Experiment Membrane Transport

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1
Experiment
Membrane Transport
Objectives ► Referring to energy, what two ways can substances enter a cell?
What is active transport?
What is passive transport?
How is osmosis related to diffusion?
How can we demonstrate active transport?
How can we demonstrate Brownian movement?
How can we demonstrate diffusion (2 ways)?
How can we demonstrate osmosis (3 ways)?
In terms of relationships between substances, how can we define
“hypertonic”, “isotonic”, and “hypotonic”?
What is the relationship between the size of a molecule and its rate of
diffusion?
____________________________________________________________________________
Supplies ► Materials Needed
Active Transport:
Baker’s Yeast
0.75% Na2CO3
0.02% Neutral Red
Erlenmeyer Flasks
Flame or Hot Plate
Brownian Movement:
India Ink (Carmine Dye or
Whole Milk may be
substituted)
Slides and Cover Slips
Diffusion:
Beaker of Distilled Water
1.5% Agar-agar (or Gelatin)
in petri plate
2
Potassium Permanganate
(KMnO4) Crystals
Potassium Dichromate
(K2Cr2O7) Crystals
Methylene Blue Crystals
Metric Ruler (to measure
in mm)
Artificial Cell:
Dialysis Tubing or Sacs
Sugar
String
Beakers Distilled Water
Scale
Osmosis and Red Blood Cells:
Distilled Water;
Salt Solutions: 12-15% Salt
Salt Solutions: 0.85% Salt
(This is human, isotonic
saline.)
Blood (Human or Animal):
Lancets and Alcohol if
Human Blood is used
Slides and Cover Slips
Microscope
___________________________________________________________________________
Key Terms ► Active Transport
Brownian Movement
Cell Physiology
Dialysis
Diffusion
Gradient
Hypertonic
Hypotonic
Isotonic
Membrane Transport
Osmosis
Passive Transport
____________________________________________________________________________
Introduction ► The individual cell is a dynamic microcosm, demonstrating in miniature
all the processes and events that occur in the macrocosm, making the apparent function of the
whole organism the actual function of many individual cells working in unison.
It is important that we understand how the individual cell, the microcosm, functions so
that we can more fully understand how the organism as a whole, the macrocosm, function. For
instance, we can say that if an action potential (or nerve impulse) is to be generated in a part of
the nervous system, a certain electrical stimulation must be present and certain ions must be
3
moving through appropriate channels. What we sometimes forget is that these events—in this
case the electrical stimulation and the ionic movement—occur at the cellular level. What seems
to be happening in the organism is happening only because the events are occurring at the level
of the individual cell. We could use similar analogies for every system in the body.
In lecture you examined the molecular intricate of the phospholipid bilayer known as the
cell membrane, and you became aware that the cell membrane is selectively permeable,
meaning that only certain substances can enter and leave the cell by freely crossing the
membrane.
You know, for instance, that the membrane is replete with channels, gates, and carrier
molecules that either facilitate, inhibit, or repel assorted ions and molecules as they randomly
approach the demarcation barrier. This demarcation barrier, the cell membrane, is functional in
maintaining cellular integrity.
You also know that since the cell is microcosm, ions and molecules must cross the
barrier, both as nutrients entering the cell and as wastes leaving the cell. In cell physiology, we
examine how events occur within the cell. Cellular functions follow the basic principles of
physiology. Many of these functions do not lend themselves to easy demonstration, particularly
this early in an introductory course. However, at this point we can demonstrate a number of
functions directly related to membrane transport. And membrane transport is one of the main
keys to cell physiology when we study the nervous system and the digestive system.)
____________________________________________________________________________
Preparation ► I.
A.
1.
Background and Protocol
Explanation of Terms
Be certain you have a working knowledge of the following terms:
Membrane Transport
Active Transport
Passive Transport
Diffusion
Osmosis
Brownian Movement
Gradient
Membrane transport is any process, active or passive, by which a substance moves from one
side of a membrane to the other side of the membrane. We have already established that the
membrane acts as a barrier that can facilitate, inhibit, or repel substances, and yet certain
substances must cross the barrier.
