Zoology 120 – Animal Physiology

EXERCISE 1: Characterization of the permeability of cell membrane through

hemolyzation methods and penetration of alkali

Abstract
The cell membrane or plasma membrane controls the entry and exit of certain chemicals and
substances into the cell. Nonetheless, in the osmotic process, it was found to be absolutely
permeable to water molecules. Due to this, bursting of the cells due to higher water potential outside
the cell than that of the inside, namely hemolysis, may occur when the cells are subjected to a
hypotonc solution. In the experiment, RBC suspensions were placed in varying concentrations of
glucose, NaCl, and KCl. Among the three, it was expected that the lowest concentration would
result to hemolysis at a much faster rate, however NaCl, with a concentration of 0.02M was the only
concentration to hemolyze, while KCl caused hemolysis of the RBCs at relatively higher
concentrations. Glucose on the other hand, did not cause hemolysis at any given concentration,
within the given period of time. The isotonic coefficients of NaCl and KCl, that is the relationship
between the hemolytic concentrations of the electrolyte with the non-electrolyte, were both found to
be 0.5. Yeast cells suspended in NaHCO3 were used in test for penetration of alkali, and the initial
color observed did not conform to the expected result, which might be due to the reagents itself or
the errors committed during the experiment. Yeast cells treated with alkalis NH4OH, KOH, and
NaOH exhibited different colors, yellow-brown, red-brown, and red-brown respectively, which was
due to their strengths as electrolytes.

