UNIVERSITY OF ZAMBIA

SCHOOL OF MEDICINE

DEPARTMENT OF NURSING SCIENCE

NAME : MWELWA CHILUFYA

COMPUTER No : 12117391

LABORATORY REPORT

SUBJECT : PHYSIOLOGY PGN 210

TITLE : THE CELL MEMBRANE

LECTURER : MRS SANDRA CHILEYA

DATE DUE : 09/05/2013

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 TABLE OF CONTENT

CONTENT NO.

SPECIFIC OBJECTIVES ………………….…………….……………………. 3

INTRODUCTION ………….……………..…………………………………. 3

METHODS …………………………………………………………………….. 4

APPARATUS ………………………………………………..………………… 4

RESULTS: HAEMOLYSIS OF CELLS WITH SOLUTIONS ………………. 5

Comment …....…………………………………………………………… 5

RESULTS: SIEVING EFFECTS OF CELL MEMBRANES ………………… 6

Comment ………………………………………………………………… 6

RESULTS: EFFECTS OF SALINE OF VARYING OSMOLALITIES ON

RED BLOOD CELL SHAPE …………………………………… 6

Comment …………….………………………………………………….. 7

DISCUSSION …………………………………………………………………. 7

CONCLUSION ……………………………………………………………….. 9

REFERENCES ……………………………………………………………….. 10

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THE CELL MEMBRANE

The aim of these experiments is to provide scientific knowledge and understanding of the
Effects of substances that penetrate and damage the cell membranes, Solute molecular size
and Cell permeability and the effect of Saline of varying Osmorarities on Red blood cell
shapes.

SPECIFIC OBJECTIVES

I. To determine the time needed to complete haemolysis after adding 0.15M Na CL,
0.3M Urea and 0.15M Na CL with a drop of soap solution added to a drop of
blood to each solution at a time.
II. To establish the Sieving effects by Membrane pores of solutions 0.3M Glucose
(C6H12O6) ,0.3M Glycerol (C3H803) and 0.3M Ethylene Glycol (C2H602) by
adding two drops of blood to each solution respectively.
III. To investigate the effect on the shape of red blood cells and thus investigate the
fragility by immersing them in Na CL solutions of varying concentrations.

INTRODUCTION

All living creatures are made up of cells which are simply small membrane bounded
compartments filled with a concentrated aqueous solution of chemicals. The cell membrane is
also referred to as the Plasma membrane; it is made up of a double layer of a semi permeable
structure that controls the passing in and out of substances. Its components include
phospholipids which are 50% by weight of membrane. The cell membrane is a lipid bilayer
with an outer hydrophilic (water soluble) portion and hydrophobic (water insoluble) portion.
This allows the heads to face towards water and all the tails to face away from water.
Phospholipids bilayer appears wherever phospholipids molecules are scattered among the
water molecules. They are made of glycerol and fatty acids, a phosphate molecule and a
hydrophilic molecule. The main lipid is phospholipids which include phosphotidylcholine
and phosphotidylserinethese molecules have amphipathic (i.e. polar and non polar) property.
Therefore, the molecular arrangement results with hydrophilic, polar head or aqueous loving
chemical groups facing the extracellular and intracellular fluid, where as the hydrophobic, on
polar and aqueous non loving fatty acid chains restricts in between the layer.

Other lipid molecules are cholesterol molecules which are interspaced among the other lipids
in both layers. Together with the number of double bonds present in the fatty acids chains

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making a kink which prevents lipids molecules from packing tightly in the membrane,
cholesterol determines the fluidity of the membrane. Proteins make up 50% of the membrane.
They are extended through the bilayer as integral or transmembrane proteins. In some
instances, however, proteins are associated with membrane lipids or integral proteins at the
inner or outer surface of the membrane, these are called peripheral proteins.

Due to its characteristic molecular make up, the cell membrane not only forms a barrier
between the extracellular and intracellular environment, but plays various roles that include:
transportation of substances into the cell which are required for metabolic reactions as well as
excretion of metabolic products and wastes. Substances move across the cell membrane
through diffusion, active transport, osmosis and also vesicular means. Hydrophilic or polar
substances like charged ions are transported by active transport through respective ion protein
channels. This process uses energy. Water molecules, however, diffuse through the
membrane due to its smaller size and may also be transported through aquaporins channels
easily through the membrane. Examples include oxygen and alcholiols.The smaller the size,
the faster the diffusion and a large surface area increase this process.

An osmotically active particle in the extracellular and intracellular environment determines

the osmotic potential across the plasma membrane. Hypertonic environment has high number
osmotically active particles where as hypotonic solution has the opposite. Isotonic solution is
one which contains same number of osmotically active particles as the intracellular
environment. Based on selective permeability of the cell membrane, this experiment assesses
the osmotic effects of some substances of different concentrations and size on the red cells in
order to understand membrane characteristics.

