Lab Exercise 5 - Transport Across a Cell Membrane

Diffusion, Osmosis, and Active Transport

Introduction:
All molecules in matter possess kinetic energy. This energy causes the molecules to collide into one
another, and constantly move about in a random manner. This random motion will cause the molecules
to move from the region where they are most concentrated to regions where they are less concentrated.
The term diffusion is used to describe this type of molecular motion. Diffusion is a non-energy
(passive) process that continues until the molecules have reached dynamic equilibrium (have been
uniformly distributed throughout the media). At dynamic equilibrium the molecules continue to move,
but because the motion is equal in all directions, no net change in concentration occurs.

Objectives:
Upon completion of this exercise, you should be able to:
1. Define and explain diffusion and osmosis.
2. Differentiate between passive and active transport.
3. Describe the effects of temperature, molecular weight and concentration gradient on the rate of
diffusion and osmosis.
4. Describe the role of Brownian movement in diffusion.
5. Describe a semi permeable membrane.
6. Define tonicity of solutions.
7. Differentiate between flaccid and turgid conditions of the cell.
8. Define all bold and underlined words in the reading.

Passive Transport:

Passive transport involves movement of molecules from the region of higher concentration to the
region of lower concentration without the expenditure of energy. Diffusion, osmosis and
facilitated diffusion are examples of passive transport.
Diffusion:
Diffusion (Figure 5.1) occurs in both living and non-living organisms. It is responsible for odors
in the air and the movement of some substances through the body. In biological systems,
diffusion usually occurs across the plasma membrane. The plasma membrane is the boundary
of the cell that separates the internal cellular environment of the cell from its non-living
surroundings. The plasma membrane is composed primarily of phospholipids arranged in a bi-
layer. This membrane is also selectively permeable, meaning that it allows certain molecules to
move freely into and out of the cell, while it restricts the movement of others.

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 Figure 5.1 Diffusion of solutes across a membrane

Movement in diffusion is predictable; always from a region of high concentration to a region of

low concentration. These regional differences within a solution or across a membrane are called
concentration gradients. The steepness of the gradient can affect the rate of diffusion. For
example, in media where there are large differences in the concentration gradient diffusion
occurs rapidly. While in media where the gradient is smaller diffusion occurs slower. Other
factors that affect the rate of diffusion include temperature, molecule size and pressure.

Osmosis:

Living organisms are composed primarily of water, thus understanding how water moves
through the plasma membrane is essential to the study of biology. Osmosis (Figure 5.2)
describes the diffusion of water through a selectively permeable membrane. In osmosis, water
will freely move from where it is most concentrated to where it is least concentrated.

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Figure 5.2 - Osmosis

Within, and around, cells there are many different substances that are dissolved in the watery
environment. This mixture of water and molecules is called a solution. In the solution the liquid
portion is called the solvent, and the dissolved molecules are called the solutes. If a cell is
placed in a solution that has a greater solute concentration than that of the cell, the solution is
said to be hypertonic. Cells placed in hypertonic solutions will always lose water. Conversely,
if a cell is placed in a solution in which the solute concentration is less than that of the cell, the
solution is said to be hypotonic. Cells placed in hypotonic solutions will take on water, and
swell. When cells are placed in solutions in which the solute concentration is equal to that of the
cell, the solution is said to be isotonic. In isotonic solution water movement into the cell is equal
to water movement out of the cell.
Figure 5.3 shows the effects of the different solutions on animal and plant cells. As you can see,
the same result is achieved in each cell type; cells in hypertonic solutions lose water, cells in
hypotonic solutions gain water, and cell in isotonic solutions gain and lose water at equal rates.
What is different about the cell types in the different solutions is the appearance of the cells.
Because plant cells have cell walls, they will feel turgid (firm) in hypotonic solutions, a little
less (firm) in isotonic solutions, and shrivel in hypertonic solutions. Conversely, because animal
cells do not have cell walls they will feel firm in an isotonic solution, swell (may burst) in a
hypotonic solution, and shrivel in a hypertonic solution.

