PRACTICAL 2: CELL MEMBRANE AND FLUIDS
The cell membrane is selectively permeable to ions and organic molecules and
controls the
movement of substances in and out of cells. The basic function of the cell
membrane is to protect
the cell from its surroundings. It consists of the phospholipid bilayer with
embedded proteins.
At the end of this experiment, one will be able to appreciate the effects of
osmotic pressure or
osmotic behaviour of human blood cells and cell permeability.
OSMOTIC EFFECTS ON A CELL CELL MEMBRANE
PRACTICAL EXERCISE
A. Osmotic effects of substances that penetrate or damage cell membranes
a) Into three clean test tubes, place relatively 2ml of each of the following
solutions.
(i) 0.15M NaCl
(ii) 0.3M Urea
(iii)0.15M NaCl plus a drop of soap solution
b) Working with one tube at a time, add a drop of blood to each solution, mixing
tube
contents immediately. Determine the time to complete haemolysis. If the tube is
held in
front of a printed page the contents will become transparent when haemolysed
(haemolysis is process whereby haemoglobin, red pigment of the red blood
corpuscles,
escape from the cell due to damage to the surface membrane) and the printing
may be
read through it.
Comment on your results and discuss the related processes.
B. Solute molecular size and cell permeability.
The three compounds used in this experiment are very similar in chemical
structure, differing
chiefly in molecular size. All three are very water-soluble. The experiment
therefore concerns
the “sieving effect” by the membrane pores.
a) Add two drops of blood to 2ml of each of the following solutions, mixing tube
contents
immediately.
(i) 0.3M Glucose (C6H12O6)
(ii) 0.3M Glycerol ( C3H8O2)
(iii)0.3 M Ethylene glycol ( C2H6O2)
Note: If you are unable to determine the time it took for haemolysis to occur or
the haemolysis
time is found to be too short, repeat the determination of time of haemolysis
until accurate
readings have been obtained, using a stop clock if necessary. If rapid haemolysis
does not occur
observe every 5 minutes for 45 minutes.
C. Effect of saline of varying osmolalities on red blood cell shape.
Investigate the effect on the shape of the red blood cells by immersing them in
NaCl solutions of
varying concentrations; thus investigate the fragility of red cells when immersed
in NaCl
solutions of varying concentrations.
NB: You will need to wear gloves for the handling of blood and blood products.
EQUIPMENT
• One test tube rack containing ten test tubes.
• A microscope with high power objective.
• 1% & 3% solutions of NaCl
• Distilled water
• A dropping pipette.
• Blood, either obtained by vein-puncture from a volunteer or supplied from
Blood Bank.
PROCEDURE
Label tubes from 1 to 10 in sequence. Into tube 1 put 40 drops of 3% NaC. Into
tube 2 put 32
drops of 1% NaCl and 8 drops of distilled water.
Similarly into test tubes 3 to10 in sequence put 28, 24, 22, 20, 18, 16, 14 & 12
drops of 1% NaCl and 12, 16, 18, 20, 22, 24, 26 & 28 drops of distilled water. To
each test tube add one drop of
blood and thoroughly mix it with the salt solution. Leave the test tubes standing
for 1 hour and
then inspect them.
If no haemolysis has occurred red cells will settle at the bottom of the test tube
and there will be
clear saline above them. Partial haemolysis will leave cells at the bottom of the
test tube and pink
fluid above and complete haemolysis will result in pink saline with no red cell
sediment. Shake
the test tubes and observe the red cells or the remnants in fluid under the
microscope. Look for
changes in red cell shape. Take note of the concentration of NaCl at which
haemolysis begins
and at which it is complete.