METHOD

As in the Physiology laboratory Manual

APPARATUS

1. Two (2) tube racks: one containing 10 test tubes, other one with three (3) tubes,
2. Light microscope
3. Glass pipette, Pasture pipette and Dropper
4. Beaker and Labelling Stickers
5. Blood sample 4 mls in EDTA tube(from an individual group member)
6. Timer(watch)

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 7. Plain paper with printed words on it
8. Latex gloves
9. Reagents:
 distilled water
 1% and 3% sodium chloride (NaCl)
 0.15 M NaCl
 0.3M urea
 Soap solution
 0.3M Glucose
 0.3mM Glycerol
 0.3 M Ethylene glycol

METHOD; As stated in the Physiology laboratory Manual.

RESULTS FOR PROCEDURE A: HAEMOLYSIS OF CELLS WITH SOLUTIONS

Test Amount of Fluid

Name of solution Time Observation
tube no in each tube
No printed letters were seen.
1 0.15m NaCl 2mls 5min
No haemolysis was seen.
Printed letters were seen.
2 0.3mUrea 2mls 17sec
Haemolysis took place.
0.15m NaCl plus a Printed letters were seen.
3 2mls 24sec
drop of soap solution Haemolysis took place.
Comment

Rapid Haemolysis was seen when a drop of blood was added to 0.3M Urea which in the
steady state is in equilibrium with cells. Its contribution to osmolality is normally about
5mosm/l each but can become quite large in hyperglycaemia or uraemia.

There was slightly delayed Haemolysis seen when a drop of blood was added to 0.15M NaCl
which had a drop of soap solution because initially with NaCl there was no Net Movement of
the osmotically active particles in the solution into cells and the particles were not
metabolized but the addition of the drop of soap solution indicates the presence of a foreign
substances in the detergent such as soap may sometimes shrink cells osmotically.

No haemolysis at 5 minutes was seen when a drop of blood was added to 0.15M NaCl
because there was no Net Movement of the osmotically active particles in the solution into

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cells and the particles are not metabolized showing that NaCl Solutions have the same
osmolality as plasma and are said to be Isotonic.

RESULTS FOR PROCEDURE B; SIEVING EFFECTS OF CELL MEMBRANES.

TITLE: SOLUTE MOLECULAR SIZE AND CELL PERMEABILITY

The three compounds used in this experiment are very similar in chemical structure, chiefly
differing in molecular size. All three are very water soluble, the experiment therefore
concerns the ‘Sieving effect ’by the membrane pores.

Add two drops of blood to 2ml of each of the solutions, mixing tube contents immediately.

Test tube solution Drops of blood Results Time in

seconds
1 0.3m glucose 2 No haemolysis 0
2 0.3m glycerol 2 Complete 15
haemolysis
3 0.3m ethylene 2 Complete 9
haemolysis
Comment

The speed by which solutes penetrate the plasma is determined by their molecule size. There
was no haemolysis in tube 1.Otherwise haemolysis rapidly occurred in tube .3 and slightly
longer in tube 2.

RESULTS FOR PROCEDURE C

TITLE: EFFECT OF SALINE OF VARYING OSMOLALITIES ON RED BLOOD

CELL SHAPE

Test tube Nacl3% Distilled Drop of Results

no Nacl1% water blood
1 40 Non 1 Clear fluid above red blood
cells
2 32 8 1 Clear fluid above red blood
cells
3 28 12 1 Clear fluid above red blood
cells
4 24 16 1 Clear fluid above red blood
cells

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 5 22 18 1 Clear fluid above red blood
cells
6 20 20 1 Test tube looks dark red
7 18 22 1 Partial haemolysis
8 16 24 1 Completely haemolysed
9 14 26 1 Completely Haemolysed
10 12 28 1 Completely Haemolysed
Comment

The table above shows the different solutions that were prepared and to each a drop of blood
which was added. Test tube 1 had a higher concentration or percentage of NaCl than the red
blood cells hence this made the outer cell environment hypertonic and this allowed water to
sip out of the cell, shrinking the cells. However this does not mean the cells are haemolysed
because naturally NaCl has the same composition as that of red blood cells.

Test tube 2-5 also showed a clear fluid on top of the cells simply because there was no
haemolysis. There was more NaCl than distilled water in those test tubes which as explained
above have the same composition as red blood cells. Test tube 6 had the same drops of NaCl
and distilled water; this gave the solution a dark red colour as the fluid around the cells was
more diluted (by equal parts) than the inner cell environment. Therefore, this fluid was
hypotonic which allowed the fluid to sip into some cells bulging them up in attempt to strike
a balance on the inside and outside cell environment.

