Transport Across
the Plasma
Membrane
Objectives:
After completing this exercise,
You should be able to:
• Describe diffusion, osmosis, filtration, and phagocytosis
• Distinguish between and define hypotonic, hypertonic, and isotonic solutions
The
plasma membrane with its unique design is responsible for selectively allowing substances in
and out of the cell (selectively permeable membrane). Table 5.1 describes the variety of methods the cell
uses to move substances across the cell membrane, either actively or passively. Active processes require
ATP (energy) to get the work done. Passive processes do not require the cell to expend energy because
the kinetic energy of the particles causes them to move from an area of their higher concentration to an
area of their lower concentration.
TABLE 5.1 Movement of Substances across Cell Membranes
TRANSPORT PROCESS
Passive Processes
•
Diffusion
•
Osmosis
•
Facilitated diffusion
•
Filtration
Active Processes
•
Active transport
•
Vesicular transport
Endocytosis
Phagocytosis
Pinocytosis
Exocytosis
DESCRIPTION
Do not use ATP
Movement of molecules or icons by kinetic energy from their greater
concentration to their lesser concentration (down a concentration
gradient) until equilibrium is reached
Movement of water molecules by diffusion across a selectively
permeable membrane; movement is from a hypotonic solution into
a hypertonic solution; water moves until equilibrium is reached
Movement of a substance down its concentration gradient using a
transmembrane protein for transport
Flow of liquid (and solutes smaller than the pores) through the pores
of a filter or plasma membrane; movement due to hydrostatic pressure
Cells use ATP to move substances across the plasma membrane
Movement of a substance against its concentration gradient using a
transmembrane protein and ATP for transport
Movement of substance in to or out of a cell using vesicles that form at
or fuse with the plasma membrane
Movement of substances into a cell via vesicles forming at the plasma
membrane
A type of endocytosis; specialized movement of solid particles into cell
by pseudopodia forming a vesicle around the particles; “cell eating”
A type of endocytosis; drops of fluid brought into a cell by an invagination
of the plasma membrane forming a small vesicle; “cell drinking”
Movement of substances out of a cell via fusion of secretory vesicles with
the plasma membrane
A. Passive Transport-Diffusion
In diffusion, there is a net movement of substances from a region of their greater concentration to a region of their
lesser concentration. Diffusion can occur in solids, liquids, or gas, and across the plasma membrane of cells. In the
following activity, diffusion of two crystals through a solid (agar) will be studied. Methylene blue has a molecular
weight of 320, and potassium permanganate has a molecular weight of 158. When placed on the agar the crystals
will dissolve and the two solutes will diffuse at different rates through the agar, which is made up mostly of water.
ACTIVITY 1 Experiment: Diffusion
1.
2.
3.
•
•
•
Prediction: With your lab group, predict which crystal will move faster. (Hint: Use the molecular
weights.) Circle your answer: methylene blue, or potassium permanganate.
Materials: Obtain the materials for diffusion.
Data Collection: Measure the diffusion rate of methylene blue and potassium permanganate.
Decide who will time, who will measure, and who will record.
Carefully using forceps, place a large crystal of methylene blue on the surface of an agar Petri dish (Figure
5.1). Be careful not to drop any extra crystals on the agar surface.
Using the same technique, place a similar sized crystal of potassium permanganate on the other side of the
Petri dish.
FIGURE 5.1 Diffusion in agar plate setup.
TABLE 5.2 Diffusion Results
TIME (MINUTES)
15
30
45
60
75
DIFFUSION OF METHYLENE BLUE
(DIFFUSION DIAMETER IN MM)
DIFFUSION OF POTASSIUM
PERMANGANATE
(DIFFUSION DIAMETER IN MM)
DIFFUSION RATE
MM/MIN
•
•
Using a millimeter (mm) ruler, measure the crystals’ diameter of diffusion at 15-minute intervals for at least 1 hour
(or longer if desired).
After each observation, record the diffusion diameter of each crystal in millimeters (mm) in Table 5.2.
4.
5.
6.
Clean up as directed by you instructor.
Data Analysis: Calculate the diffusion rates for each time period for the two crystals using Table 5.2.
Complete the Experimental Report with your lab group.
EXPERIMENTAL REPORT: DIFFUSION
Results: Which substance moved faster?
Discussion: Discuss why the diffusion rate of the two substances differed.
Conclusion: State how molecular weight affects diffusion rate.
