THE EXPERIMENTAL COURSE

OF CELL BIOLOGY

Welcome to Study on ECCB2015

Huang Zhi; Ling Qinjie
 Exp4-Permeability of cell membrane
Objectives:
 To understand the permeability of plasma
membrane to different solutes.
 To observe the passive transport speed of
various substances through the cell membrane.
 To understand the hemolysis and the
mechanism of osmosis.
 Introduction

 The internal environment of the human body largely consists

of water-based solutions. A large number of different solutes
are dissolved in these solutions.
 Two qualifications of a solute are needed in the passive
transport through a plasma membrane. It must be present at
higher concentration on one side of the membrane than the
other, and the membrane must be permeable to it.
 A membrane is permeable to a given solute either (1) because
that solute can pass directly through the lipid bilayer, or (2)
because that solute can traverse an aqueous pore that spans
the membrane.
 Introduction
 Water moves much more rapidly through a cell membrane
than do dissolved ions or small polar organic solutes. Because
of this difference in the penetrability of water versus solutes,
membranes are said to be semipermeable.
 Water moves readily through a cell membrane from a region
of lower solute concentration to a region of higher solute
concentration. This process is called osmosis, which is readily
demonstrated by placing a cell into a solution containing a
nonpenetrating solute at a concentration different than that
present within the cell itself.
 Aquaporins allow the passive movement of water from one
side of the plasma membrane to the other.
The Nobel prize in chemistry 2003 awarded jointly to Peter Agre “for the discovery of
water channels" and Roderick MacKinnon "for structural and mechanistic studies of ion
channels". (http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2003/)
The effects of differences in the concentration of solutes on opposite
sides of the plasma membrane.
 When the erythrocytes are placed in some isotonic salt
solutions, because the cell membrane permeability is different
for various solutes, some solutes can enter erythrocytes, some
solutes can not.
 Solutes entered can increase the cell osmotic pressure and
permit water to enter cells, causing hemolysis.
 As the solutes penetrate at different speeds, hemolysis time is
different, therefore, the time length required for occurrence
of hemolysis can be used to measure the speed of the
substance across erythrocytes.
 Erythrocyte equilibrium
• No osmotic pressure
- cell is in an isotonic solution
- Water does not cross
membrane
• Increased [Osmotic] in cytoplasm
- cell is in an hypotonic solution
- Water enters cell, swelling

• Decreased [Osmotic] in cytoplasm

- cell is in an hypertonic solution
- Water leaves cell, shrinking
Erythrocyte ghost
 Cell Lysis
• Using hypotonic solution
• Or interfering with Na+
equilibrium cause cells to
burst
 3. Materials and Methods

• Instrument: light microscope

• Reagents: 0.17mol/L NaCl 、 0.17mol/L
NH4Cl 、 0.17mol/L NH4Ac 、 0.17mol/L
NaNO3 、 0.12mol/L (NH4)2C2O4 、 0.12mol/L
Na2SO4 、 0.32mol/L glucose 、 0.32mol/L
glycerol 、 0.32mol/L ethanol 、 0.32mol/L
propanol 、 H2O 。
• Material: rabbit erythrocyte
1 、 10% Preparation of rabbit erythrocyte suspension ：
8% Heparin Sodium 肝素钠 2 mL+ rabbit blood 20 mL+0.17 mol/L NaCl
200 mL
2 、 Observation of hemolysis:
Take a test tube, add 9 mL isotonic solution, then add 1 mL prepared 10%
rabbit red blood cell suspension, mix quickly and place in the incubator,
observe whether the hemolysis in test tube and record the time of hemolysis,
analysis reasons. Experimental procedures in Table 1.
Note: There are red blood cells and test solution, should not shake vigorously,
avoiding red blood cell rupture.
3 、 Determine hemolysis:
Hemolysis, the opaque suspension becomes transparent red hemoglobin
solution. Red blood cells rupture, causing 100% hemolysis, so that the light
can easily pass through the solution.
 Table. Hemolysis of isotonic solutions of different solutes (pure water is not isotonic solution )

No. of test tube Hemolysis Time Result analysis

Yes/No
(0) 9 mL H2O +1 mL erythrocyte

(1) 9 mLNaCl +1 mL erythrocyte

(2) 9 mLNH4Cl+1 mL erythrocyte

(3) 9 mLNH4Ac+1 mL erythrocyte

(4) 9 mLNaNO3+1 mL erythrocyte

(5) 9 mL(NH4)2C2O4+1mL erythrocyte

(6) 9 mLNa2SO4+1 mL erythrocyte

(7) 9 mLGlucose+1 mL erythrocyte

(8) 9 mLglycerol+1 mL erythrocyte

(9) 9 mLethanol+1 mL erythrocyte

(10) 9 mLpropanol+1 mL erythrocyte

 4. Results and Discussion

Compare and analyze the difference of hemolysis time of

isotonic solutions with similar solutes.

Osmotic concentration: concentration of osmotically active

particles in solutions.