Test tube 7-8 had partial haemolysis resulting from more concentration of distilled water than
NaCl in the test tube. Test tubes 9-10 were completely haemolysed due to the above mention
reason.

DISCUSSION

All fluid compartments of the body are in or nearly in osmotic equilibrium. The term Tonicity
is used to describe the osmolality of a solution relative to plasma. Solutions that have the
same osmolality as plasma are said to be Isotonic. Those with greater osmolality are
Hypertonic. Those with lesser osmolality are Hypotonic. All solutions that are initially
Isosmotic with Plasma, (that have the same actual osmotic pressure or freezing –point
depression as Plasma), would remain Isotonic if it were not for the fact that some solutes
diffuse into cells and others are metabolised. The freezing point of normal human plasma is
-54 degrees Celsius which corresponds to osmolar concentration in plasma of 290 mosm/l.
This is equivalent to an osmotic pressure against pure water of 7.3atm.The osmolality might

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be higher than this because the sum of all the cations and anions equivalents is not an ideal
solution and ionic interactions reduce number of particles free to exert an osmotic effect.

Thus 0.9%Saline solution remains Isotonic because there is no Net Movement of the
osmotically active particles in the solution into cells and the particles are not metabolised. On
the other hand 5% Glucose solution is isotonic when initially infused intravenously but
Glucose is metabolised, so the Net effect is that of infusing a hypotonic solution.

All but about 20 of the 290 mos in each litre of normal Plasma are contributed by Sodium and
its accompanying anions, principally and Carbohydrates. Other cations and anions make a
relatively small contribution .Although the concentration of the Plasma proteins is large when
expressed in grams per litre; they normally contribute less than 02 mosm/l because of their
very high molecular weights.

The major non electrolytes of Plasma are Glucose and Urea which in the steady state are in
equilibrium with cells. Their contribution to osmolality is normally about 5mosm/l each but
can become quite large in hyperglycaemia or uraemia. The total Plasma osmolality is
important in assessing dehydration, over hydration and other fluid and electrolyte
abnormalities.

Hyperosmolality can cause coma (hyperosmolar coma). Because of the predominant role of
major solutes and the deviation of Plasma from each ideal solution, Plasma osmolality can be
estimated within a few mosm per litre by using a formula in which the constants convert the
clinical units to mill moles of solute per litre-:

Osmolality =2(Na) + 0.055 (glucose) + 0.36 (BUN)

(Mosm/l) (mEq/l ) (Mg/dL) (MG/dl)

BUN is the Blood Urea Nitrogen.

An observed plasma osmolality (measured by freezing point depression) that greatly exceeds
the value predicted indicates the presence of a foreign substances such as Ethanol, Manitol
(sometimes injected to shrink swollen cells osmotically) or poisons such as Ethylene glycol
or Methanol (components of ant- freeze).

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Animal cell membranes are flexible and therefore swell when exposed to extra cellular
Hypotononicity and shrink when exposed to extra cellular Hypertonicity.

However cell swelling activates channels in the cell membrane that permit increased efflux of
Potassium, Chloride and small organic solutes referred to collectively as Organic Osmolytes.
Water follows these osmotically active particles out of the cell and the cell volume returns to
normal.

CONCLUSION

Osmotic Effects of Substances That Penetrate or Damage Cell Membranes revealed that there
was haemolysis in urea and in NaCl plus a soap solution than in NaCl. Effect of saline of
varying Osmolalities on red cells shape after adding varying Concentrations of NaCl and
adding a drop of blood to each solution resulting in no haemolysis in the test tubes 1-6 then
there was partial haemolysis in test tubes 7-8 and in test tube 9-10 there was complete
haemolysis.

Finally checking under the Microscope of some cells from the first tube contents where there
was no haemolysis after mixing revealed the shape of the red blood (erythrocytes) cells which
were biconcave discs which seemed to be highly motile.

Solute molecular size and cell permeability revealed that 02 drops of blood added revealed no
haemolysis in 0.3m glucose. Haemolysis occurred with Glycerol and Ethylene glycol.

REFERENCES

1. Ganong .W. (2005) Review of Medical Physiology: 2ndEdition, Lange Medical

Publication, California.USA.
2. Guyton.A.C. (1986) Text book of Medical Physiology: WBS Saunders Company,
New York.USA.

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3. Tortora.J. etal.Principles of Anatomy and Physiology: 9th edition, John Wiley and
Sons, Inc. NewYork.USA.
4. Boron.W.F. and Boulpaep.E.L. (2009)Medical Physiology; A cellular and molecular
approach, 11th edition, Biological sciences Text books Inc and Bryan Derrickson,
USA.

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