B. Passive Transport -Osmosis
A solvent is dissolving medium, typically a liquid and always water in living organisms. Solutes are the substances
dissolved in a solvent. When two different solute concentrations are on either side of a selectively permeable membrane,
there is a concentration gradient of the solute.
Osmosis is the net movement of water molecules through a selectively permeable membrane from a solution with
a lower solute concentration (hypotonic) into a solution with a higher solute concentration (hypertonic). The term
hypotonic (hypo- = deficient; -tonos = stretching) solutions or hypertonic (hyper- = excessive) solutions are used only when
two solutions are compared. Water will move into the hypertonic solution until the solute concentrations of the two
solutions equalize to become isotonic solutions (iso- = same) or if enough pressure is applied to stop the flow of water.
Red blood cells (RBCs) are good examples to use for this osmosis experiment because their shape changes
dramatically when they are exposed to hypotonic or hypertonic solutions. Their normal shape is a round biconcave disk
with a smooth plasma membrane. Under the microscope, they look two-toned, with the center being lighter because of the
concavity and thinness of the center. If the cell loses most of its water by osmosis when put in a hypertonic solution, it
becomes crenated or shriveled with spiked edges. If the cell gains a significant amount of water by being placed in a
hypotonic solution, it swells and may eventually burst – a process called hemolysis (hemo- = blood; -lysis = break down).
As a basis for comparing various solutions to blood, the salt (NaCl) content of blood is 0.9% and is the same salt content as
a physiologic saline solution.
V.I.P. NOTE Remember… salt shrivels and water swells (a cell).
Safety Precautions for working with human and Blood and Body Fluids:
•
Clean laboratory tabletops before and after conducting your experiments.
•
You need to review these precautions every time you are working with blood, or any body fluid for that matter.
Like all other body fluids, blood can house viruses and bacterial agents -- pathogens that can cause disease. AIDS
(HIV) and Hepatitis are just two examples of such contaminants that you are probably familiar with. You need not
worry about exposure to these contaminants from your own blood -- you are already exposed. But you must be
much more diligent when working with the fluids from someone else. There are a few precautions that must be
taken when working with the body fluids from someone else. You must wear gloves. Do not assume a sample is
disease-free, instead assume it is contaminated. Do not re-use any lancet, needle or object that has been exposed
to the blood sample. Dispose of all materials according to the lab instructions.
•
If at anytime during the course of the lab the sight of blood makes you feel dizzy, faint or if your lab partner
notices that you are "very absent minded", contact your lab instructor and then sit down on the floor. The easiest
way to minimize the risk to your body, if you do faint, is to minimize the distance your body must travel to reach
the floor. That is why you are asked to sit down.
ACTIVITY 2 Experiment: Osmosis in living RBCs
1.
With your lab group, discuss and identify which solutions for this experiment are hypotonic, hypertonic, or
isotonic to the RBCs. The isotonic solution is also called physiologic saline.
• 0.9% saline solution ____________________________________
• 5% saline solution______________________________________
• Distilled water_________________________________________
2.
In Figure 5.2, identify the shape of the RBC (normal, swollen, or crenated) and the type of solution the RBC is in
(isotonic, hypertonic, or hypotonic).
Prediction: With your lab group, predict the net movement of the water in all three types of solutions. Circle your
answer for all three situations:
• In the isotonic solution, the RBCs will swell, crenate, or not change shape.
• In the hypotonic solution, the RBCs will swell, crenate, or not change shape.
• In the hypertonic solution the RBCs will swell, crenate, or not change shape.
Materials: Use a plastic dropper to obtain a few drops of blood from the vacutainer when your slides and
coverslips are set up as described below.
Date Collection:
• Decide who will set up and initially observe each slide. Every member of your group should observe each
slide.
3.
4.
5.
Slide # 1: 0.9% saline solution
• Place a drop of blood on a slide with a medicine dropper and cover with a coverslip. With a different
medicine dropper, add a drop of 0.9% saline solution to the blood. Tilt the slide to intermix the two solutions,
and cover with a coverslip.
• Using high power, observe the slide for changes in cell shape. Blood cells are very tiny and difficult to see
under low power.
FIGURE 5.2 Osmosis in RBCs.
• Record shape of RBCs and type of solution in which they were placed.
shape_________________ type of solution______________________
Slide # 2: 5% saline solution
• Place a drop of blood on a second slide and cover with a coverslip. Observe the RBCs using high power. On
one slide of the coverslip, add one drop of 5% saline with a clean medicine dropper. Place a small piece of
paper towel on the opposite side of coverslip to absorb the liquid and pull the 5% saline solution into the
RBCs
• Immediately observe the second slide under high power and watch for changes in cell shape. You may have
to wait a few minutes for cell changes to occur.
