LAB EXERCISE 1

CELL MEMBRANE PERMEABILITY

(Martini, p 90 - 100)
Work in pairs
Objectives:

Describe and account for the effects of hypotonic, isotonic and hypertonic solutions on red blood cells
Differentiate between tonicity, osmolarity and molarity of a solution.
Describe the relationship between molecular weight and permeability of penetrating solutes, and relate
to cell membrane structure
Define the partition coefficient of a compound and relate it to molecular structure, permeability and
structure of the cell membrane

SAFETY PROCEDURES
Handle blood and blood by-products with care because they can transmit infectious diseases.
All students must wear latex gloves.
If you have any open cuts on your hands, do not handle blood.
Never pipette blood or blood by-products by mouth.
Work over tray to avoid spilling chemicals and blood on the bench.
If you spill blood on your tray or on the bench wipe it up immediately.
At the end of the lab:
Discard all the material that has been in contact with blood in the heavy black plastic bags labeled "to
be autoclaved";
Wipe your bench, your microscope and your tray to remove ALL blood stains
Wash the glassware with detergent and rinse it thoroughly with tap water; let it dry on
paper towels
on your tray; make sure that you leave clean glassware for students in the next section;
Remove your gloves and wash your hands.
All activity, and therefore life itself, is dependent on the ability of cell membranes to
selectively permit or promote the transfer of some substances in or out while preventing
other substances from entering or leaving the cell. This exercise is concerned with some of
the factors that determine the selective permeability of the plasma membrane of the red
blood cells.
Water molecules pass through cell membranes by osmosis with relative ease. The permeability of solute
molecules depends on a number of factors. Molecular size is important; small polar molecules pass
through cell membranes more readily than larger molecules. Electrolytes generally pass through less
readily than non-ionized polar molecules of similar size since ions carry electrical charges that attract and
bind water molecules forming larger hydrated spheres. Furthermore, active transport pumps in the cell
membrane transport some ions against their concentration gradients (e.g. Na+, K+). Because of this, there is
no net movement of these ions into or out of cells even when the membrane is permeable to them.
Solubility in fat or fat solvents is also an important factor in determining cell membrane permeability.
Nonpolar compounds are highly soluble in fats but have very low solubility in water. Some polar
compounds are freely soluble in water but may also be soluble in oil if they have a number of nonpolar
bonds (e.g. ethyl alcohol). Lipid soluble (nonpolar) molecules pass through cell membranes by dissolving
in and diffusing through the lipid portion of the membrane.

I. PREPARATION OF A STOCK BLOOD SUSPENSION

Equipment:
bovine 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 bovine 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 bovine 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.
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
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
WEIGHT (g)

MOLECULAR
DIAMETER (A)

FORMULA

urea

60

3.6

H2N
H2N

C=O

ethylene glycol

62

~3.6

CH2OH
CH2OH

glycerol

92

6.2

CH2OH
CHOH
CH2OH

glucose

180

8.6

CHO
CHOH
CHOH
CHOH
CHOH
CH2OH

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 monoacetin
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.
1. Determine times to hemolysis for each solution, following the procedure outlined for EXERCISE B.
(Repeat glycerol test - do not use previous results).
2. Record hemolysis times in Table 4 and on the blackboard.
Table 3: Partition coefficients and formulas.
SUBSTANCE

MOLECULAR
WEIGHT (g)

PARTITION
COEFFICIENT

FORMUL
A

glycerol

92

0.00001

CH2OH
CHOH
CH2OH

monoacetin

134

0.01

CH2-OCOCH3
CHOH
CH2OH

diacetin

176

0.1

CH2-OCOCH3
CHOH
CH2-OCOCH3

Table 4: Your results.

SUBSTANCE
glycerol

Monoacetin

diacetin

TIME TO
HEMOLYSIS

V. OBSERVATION OF OSMOSIS UNDER MICROSCOPE

EXERCISE D
Equipment:
bovine 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
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 bovine 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
bovine 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.

NAME & section #:

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Biology 153 LAB REPORT (2002-2003)

EXERCISE 1: CELL MEMBRANE PERMEABILITY
INTRODUCTION
State the purpose of this exercise

MATERIALS AND METHODS

See the lab manual
If you made any changes from the lab manual, note them below.