According to this model, the membrane

Introduction
Penetration of different substances into
the cell is one of the most difficult problems
that a cell can encounter. As an answer to this,
a cellular structure, which is the plasma
membrane, controls the entry and exit of
certain chemicals and substances into the cell.
The plasma membrane not only defines the
border of the cell but also interacts with its
environment in a controlled way. One of the
major roles of the plasma membrane is to
regulate materials into the cell and from the
cell to its environment. In order to carry out
this responsibility, the membrane is composed
of phospholipid bilayer. The main model in
understanding the structure of the plasma
membrane is called the fluid mosaic model
(Khan Academy, 2015).
is primarily composed of phospholipids,
cholesterol and proteins that move freely in the
plane of the membrane. The phospholipid is
generally composed of a hydrophobic tail and a
hydrophilic head (Khan Academy, 2015). The
protein component of the membrane aids for
the transport some nutrients against the
concentration gradient, which requires
additional energy. Lastly, cholesterol is tucked
between the hydrophobic tails of the membrane
phospholipids providing additional flexibility
of the membrane. All of these work together
for the membrane to keep the inside and
outside of the cell regulated. The permeability
of the plasma membrane allows the cell to
reach equilibrium and is also one of the reasons
why difference in concentration occurs, which
in turn produces hemolysis. Osmosis in a
hypotonic solution causes the hemolysis of
cells. Also, the blood tonicity of the difference
in the concentration of substances inside and
outside the cell determines whether the cell
will shrink, swell or remain its normal size. In
addition, another factor that affects the rate of
diffusion is the temperature. Higher
temperature increases the rate of diffusion
across a membrane. The mass or the size of the
diffusing particle also affects rate of diffusion
in the sense that as the mass becomes larger,
the diffusion rate becomes slower. For a
smaller particle can move faster than a larger
particle. Furthermore, larger membrane surface
area available for diffusion facilitates a faster
diffusion rate and a greater distance over which
diffusion must take place results in a slower
diffusion rate.
There are several factors affecting the
permeability of the plasma membrane such as
the temperature, chemicals, size of the
molecule passing across it and its nature. The
cell membrane is known to be selective in
terms of allowing substances to pass through it,
but with certain chemicals, it is possible that
the membrane will be destroyed together with
its selectivity. This also applies to temperature.
The size of the molecule passing through the
membrane is also a factor that affects its
permeability. The larger the molecule the
harder will it pass through the membrane,
unless it undergoes the process of endocytosis.
The nature of the molecule is also a big factor
that affects the permeability of the cell
membrane. Water soluble and fat-soluble
substances can easily pass through the
membrane but substances, which are
electrolytes in nature, would be slower than the
non-electrolytes due to the fact that electrolytes
with high valency cannot penetrate the
membrane easily (Wong, nd).
Penetration of alkali simply refers to the
entry of dissociated alkali metals, and/or
ammonium into the cell by diffusion (Brooks
1923). Alkali penetration is dependent on their
degree of dissociation, since only ionized form
of the alkali are transported through the
membrane, may it be passive or active
(Burggren 2002). Ionization degrees are higher
with strong acids and bases and lower with the
weak ones (Campbell & Farrell 2013).
Penetration of alkali is commonly observed
with the use of dyes, like the neutral red dye,
which stains the golgi apparatus red, to stain
cells, and subjecting the set-ups to alkali
compounds, then observing the color changes
before and after dissociation by the liquid.
In the experiment alkali metals such as
sodium and potassium were present in the form
of NaCl and KCl. Sodium Chloride (NaCl) and
Potassium Chloride (KCl) are both solutions
that are classified as non-penetrating
electrolytes. Both solutions cause hemolysis at
lower molar concentrations than the non-
penetrating nonelectrolyte, which are glucose
and sucrose, to name a few, that cause
hemolysis at the same molar concentrations.
This is because an electrolyte can dissociate
into two ions, and every ion in the dissociated
solution exerts the same osmotic pressure as is
produced by the entire molecule. Thus, at the
same molar concentrations, there would be
more molecules per liter in the electrolyte
solution than in the nonelectrolyte solution and
the solutions would exhibit different osmotic
pressures. The osmotic pressure exerted by
particles in a solution, whether they are
molecules or ions, is determined by the number
of particles per unit volume of fluid, not by the
mass of the particles. This is because each
particle in a solution exerts the same amount of
pressure against the membrane (Guyton &
Hall, 2006). In addition, depending on the
degree of dissociation of the particular
electrolyte in the solvent, different electrolyte
solutions can differ in the osmotic pressure
they exert (Abramoff and Thomson, 1982).
A coefficient called isotonic coefficient
(i) expresses the relationship of the electrolyte
and the nonelectrolyte. It can be computed
using the following equation:
(Equation 1)
Isotonic Coefficient
Hemolytic point of electrolyte (molar concentration)
= 10 ml of neutral red solution was added to 15
Hemolytic point of non electrolyte
This experiment aims to help the
researchers understand osmosis, the selective
permeability of the cell, and the factors that
determine it.
Materials and Methods
The red blood cell suspension used for
the hemolysis experiment was prepared by
mixing ten drops of blood from the fingertips
of the donor, and isotonic saline solution to a
test tube. Isotonic saline solution was
continuously added to the suspension until the
color of the suspension became reddish pink.
For the first part of the hemolysis
experiment, one test tube was filled with 5 ml