Active transport requires cellular energy because the substance traversing the barrier
either cannot do so without a push or cannot do so in sufficient quantity to maintain cellular
integrity. Active transport is absolutely essential in maintaining the functioning organism.
Active transport is accomplished via ionic pumps. Manu of our carrier systems work because of
active transport. Gradients, which enable work to be done, are maintained because of active
transport. Active transport can best be demonstrated by visualizing some very common events,
both in the laboratory and in daily life.
Passive transport requires no input of cellular energy. Several categories of passive
transport can be identified. Nevertheless, the most common readily identifiable form of passive
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transport is diffusion, the net movement of molecules from an area of higher concentration to
an area of lower concentration. Osmosis is form of diffusion. In the human system, osmosis is
the diffusion of water. Passive transport can be easily demonstrated by both diffusion and
osmosis experiments.
Brownian movement, while technically not diffusion, is covered here with the passive
transport experiments because Brownian movement demonstrates molecular motion. It is
precisely because of this innate movement that molecules are able to move passively from one
point to the next point—including from one side of a membrane to the other side.
A gradient is any difference in intensity between two sides of a demarcation. For
instance, a concentration gradient would be a difference in concentrations of a substance on
each side of a demarcation. A physical barrier may or may not be present. We can also have
temperature gradients, electrical gradients, flow gradients, and pressure gradients.
B.
General Procedural Points
Your instructor may set up parts of this experiment as a demonstration. If so, answer the
questions from your observations.
1.
Work with a partner in Part II.
2.
Divide the work between you and your partner in Part III so that each of you is doing one
of the experiments.
3.
If you use human blood, READ THE CAUTIONS GIVEN IN Anatomy of the Blood
AND Blood Testing Procedures.
____________________________________________________________________________
Experimentation ► II.
Active Transport
Active transport is the energy-requiring movement of a substance across a barrier or a gradient.
This transport could not take place without the input of energy. In the biological world, energy
is supplied by the living cell. We can observe the principles of active transport by examining
whether or not the uptake of a substance will take place in living and/or nonliving cells.
Neutral red is a dye that is actively transported into the living yeast cell. Neutral red is red
in an acidic solution and yellow in a basic solution. A sodium carbonate (Na2CO3) solution is
basic.
A.
Experiment #1: Active Transport
1.
Obtain two Erlenmeyer flasks, 2 g yeast, 50 ml solution carbonate, and 50 ml neutral red.
2.
Into Flask #1 place half the yeast and half the sodium carbonate. Mix well and heat
gently. Boil the solution for 2 or 3 minutes. When the solution is cool, add about half the
neutral red. The solution should be yellow.
5
3.
To Flask #2 add the remaining Na2CO3 and neutral red. Gently swirl in the remaining
yeast and watch for a color change.
Concept Check 1
Jot down your observations and discuss with your partner the differences in color between
Flask #1 and Flask #2.
B.
Additional Observations
Tip: In Flask #2 either the dye
entered the cell or some substance
was given off by the cell to change
the color to red. Which answer
seems most logical? Why? How
might you test the solutions to see
exactly what happened?
Concept Check 2
Discuss with your partner the involved in windmills and water pumps. What is the role of
energy in these apparatuses? How can you relate these functions to the role of active transport
in the cells?
III.
Passive Transport (Diffusion)
Passive transport is the movement of a substance across a membrane or a gradient without the
expenditure of cellular energy. We have several experiments that demonstrate passive
transport.
A.
Experiment #2: Brownian Movement
All molecules with a temperature above absolute zero are in a state of constant motion.
Because of this motion, molecules collide regularly and randomly. The manifestation of these
collisions is called Brownian movement. Some disagreement exists as to the exact nature of
Brownian movement. Nevertheless, for our purposes, the exact results are the same and we can
see Brownian movement whenever we have a liquid mixture composed of differently sized
molecules.
Brownian movement is not actually a part of diffusion, but if the molecules were not in
motion, they could not diffuse across a membrane or a gradient. Thus, in order to understand
diffusion we should gain some insight into molecular movement.
India ink is a suspension of carbon in water, along with ethyl alcohol and acetone.
(Carmine dye and whole milk, the alternate substances for this experiment, are also molecular
suspensions.)