• Record shape of RBCs and type of the solution in which they were placed.
shape___________________ type of solution ________________________
Slide #2 again: distilled water
• Using slide #2 again, add one drop of distilled water to the same edge of the coverslip that was used for the
saline solution.
• Hold a piece of paper towel at the opposite edge of the coverslip, observe the paper absorb the saline solution,
and which as the distilled water is drawn under the coverslip and into the RBCs
• Immediately observe the slide under high power and watch if the RBCs change shape.
• Record shape of RBCs and type of solution in which they were placed.
shape___________________ type of solution_________________________
6. Clean up your lab area.
• Place blood-stained items (slides, coverslips, and droppers) in a 10% bleach solution of as directed by your
instructor.
• Place gloves in an autoclavable bag or location indicated by your instructor.
• Wash down the lab counters with 10% bleach solution in a squirt bottle and wipe with paper towels wet
with 10% bleach solution. Allow to air dry.
• Wash your hands with soap and water before leaving the lab area.
7. Complete the Experimental Report with your lab group.
EXPERIMENTAL REPORT: OSMOSIS IN LIVING RBCs
Results: Describe how the RBC shape changes when placed in each solution.
• 0.9% saline solution ___________________________________
• 5 % saline solution ____________________________________
• distilled water _______________________________________
Discussion:
1. Describe how solute concentration affects osmosis.
2. Describe how water movement affects the cell shape.
Conclusion: State which solution(s) caused osmosis in RBCs. Compare what would happen if the RBCs were placed in tap
water instead of distilled water.
C. Osmosis and Diffusion Across a Dialysis Membrane
In this activity, a sucrose solution is added to a dialysis membrane bag which is placed in distilled water. Dialysis
membranes contain small pores which allow water and small solutes to cross. If water enters the bag, the weight of the bag
will increase and if water leaves the bag, its weight decreases.
Selective Permeability refers to the fact that some molecules may be unable to cross through the pores of a membrane and
diffuse down their concentration gradients. If the solute molecule is large and pore is small the diffusion of the solute is
‘selectively’ excluded because the solute cannot fit through the pore. The dialysis tubing used in this lab has pores that
exclude particles with a molecular weight of about 13,000 or larger. The molecular weights of the molecules used in this
lab are as follows: water (18 g/mole), Sucrose (342 g/mole), Congo red dye (697 g/mole), and starch (a very large
polysaccharide with a MW larger than 13,000).
ACTIVITY 3 Experiment: Osmosis and Diffusion Across a Dialysis Membrane
NOTE: This experiment should be set up at the beginning of class, so that changes can be observed and recorded through out the lab
time. The results become more dramatic the longer this experiment runs.
1.
2.
3.
Label Figure 5.3.
Prediction: With your lab group, predict the direction of water and sucrose movement in Figure 5.3 by circling
the correct italicized choice.
• The net movement of water will be into or out of the dialysis bag (see Figure 5.3).
• The net movement of sucrose will be into or out of the dialysis bag (see Figure 5.3).
Materials: Obtain materials for osmosis and diffusion across a dialysis membrane.
Figure 5.3 Dialysis bag setup.
•
Hypertonic solution
(hy-per-TOHN-ik)
•
Hypotonic solution
(hy-poh-TOHN-IK)
1._______________________________
2._______________________________
TABLE 5.3 Dialysis Bag Results
TIME(MIN)
0 min
COLOR OF
BEAKER WATER
CHANGE IN WEIGHT OF
SUCROSE SOLUTION (GRAMS)
Start Weight:
15 min
30 min
45 min
60 min
4.
Data Collection: Observe osmosis and diffusion through a dialysis membrane and record your results in Table
5.3.
• Decide who will set up the experiment, who will time, who will weigh and observe the color, and who will
record.
•
5.
6.
Obtain pre-filled dialysis bag from the instructor. This bag contains 40% sucrose, Congo Red Dye, and starch
(1%).
• Check to see that no liquid is leaking out of either end of the dialysis bag.
• Rinse the bag to remove excess sucrose solution, dry the bag, and weigh it. Record the beginning weight in
Table 5.3.
• Submerge the dialysis bag in a beaker of distilled water.
• Record the time in Table 5.3.