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 Figure 5.3 - Hypotonic, Isotonic, and Hypertonic Solutions
Plasmolysis:
Plant cells have rigid external cell walls that function to provide protection and structural support
to the cells. The wall, like the plasma membrane, is porous and allows materials to enter and
leave the cell. When plant cells are placed in high salt water, the cells shrivel, and the plasma
membrane pulls away from the cell wall. The condition in which the plasma membrane and cell
wall are separated is call plasmolysis (Figure 5.3).

Active Transport:
Certain molecules do not cross the plasma membrane easily. For example, ions cannot enter or
leave the cell by diffusion. Instead these charged atoms must use a protein channel, and energy.
Molecular movement that requires the expenditure of energy is called active transport (Figure
5.4). In active transport molecules are moved against their concentration gradient, that is, from
an area of low concentration to an area of high concentration.

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Figure 5.4 - Passive and Active Transport

Liquid and gas molecules are capable of constant, random motion. The molecules move in a
straight line until they collide with another molecule and change their path. This random and
irregular movement of very small particles suspended in liquid or gas medium is termed
Brownian movement. With more energy, particles move faster and collide more often and using
the microscope one can observe very small particles suspended in liquid medium as they move
randomly.

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Lab Activity 5 - Transport Across a Cell Membrane
Diffusion, Osmosis, and Active Transport

Required Materials:

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 250ml beakers 6 numbers
Water Bottle
40% Salt Solution
Celery sticks
Forceps 2 numbers
dialysis tubing approximately 20-25 cm
20% starch solution
20% sugar solution.
Iodine solution
Test Tubes 6 numbers
Test Tube Holder
Droppers
Benedicts solution
Hot plate
Tongs
Compound microscope
Carmine dye powder
Plain Slides
Cover glass
Dissecting needle 2 numbers
2 thermometers
Ice cold water
Potassium permanganate granules
Spatula
Agar plate
Methylene blue powder
Ruler
Marker

Assignment 1
Tonicity
The purpose of this experiment is to show the relationship between osmosis and tonicity.
Procedure:
1. In the 1st beaker, place 50ml distilled water.
2. In the second beaker, place 50ml of salt solution.
3. In each beaker, place 1 stick of celery.
4. Set these beakers aside and observe at the end of class.
Remove the celery stick from each solution and describe the relative firmness of the stick from the distilled
water compared to that of the stick from the salt solution. Record your result, the relative turgidity
(firmness) of the celery sticks in distilled water and salt solution and answer the questions under
Assignment 1 in the Lab Report

Assignment 2

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 Osmosis

The purpose of this experiment is to demonstrate the diffusion of small molecules through dialysis
tubing, an example of a selectively permeable membrane. The tubing will allow water and small
molecules to pass through the membrane. Larger molecules may cross the membrane slowly, or
perhaps may not cross at all.

Procedure:

1. Place dialysis tubing in a beaker of water to moisten. Tie a knot at one end of the moistened tube to
form a bag. Open the top of the other end by rubbing the ends between your fingers (it may be helpful
to wet your fingers).
2. Add 3 droppers of 20% starch solution to the bag and then add 2 droppers of 20% glucose solution.
Tie off the other end of the bag.
3. Gently rinse the outside of the bag with water. Record the color of the solution inside the bag under
Assignment 2 in the Lab Report.
4. Fill a 250 ml beaker about two thirds full with water. Add 1 dropper of iodine solution to the beaker of
water and record the color of the solution under Assignment 2 in the Lab Report. Submerge the
dialysis bag into the beaker of iodine and water.
6. Allow the beaker-bag assembly to sit for about 30 minutes or until you see a distinct color change in
either the beaker or the bag. Record the final color of the solutions in the bag and in the beaker under
Assignment 2 in the Lab Report.
7. Test the contents of the beaker for the presence of glucose. Add 2 droppers (about 2 ml) of the beaker
solution to a test tube. Add 6 drops of Benedicts solution to the test tube. Heat the test tube in water
bath (beaker with water) for about 2-3 minutes. If glucose is present, light green, yellow, orange, brick
red colored solution will be observed.

Assignment 3

Brownian movement

The purpose of this experiment is to observe Brownian movement.