Calculate the amount of solute that you have to dissolve in order to make 1 liter of the solutions used
throughout the exercises. Calculate also the osmolarity of these solutions.
Write your answers in the following table.
SOLUTION

MOLECULAR
WEIGHT OF
SOLUTE (g)

0.15M of NaCl

58

0.3M of NaCl

58

0.3M of urea

60

0.3M of ethylene glycol

62

0.3M of glycerol

92

0.3M of glucose

180

0.3M of monoacetin

134

0.3M of diacetin

176

QUANTITY OF
SOLUTE (in g) PER
LITER OF
SOLUTION

RESULTS
Use separate pages to present your results.

OSMOLARITY OF
THE SOLUTION
(in osmol/L)

A. EFFECT OF MOLECULAR SIZE (Exercise B.)

- Table: tabulate class mean hemolysis times for each solute. Include in the table:
1. name of solute
2. molecular weight
3. mean hemolysis time
- Graph molecular weight (horizontal axis) vs. mean hemolysis times (vertical axis).
- Describe the results in no more than three lines. What trend do you observe?
B. EFFECT OF LIPID SOLUBILITY (Exercise C.)
- Table: tabulate class mean hemolysis time for each solute. Include in the table:
1. name of solute
2. its molecular weight
3. its partition coefficient
4. mean hemolysis time
- Make two graphs:
First graph: graph molecular weight (horizontal axis) vs. mean hemolysis times (vertical axis).
Second graph: graph partition coefficient (horizontal axis) vs. mean hemolysis times (vertical axis).
- Describe the results in no more than three lines. What trend do you observe?
C. OBSERVATION OF OSMOSIS UNDER MICROSCOPE (Exercise D.)
- Sketch and label red blood cells from each of the three slides.
- Describe your results in 3 lines. Describe the appearance of the cells. If you didnt observe anything, or
your observations are unexpected, describe what you would have expected to see.
DISCUSSION
Use only spaces provided for your answers; do not use extra pages; do not copy word for word from the
book.
A. DEMONSTRATION OF OSMOSIS
Helpful information: 1) the osmolarity of the intracellular liquid of the red blood cells is 0.3 osmol/L; 2)
solutes inside the red blood cells are non-penetrating; 3) NaCl in water dissociates into 2 particles Na+ &
Cl-; 3) urea, ethylene glycol, glycerol, glucose, monoacetin, diacetin do not dissociate in water.
1. Explain what happens to RBCs in each of the three solutions and why. Include in your explanation:
solute concentration; penetrating characteristics of the solutes; movement of solute; movement of water;
the tonicity of the solution; change in shape of cells.
- Distilled water:

- 0.3 M NaCl:

- Isotonic saline (0.15M):

2. Explain what happens to RBCs in the solution of 0.3M urea and why (consider solute
concentration; penetrating characteristics of the solutes; movement of solute; movement of water; change
in shape of cells).

Can the above explanation be used to describe what happens to RBCs in the solutions of ethylene glycol,
glycerol, monoacetin, diacetin and glucose? Briefly explain your answer.

3. From these exercises we observe that two isosmotic solutions can have two very different
effects on the red blood cells:
- Solutions of 0.15 M of NaCl and solutions of 0.3M of glucose are isosmotic and isotonic
with blood.

- 0.3M solutions of urea, ethylene glycol, glycerol, monoacetin and diacetin are isosmotic
with blood but not isotonic.
Define the following terms:
- Isosmotic:

- Isotonic:

What single factor determines if an isosmotic solution will be isotonic or hypotonic with blood?

B. EFFECT OF MOLECULAR SIZE

Helpful information: urea, ethylene glycol, glycerol, glucose have similar partition coefficients.
1. What can you conclude from the results from Exercise B? (State the trend observed in hemolysis times
when testing solutions of varying molecular size)

2. How do the results from Exercise B relate to cell membrane structure? (Describe the structure of the
cell membrane to explain the trend observed in testing molecules of varying molecular size; consider the
properties of the solutes used in exercise B)

C. EFFECT OF LIPID SOLUBILITY

1. What can you conclude from the results from Exercise C? (State the trend observed in hemolysis times
when testing solutions of varying lipid solubility)

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2. Explain your results on the basis of cell membrane structure and the chemical nature of nonpolar
solutes tested in Exercise C.

Account for the different effects of molecular size on time to hemolysis in Exercise B and C.

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