• Dry and weigh the dialysis bag after 15-minutes, 30-minute, and 45-minute intervals. If your lab lasts longer
you may also do a 60-minute measurement.
• Record each measurement in Table 5.3.
• After the final weight measurement, return the dialysis bag to the beaker. Add three drops of iodine solution to
the beaker and record the color of the bag and beaker.
Clean up as directed by your instructor.
Complete the Experimental Report with your lab partners.
EXPERIMENTAL REPORT: DIALYSIS BAG
Results:
1. Describe how the weight of the bag changed over time.
2.
Describe how the color of the beaker water changed over time
3.
Describe how the color of the solution in the bag and beaker changed when iodine was added.
Discussion:
1. Why was distilled water used instead of tap water?
2.
Why was the red dye added to the sucrose solution?
3.
Which direction did osmosis occur?
4.
Which direction did diffusion of solutes occur?
5.
Did net osmosis and diffusion of solutes stop? Did the two solutions become isotonic? Explain.
6.
Explain why the bag gained weight since sucrose, Congo red, and water crossed the dialysis membrane.
(Hint: The molecular weight of sucrose is 342; Congo red, 697; and water, 18.)
7.
Why was the starch unable to leave the dialysis bag?
Conclusion: State what drives the osmosis and the diffusion of solutes, and when does the net movement of both stop?
How does pore size determine selective permeability?
D. Passive Transport-Filtration
DEMO- Lab Instructor will show you this in lab.
Filtration is the separation of substances passed through a filter or membrane from a region of higher pressure to a region
of lower pressure. In the human body, filtration occurs in the kidneys and capillaries. Liquid and dissolved solutes flow
through the pores of a filter or membrane, keeping solutes larger than the pores on the original side. In this activity, you
will observe selectivity of the filtration membrane and a change in filtration rate. Keep in mind that the plasma
membrane is part of a living cell and can have active processes going on simultaneously with passive processes, such as
filtration.
ACTIVITY 4 Filtration
1.
Prediction: With your lab partners, predict which substances will pass through the filter: water, copper sulfate
(blue), or charcoal.
_________________will pass through the filter.
_________________will not pass through the filter.
2.
Materials: Obtain materials for filtration.
3.
Data Collection:
• Decide who will set up the experiment, who will time, who will count, and who will record.
• Fold the circular filter paper in half twice, and open it to form a cone (Figure 5.4). Place the cone in the
funnel and place the funnel in a ring on the ring stand. Put the beaker under the funnel.
FIGURE 5.4 Filtration setup.
TABLE 5.4 Filtration Results
TIME (SEC)
# DROPS
FILTRATION
RATE (DROPS/SEC)
15
30
45
60
90
•
4.
5.
Stir or mix the filtrate solution comprised of water, copper sulfate (blue), and powdered charcoal and pour it
into the funnel to just below the top of the filter paper. Watch so the edges of the filter paper do not capsize or
that the solution doesn’t go over the top.
• As soon as the liquid slows down enough to count individual drops, count number of drops in 15, 30, 45, 60,
and 90 seconds.
• Record your information on Table 5.4
• Observe the filtrate (liquid) in the beaker. If it is blue, the copper sulfate was filtered; if it is black, the
charcoal was filtered.
• Compare your results with you predictions.
Clean up as directed by your instructor.
Complete the Experimental Report with your lab partners.
EXPERIMENTAL REPORT: FILTRATION
Results: Name the substance that passed through the filter paper.
Discussion:
1. What cell structure does the filter paper represent?
2.
What determines which substance cross the filtration paper?
3.
What is the force that drives the filtration?
4. Did you see a difference in the filtration rate during the activity? Explain.
Conclusion: State how cell membranes in kidneys control filtration.
E. Active Transport-Phagocytosis: DEMO-Lab Instructor will show you this in lab.
Phagocytosis (phag- = to eat), or “cell eating,” is an active process that involves bringing solid material into the cell via
vesicles that form from cellular extensions of the plasma membrane called pseudopodia (pseudo- = false; -podia = feet).
Although one-celled organisms like amoebas are involved in this activity, the human body also has cells that move in an
amoeboid fashion and are involved in phagocytosis.
ACTIVITY 5 Phagocytosis in Amoebas
1.
•
•
•
•
•
Observe phagocytosis in amoebas.
Obtain a compound microscope, microscope slide, coverslip, amoeba culture, and amoeba food.
Using a medicine dropper, place one drop of amoeba (starved) culture on a clean microscope slide.