1.
Obtain a slide, cover slip, and microscope.
2.
Place a drop of India ink on a slide with a small drop of water. Cover carefully with a
cover slip and allow the solution to rest for 2 or 3 minutes.
6
3.
Observe the suspension under both low and high power. If possible, also observe the
suspension under oil. Keep the light as low as possible so you manitian maximum contrast. If
your suspension is too dark, you may wish to start over using a larger drop of water.
4.
Sketch what you see.
5.
Describe Brownian movement based on your observations.
B.
Experiment #3: Diffusion in Liquids
Diffusion—the net movement of molecules from an area of higher concentration to an area of
lower concentration—can be demonstrated in two different ways.
1.
Obtain a beaker of water and a crystal of potassium permanganate (KMnO4).
2. Drop the crystal into the water and watch the diffusion process. Use Figure 1 to sketch the
initial diffusion path. Note the time and observe the beaker at 5-minute intervals. Note the time
when you think diffusion is complete. How long did it take?
Table 1 Petri Plate Diffusion Time
Time
KMnO4
15 min
30 min
45 min
60 min
75 min
90 min
____
C.
Experiment #4: Diffusion In Solids
K2Cr2O7
Methylene Blue
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1.
Obtain an agar-agar or gelatine petri plate and ruler. Also obtain equal crystals of
potassium permanganate (KMnO4), potassium dichromate (K2Cr2O7), and methylene blue.
2.
Construct an imaginary equilateral triangle on the petri plate and put one crustal at each
angle, per Figure 2. At 15-minute intervals measure the radius (in millimeters) that each
substance has diffused from the original crystal mark. Use Table 1 to record these distances.
Figure 2 Petri Plate.
Concept Check 3
The molecular weight for KMnO4 is 158, for K2Cr2O7 is 294, and for methylene blue is 320. In
general terms, what is the relationship between the distance traveled and the molecular weight
of each crystal?
[Challenge: Although your measurements are probably not exact enough to derive a
mathematical formula, do you see that the diffusion rate of any substance might well be
inversely proportional to the square root of the molecular weight?]
D.
Osmosis Overview
Osmosis is the movement of water from an area of higher water concentration, across a
selectively permeable membrane, to an area of lower water concentration. That is, from an area
of lower solute concentration to an area of higher solute concentration. Movement is always
toward equilibrium. Dilution is toward equilibrium.
Dialysis is the movement of a nonwater particle across a barrier. This movement is based
on the size of the particle in relation to the size of the pores within the barrier. In the living
system this barrier is the cell membrane.
We can perform three experiments to demonstrate the principles of osmosis and dialysis.
Work with a partner.
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Table 2 Artificial Cell Results
____
Concept Check 4
Explain your results by answering these questions: At what height did each solution stop
rising? Then what happened? What differences did you notice between the sugar and salt
osmometers? How do you explain those differences? If you had tasted the water at the end of
the experiment, what would you have noticed? How would you explain this?
9
E.
Experiment #6: Osmosis (Artificial Cell)
This experiment is outlined in Figure 4 and Table 2
1.
Tip: keep in mind the sizes of
various ions, molecules, and
membrane pores.
Obtain 5 beakers, 5 artificial cells (dialysis tubing or sacs), string, sugar, scale.
2.
Make 3 sugar solutions, one 40%, and one 10%. Set up 5 artificial cells. Into cell #1 put
some of the 40% solution, into cell #2 the 20% solution, into cell #3 the 10% solution, and into
cell #4 and cell #5 put distilled water.
3.
Take the 5 beakers. Into beaker #1 and #2 put distilled water, into #3 put 10% sugar, into
#4 put 20% sugar, and into #5 put 40% sugar. Weigh each artificial cell and place it into its
corresponding beaker.
4.
At the end of the laboratory period—or later in the day, depending in the directions from
your instructor—remove the cells from the beakers, wipe the cells with a paper towel, and
reweigh. Calculate the percent differences in weight for each cell. Chart you results on Table2.
What type of differences do you see in the different cells? How would you explain these
differences?