Procedure:
1. Select and clean one microscope slide.
2. Place 3 drops of water on the clean microscope slide.
3. Dip the tip of a dissecting needle into carmine powder.
4. Tap the tip of the needle into the drop water so that a few particles of the dye fall into the drop.
5. Gently place the cover slip over the drop.
6. Using the magnification of 400X, observe the movement of the particles in the solution. This observed
movement of the dye particles is called Brownian movement. Water molecules are in constant motion
due to their kinetic energy and as a result they bump into the dye particles which we can observe.
Answer the question under Assignment 3 in the Lab Report.

Assignment 4
Diffusion and Molecular Weight

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 The purpose of this experiment is to show the relationship between the rate of diffusion and
molecular weight
Procedure:
1. Using a dissecting needle, insert the tip into the agar on the bottom of a Petri dish. Gently remove and
dip the needle into potassium permanganate. Reinsert the coated tip into the original hole made in the
agar, depositing the potassium permanganate and gently remove the needle. Wipe the needle point with a
tissue. Choose another spot, 5 cm away from the first spot, and repeat the process using methylene blue.
2. Record the time immediately under Assignment 4 in the Lab Report.
3. After 20 minutes determine the rate of diffusion by turning the plate upside down and measuring the
diameter of each colored circle in the agar. Record your results and answer the questions under
Assignment 4 in the Lab Report

Assignment 5
Diffusion and Temperature

The purpose of this experiment is to show the relationship between the rate of diffusion and
temperature.

Procedure:
1. Fill one beaker full with water and heat it on a hot plate to about 50C. Use the beaker tongs to place
the beaker containing the heated water on a pad of paper towel.
2. In a second beaker, place a similar amount of chilled water.
3. Record the temperature of water in each beaker under Assignment 5 in the Lab Report
4. Keeping both beakers motionless so that the water is also motionless, add a small amount of potassium
permanganate to each beaker.
Observe the rate of diffusion of the potassium permanganate molecules in hot and cold water for 2
minutes and record your results and answer the questions under Assignment 5 in the Lab Report

Lab Report 5 - Transport Across a Cell Membrane

Diffusion, Osmosis, and Active Transport

Name: ________________________________ Date: ____________________________

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Class Index: ___________________________ Instructor: ____________________________

Before you begin filling out this lab report you must read Exercise 5 - Transport Across a Cell Membrane in
your lab manual. Complete Assignments 1-6 below. You can use your Lab Manual results and Textbook to
complete the information below.

Assignment 1
Tonicity

a) Describe the relative turgidity (firmness) of the two celery sticks.

i. Celery stick in distilled water _____________________.
ii. Celery stick in salt water _________________________.

b) The tonicity of the salt solution relative to the celery stick is _________________________
c) The tonicity of the distilled water relative to the celery stick is _______________________.

Assignment 2
Osmosis
a.
Test Initial contents Color observed at the Color observed at Positive test for
beginning of the test the end of the test starch or sugar?
20% Glucose & In bag: In bag:
1. BAG
20% Starch
In test tube:
2. BEAKER H2O & Iodine In beaker:

b. Which substances diffused into the sac? _____________________________

c. Which substances diffused out of the sac? ___________________________

d. Osmosis is defined as
________________________________________________________________
____________________________________________________________________________________
e. Considering this experiment, what can be said regarding the diffusion of large molecules
relative to small molecules? _____________________________________________________________
____________________________________________________________________________________

Assignment 3
Brownian movement

Define Brownian movement based on your experiment and reading.

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 _________________________________________________________________________________
_________________________________________________________________________________

Assignment 4
Diffusion and Molecular weight

a) Molecular Diameter of colored circle

Test # Material used Start Time
Weight after 20 minutes.

1 Methylene Blue 320 _________ mm

2 Potassium permanganate 158 _________ mm

a)

b) Is there a difference in the extent of diffusion in agar plate relative to molecular weight? Yes/No
______________.
c) If yes, explain the correlation between the rate of diffusion and molecular weight.
________________________________________________________________________________

_________________________________________________________________________________
Assignment 5
Diffusion and Temperature

Test # Potassium permanganate in Temperature (oC) Rate of diffusion (slow/fast)

1 Hot Water

2 Cold Water

a) Diffusion is defined as ____________________________________________________________

_________________________________________________________________________________
b) What is the relationship between temperature and the rate of diffusion?
_________________________________________________________________________________
_________________________________________________________________________________

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