table 4.

0.15M NaCl 0.3M Sucrose 0.3M Glycerol 0.3M Urea 0.3M Ethanol 0.5M Urea

RESULTS

Table 4. Results of NaCl, Sucrose, Glycerol, Urea, and Ethanol solution relative to the red blood cells Solution Osmolarity of

the solution relative to the RBCs before blood is added. Select one: isoosmotic, hypoosmotic, or hyperosmotic

Appearance of the solution after blood is added. Select one: cloudy, semi-cloudy, or clear

Osmolarity of the solution relative to the RBCs after blood is added. Select one: isoosmotic, hypoosmotic, or hyperosmotic

Tonicity of the solution relative to the RBCs. Select one: isotonic, hypotonic, or hypertonic

0.15M NaCl

0.3M Sucrose

0.3M Glycerol

0.3M Urea

0.3M Ethanol

0.5M Urea

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POSSIBLE QUESTIONS TO THINK ABOUT

1. Can you determine if the membrane of the RBC is permeable to each of the substances tested based on the results obtained from experiment 4? Explain your answer.

2. If the membrane of the RBC is permeable to BOTH water and solutes of a given solution, what is the osmolarity before and after the blood is added? Provide an example and explain.

3. If the membrane of the RBC is permeable ONLY to water and impermeable to the solutes of a given solution, what is the osmolarity of the solution before and after the blood is added? Provide an example and explain.

4. List the characteristics of the following solutions relative to the RBCs.

a. Hypertonic solution Characteristics Direction of flow of water

Appearance of the cell

Appearance of the solution

Osmolarity of the solution

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b. Hypotonic solution Characteristics Direction of flow of water

Appearance of the cell

Appearance of the solution

Osmolarity of the solution

c. Isotonic solution Characteristics Direction of flow of water

Appearance of the cell

Appearance of the solution

Osmolarity of the solution