Test condition
#1 Sac—40% Sugar Beaker—Water
#2 Sac—20% Sugar Beaker—Water
#3 Sac—10% Sugar Beaker—10% Sugar
#4 Sac—Water Beaker—20% Sugar
#5 Sac—Water Beaker—40% Sugar
Weight at start
Weight at finish
5.
Below is a partially constructed bar graph. Complete this bar graph to show the relative
changes in the weights of the sacs.
6.
Repeat #1-4 above using time as a variable. Use the space at the end of this experiment to
construct a graph demonstrating your findings.
F.
Experiment #7: Osmosis (Red Blood Cells)
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A solute is any substance dissolved in any solvent. In biology, the solute is usually ionic,
molecular, or particulate. The solvent is generally water or water-based. Hypertonic, isotonic,
and hypotonic are terms used to describe the relationship between the solute concentrations on
two sides of a membrane or gradient. Keep in mind that these are relationship terms. We
shouldn’t simply state, “Solution X is hypertonic.” To show the relationship, we should say,
“Solution X is hypertonic to Y.”
Hyper-means greater (more, larger), so a hypertonic solution has a higher particulate
concentration than the substance with which it is being compared. Iso- means same, so the two
isotonic solutions would be of the same concentration. Hypo- means beneath (less, below), so a
hypotonic solution has a lower particulate concentration than the substance with which it is
being compared.
When particulate movement occurs between two concentrations, the net movement is
always toward equilibrium. Note the words “net movement”. If pore size is adequate, some
movement against equilibrium does occur because molecules are in constant motion. We are
concerned here with net movement. We are also concerned here only with passive movement.
(Occasionally, cell membrane integrity or active transport mechanisms may influence net
movement. We are not considering those aspects of movement here.)
Note: Diseases such as cholera disrupt osmotic equilibrium. The cholera toxin causes
changes in the membrane of the cells of intestinal epithelium. These cells lose fluid to the
lumen. Because the intestinal cells have become more hypertonic to their environment, water
from the interstitium enters these cells. This water is also lost to the lumen. Because of the
increased hypertonicity of the interstitium, water leaves adjoining cells and enters the
interstitium and eventually the epithelial cells. Dehydration progresses rapidly. Diarrhea is a
characteristic of cholera because of the massive water loss. The large intestine (which is
basically unaffected by the cholera toxin) cannot reabsorb the water as rapidly as it is lost from
the small intestine.
I. PREPARATION OF A STOCK BLOOD SUSPENSION
Equipment:
 ovine blood
 isotonic saline (0.15M NaCl; 0.9% NaCl is another way of expressing the same
concentration)
 1 box of parafilm
 1 pipette (10cc)
 1 pipette suction pump
 1 test tube
 1 Pasteur pipette
Each of the experiments in this lab requires a few drops of a stock suspension of ovine red
blood cells, prepared as follows:
1. Put 10 ml of isotonic saline solution (0.9 % NaCl) in a clean test tube.
2. Add about 10 drops of ovine blood.
3. Seal the opening of the test tube with parafilm.
4. Mix thoroughly by putting your finger over the end of the tube and inverting twice gently.
11
II. DEMONSTRATION OF OSMOSIS
The effects of osmosis can be observed when red blood cells are exposed to salt solutions at
concentrations different from that of blood plasma. Hemolysis or crenation results when water
molecules enter or leave the cell.
EXERCISE A
Equipment:
 stock suspension of blood prepared in I.
 distilled water
 isotonic saline (0.15M NaCl))
 2 pipettes (10cc)
 1 Pasteur pipette
 1 pipette suction pump
 2 test tubes
1. Add 5 drops of stock blood suspension to each of 2 test tubes containing the following solutions:
i) 5 ml distilled water
ii) 5 ml isotonic NaCl.
2. Observe the print on this page through each of the test tubes.
The solution of isotonic saline and blood is turbid and the printed characters appear blurry: this
indicates that there is no hemolysis of the red blood cells (the print seen through the solution in
the test tube appears blurry because the red blood cells are intact and thus diffract the light).
The solution of distilled water and blood is clear and the printed characters can be seen very
clearly: this indicates that hemolysis of the red blood cells has occurred (the print seen through
the solution in the test tube appears very clear because the red blood cells have exploded and
thus do not diffract the light anymore).