Place one drop of amoeba food in the drop of amoeba culture on the slide. Cover with a coverslip.
Observe the amoebas first under low power and than under a higher power
Watch the amoebas change shape as they move. Observe them as they extend out their “false feet” to engulf the
food.
2. Observe phagocytosis of amoebas at www.wiley.com/college/allen.
3. Answer the discussion question with your lab partners.
? DISCUSSION QUESTION: PHAGOCYTOSIS
How would you describe the shape of an amoeba?
Reviewing Your Knowledge from the Laboratory Exam:
A.
Transport across the Plasma Membrane
Match the definition with the term
a. to shrink or shrivel
b. water moving through selectively permeable membrane
c. false feet of an amoeba or phagocytic white blood cell
d. substance dissolved in a solution
e. to burst a red blood cell
f. difference between solute concentrations across a membrane
g. same solute concentration on both sides of membrane
h. cell eating of large solid particles
i. lower concentration of solutes that in cytosol of cell
j. a fluid that can contain dissolved substances
k. higher concentration of solutes than in cytosol of cell
l. random movement of particles from their grater concentration to their lesser concentration
m. molecule is unable to pass through a pore because it is too large.
___1. concentration gradient
___2. crenate
___3. diffusion
___4. hemolysis
___5. hypertonic solution
___6. hypotonic solution
___7. isotonic solution
___8. osmosis
___9. pseudopodia
___10. phagocytosis
___11. solute
___12. solvent
___13. selective permeability
B.
Diffusion
Select the correct lettered answer.
1. A larger molecular weight substance will diffuse through agar ______ compared to a smaller molecular weight
substance
(a) faster
(b) slower (c) at the same rate
2. If there is no concentration gradient, a substance will not have a new movement.
(a) true
(b) false
3. Passive process use ________ to move substances across a cell membrane.
(a) ATP
(b) kinetic energy
4. Diffusion of a substance ceases when:
(a) the cell runs out of ATP
(b) too much pressure is applied to the cell
(c) osmosis stops
(d) equilibrium of the substance is reached
C.
Osmosis
Select the correct lettered answer.
1. An isotonic solution will _________ an RBC.
(a) crenate (b) hemolyze (c) cause no change to
2. A hypertonic solution will ________ an RBC.
(a) crenate (b) hemolyze (c) cause no change to
3. A hypotonic solution will _________an RBC.
(a) crenate (b) hemolyze (c) cause no change to
Using Your Knowledge
Select the correct lettered answer in questions 1 – 8
A. Diffusion
____ 1. White blood cells engulf bacteria. This is an example of diffusion.
(a) true
(b) false
____ 2. Food cooking on the stove in the kitchen can be smelled in the living room. This is an example of
diffusion.
(a) true
(b) false
____ 3. Oxygen in the lungs moves into the bloodstream and carbon dioxide moves in the opposite direction.
This is an example of diffusion.
(a) true
(b) false
B.
Osmosis
____ 4. A test tube with blood in it has a particular solution added to it. After several minutes, the solution is
not clear anymore, but becomes red. Which solution was added to the blood to obtain this result?
(a) 0.9% saline (b) 5% saline (c) distilled water
____ 5. A 0.8% saline solution would be ______ to the cytosol of a cell.
(a) hypotonic (b) hypertonic (c) isotonic
____ 6. If a 50% sugar solution had been used in the dialysis bag in Activity 3, there would be a faster rate of
osmosis.
(a) true
(b) false
____ 7. If you placed a peeled apple or potato in a 5% salt solution, it would:
(a) gain weight (b) lose weight (c) stay the same weight
____ 8. A person’s hands become wrinkled after spending a long relaxing time in the tub. Tub water does
not have as many solutes in it compared with the human body. Explain why the hands look wrinkled.
(a) The tub water is hypotonic to body cells and water enters the cells.
(b) The tub water is hypotonic to body cells and water leaves the cells
(c) The tub water is hypertonic to body cells and water enters the cells.
(d) The tub water is hypertonic to body cells and water leaves the cells.
C.
Filtration
9. In the filtration experiment, hydrostatic pressure was the force driving filtration. What is the force driving
filtration in the kidney?
10. What prevents blood cells from being filtered into the urine?
This is a laboratory activity modified from the original: Allen, C and Harper V. Laboratory Manual for
Anatomy and Physiology 2nd Edition. John Wiley & Sons. 2006, for use and instructional purposes only.