Note: Keep these tubes for future reference: they will be your control tubes for the
exercises B & C.
III. EFFECT OF MOLECULAR SIZE ON PERMEABILITY OF THE CELL
MEMBRANE
The cell membrane is differentially permeable. Ionized solutes such Cl--, in spite of their small
molecular size, do not pass through rapidly. Moreover, active transport pumps for Na+ and
Ca++ remove these ions from cytoplasm as fast as they enter cells. The cell membrane is more
permeable to some of the unionized polar solutes used in this experiment.
EXERCISE B
Equipment:
 stock blood suspension
 distilled water
 0.3 M urea
 0.3 M ethylene glycol
 0.3 M glycerol
 0.3 M glucose
 5 test tubes
 5 pipettes
 1 pipette suction pump
 1 timer
12
Relevant properties and formulas for the solutes used in this experiment are given in Table 1.
Note that molecular size (diameter) roughly parallels molecular weight. All of these
compounds are polar since they have either -OH (hydroxyl) or -NH2 (amino) groups.
1.
Put 5 ml of 0.3 M urea in a test tube.
2.
Add 5 drops of red cell suspension and mix gently.
3.
As the first drop of blood reaches the solution, start the timer (or note the exact time if
you prefer using your watch).
Determine the time required for hemolysis to occur. You do this by holding the tube in front of
the print on this page; as hemolysis occurs, the print will appear more and more clear.
The hemolysis time is the time required for the solution in the tube to go from turbid to clear
(and the print to go from blurry to clear). You should stop your timer when the print seen
through the test tube containing urea is as clear as the print seen through the control tube
containing distilled water.
4.
Repeat the procedure using ethylene glycol, glycerol and glucose.
5.
Record hemolysis times in Table 2 and on the blackboard.
Table 1: Properties and Formulae of Solutes and Solvents
SUBSTANCE
MOLECULAR
WEI WEIGHT (g)
Urea urea
urea
3.6
MOLECULAR
DIAMETER (A)
FORMULA
H2 N
C=O
H2 N
ethylene glycol 62
~3.6
CH2OH
CH2OH
glycerol
92
6.2
CH2OH
CHOH
CH2OH
glucose
180
8.6
CHO
CHOH
CHOH
CHOH
CHOH
CH2OH
13
Table 2: Your results.
SUBSTANCE
Urea
Ethylene
glycol
glycerol
glucose
TIME TO HEMOLYSIS
IV. EFFECT OF LIPID SOLUBILITY ON PERMEABILITY OF THE CELL
MEMBRANE
This experiment examines the relation between lipid solubility and cell membrane permeability. Recall the structure of the plasma membrane. Ions and water-soluble molecules must pass
through pores in the membrane because they cannot dissolve in the central lipid layer of the
membrane. On the other hand, lipid-soluble molecules can dissolve in the lipid portion of the
membrane and diffuse across easily.
Many organic molecules contain both non-polar bonds, which confer lipid solubility, and polar
bonds, which confer water solubility. Hydroxyl (-OH) and amino (-NH2) groups promote
water solubility by virtue of the polar bonds between hydrogen and oxygen (in -OH groups) or
between hydrogen and nitrogen (in -NH2 groups).
On the other hand, carbon-carbon or carbon-oxygen bonds are nonpolar. When molecules
contain bonds of both types they are soluble (to a greater or lesser extent) both in water and in
lipids. The more polar bonds present, the greater is the water solubility, and the less soluble the
compound is in lipids.
EXERCISE C
Equipment:
 stock blood suspension
 0.3 M glycerol
 0.3 M triacetin
 0.3 M diacetin
 3 test tubes
 3 pipettes
 1 pipette suction pump
 1 timer
The partition coefficient of a compound is a measure of its relative solubility in water and
lipids.
Partition Coefficient = Solubility in Olive Oil / Solubility in Water
As the partition coefficient increases, fat solubility increases and water solubility decreases.
Note in Table 3 that the partition coefficient increases as the number of polar hydroxyl groups
decreases.
14
Determine times to hemolysis for each solution, following the procedure outlined for
EXERCISE B. (Repeat glycerol test - do not use previous results).
Record hemolysis times in Table 4 and on the blackboard.
Table 3: Partition coefficients and formulas.
SUBSTANCE
MOLECULAR
WEIGHT (g)
PARTITION
COEFFICIENT
FORMULA
glycerol
92
0.00001
CH2OH
│
CHOH
│
CH2OH
triacetin
218
0.36
diacetin
176
0.1
CH2‐O‐COCH3
│
CHO‐COCH3
│
CH2‐O‐COCH3
CH2-O-COCH3
│
CHOH
│
CH2-O-COCH3
Table 4: Your results.
SUBSTANCE
glycerol
Triacetin
diacetin
TIME TO
HEMOLYSIS
V. OBSERVATION OF OSMOSIS UNDER MICROSCOPE
EXERCISE D
Equipment:
 ovine blood
 distilled water
 isotonic saline (0.15M NaCl)
 0.3 M NaCl
 1 box of Kimwipes
 3 microscope slides and cover slips
 1 microscope
 4 Pasteur pipettes
 masking tape
15
1.
Label 3 microscope slides: "dist. water", "saline" and "0.3 M NaCl".
2.
Label the 4 Pasteur pipettes: "dist. water", "saline", "0.3 M NaCl" and "blood".
3.
Dilute the stock ovine blood 2:1 with isotonic saline (10 drops isotonic saline to 5 drops
of blood).
4.
Place a drop of isotonic saline on the appropriate microscope slide. Add a small drop of
the diluted ovine blood on the top of the drop of isotonic saline. Add a cover slip and examine
immediately under the microscope.
In the center of the slide, the red blood cells are piled up several layers thick and thus it is very
hard to determine the shape of individual red blood cells. Make sure that you look where the
layer of blood is thinner and try to find a spot where you see individual cells clearly. Use high
power and reduced light intensity. Sketch outlines of cells.
5.
Repeat this procedure twice, but use distilled water and 0.3M NaCl instead of isotonic
saline.
CLEAN-UP
 See “At the beginning of the lab” on first page.
 Leave your space clean and tidy.
16
WORKSHEET
Name:
ID No.:
Instructor:
Course / Section:
Partner’s Name (if applicable):
Date:
Additional Activities
1.
Try any of the diffusion experiments again. This time using exact measurements.
Compute the exact rate of diffusion for each substance. Use a variety of mathematical and/or
statistical formulas to discover whether or not other relationships exist.
2.
Design additional experiments to demonstrate movement across a membrane—either
active or passive.
3.
Refer to the experiment on red blood cell osmosis. What would you need to know if you
wanted to determine the relative amount of water moving across the cell membrane?
Answer to Selected Concept Check Questions
1.
In flask #2 the neutral red must have been transported into the living yeast cell. In Flask
#1 the yeast cells were killed so no transport could occur.
2.
These pumps correspond to the sodium/potassium pump in the cell membrane. The
sodium/potassium pump actively pumps three sodium ions out of the cell for every two
potassium ions pumped into the cell, thus maintaining the membrane potential. You may wish
to figure out other macroscopic situations where active energy input is required.
3.
The potassium permanganate should have traveled about twice as far as the potassium
chromate. The potassium chromate should have traveled just a bit further than the methylene
blue.
Name:
General Questions
Date:
17
Use your own paper if necessary.
1.
Define active and passive movement across a cell membrane.
2.
Explain what happened to the yeast solutions. Why was the solution in Flask #2 a
different color than the solution in Flask #1?
3.
What caused the Brownian movement you observed?
4.
Explain what happened when you dropped the potassium permanganate (KMnO4)
Crystals into the water.
5.
If your diffusion rates do not match the expected diffusion rates, how might you explain
the discrepancy?
6.
In which of the experiments did osmosis occur? Dialysis? In which direction did these
events occur?
7.
Check your lecture text and find out what effective osmotic pressure is? How does it
relate to cellular osmosis?
8.
Explain hypertonic, isotonic, and hypotonic in terms of the red blood cell experiment.
9.
List several factors (other than the ones we demonstrated by these experiments) that
might influence the rate of either active or passive transport.
10.
Why are sugar and salt used for food preservations?
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