of distilled water, while another test tube was
filled with 5 ml of 0.2M NaCl. Each test tube
was added with five drops of the prepared red
blood cell suspension. The test tubes were then
mixed gently by inverting the test tube from
side to side, several times. The test tubes were
placed against a proper background and were
observed for hemolysis.
The last part of the hemolysis experiment
established the hemolyzing concentration of
glucose, NaCl, and KCl. 5 ml of each test
solution or reagents having different
concentrations were transferred to different test
tubes. Each test tube was added with five drops
of the prepared red blood cell suspension.
Hemolysis reading was done at 5-minute
intervals. The values obtained from the
hemolysis reading were used to calculate the
isotonic coefficients of the salts by using
Equation 1.
For the penetration of alkali experiment,
ml of yeast suspension. Color changes of the
mixture in a span of 5 min were noted. Each of
the four labeled test tubes (A, B, C and D) was
added with 2 ml of the prepared mixture of
neutral red solution and yeast suspension. 0.5
ml of 0.01 N NH4OH, 0.5 ml of 0.01N KOH,
0.5 ml of 0.01N NaOH and 0.5ml of distilled
water were added to test tubes A, B, C and D,
respectively. Each mixture was filtered. The
color of yeast cells and the filtrate was then
observed under the microscope.
Results and Discussion
In the first part of the experiment,
experimenters made red blood cell
suspensions. Making RBC suspension is
especially important for this experiment to
make sure that the cells are purified; so that no
plasma is attached to the cells.
Hemolysis of RBC (Red Blood Cells)
As RBC suspensions were placed in
different solutions with varying
 concentrations, the first setup to exhibit
hemolyzation was 0.02 M NaCl with a
duration of 4 minutes. The intracellular fluid
of erythrocytes is a solution of salts, glucose,
protein, and hemoglobin. According to
Koeppen & Stanton (2009), a saline solution
that is isotonic to red blood cells has a
concentration of 0.85% NaCl. This greatly
affected the rate of diffusion due to the
difference of concentration gradient of the
external and internal environment. The
relationship of the steepness or the difference
in the concentration gradient to the rate of
diffusion is that, the greater the difference
between concentrations of two sides the
higher the rate of diffusion (Tortora, 2009).
Moreover, this explains why the duration of
hemolysis of RBC also increases as the
concentration of NaCl increase (As seen in
table 1).
Table 1. Time of hemolyzation of RBC in different
solutions with varying concentrations; (-)
RBC did not hemolyze after 30 minutes
Reagent Duration of
Hemolyzation
0.9% NaCl 29 minutes
0.1 M NaCl 26 minutes
0.02 M NaCl 4 minutes
0.3 M Glucose - almost equal.
0.2 M Glucose - Furthermore, five milliliters of each of the
0.1 M Glucose - glucose with the following concentrations (0.3,
0.05 M KCl 23 minutes
0.1 M KCl 25 minutes
0.2 M KCl 22 minutes
In the 0.9% NaCl solution, it was
expected that the red blood cells will not
hemolyze, for as mentioned before, the
physiological salinity of plasma is 0.85%
NaCl, which is quite close, and was suppose
to provide an isotonic environment. However,
after 29 minutes the RBC in the solution
hemolyzed. This might be due the RBC itself,
since the blood suspensions added to the
solutions cleared up as soon as the test tubes
where it was placed were shaken. The
duration of the hemolyzation of the cells as
compared to other groups with different blood
source was unusually faster.
Low concentration of NaCl in the
extracellular environment induces hypotonic
condition to the RBCs. The 0.2 M NaCl
solution exhibited a turbid solution, which
suggests that the water is still flowing into the
cell and might or might not hemolyze through
time. Suspension in distilled water, on the
other hand, hemolyzed for the solution
became transparent as observed against a
white background. Since distilled water is
permeable, it does not contribute to osmotic
pressure (Guyton & Hall, 2006). Thus, a cell
will swell in distilled water and the plasma
membrane may rupture due to an excessive
internal pressure that is much higher than that
of the NaCl. The 0.1 M NaCl solution, as
expected hemolyzed but at a much slower
rate for the isosmotic concentration for NaCl
and plasma membrane is 0.154 M, which is
0.2, 0.1) and five milliliters of each of the
potassium chloride with varying
concentrations (0.05 M, 0.1 M, 0.2 M) were
placed into different test tubes and added with
five drops of the red blood cell suspension.
The table above shows the results in which the
blood hemolysed faster. Hypotonic solutions
are solutions in which their osmolarity is lower
than the suspended cells (Betteilheim, 1989).
In this set up, the Potassium chloride solution
is a hypotonic solution. Just like the sodium
chloride, potassium chloride is also an
electrolyte, which dissociates into K+ and Cl-
exerting the same osmotic pressure inside and
outside the cell resulting now to swelling. This
swelling results to the chloride interaction with
the potassium channels which further leads to
the opening of the channels allowing the entry
of the potassium ions into the cell. Hence, the
chloride anion can now be inferred to be the
reason of the potassium ion influx into the cell
which further swells, releasing hemoglobin
and dragging water into the cell leading to an
early hemolysis (Nepal, 2012). Additionally,
the higher the concentration of the potassium
chloride the faster will be the action of
hemolysis. In the results gathered above, it can
be inferred that a minimal error has occurred
into the setup since the 0.1 M concentration of
potassium chloride was the first one to
hemolyze instead of the solution with a 0.05
molar concentration. This may be due to the
chemicals used in the experiment and/or the
measurement of the required chemicals used
was not accurate.
The isotonic coefficient for NaCl and KCl
were computed and were found to be both 0.5.
The solutions can be seen below:
0.02 𝑀
Isotonic Coefficient of NaCl = = 0.5
0.04 M
So that in every 100 molecules of
glucose, 50 molecules of NaCl should be
present to maintain an isotonic environment
and prevent the bursting of the cell. Since,
NaCl will exert same osmotic pressure when
it dissociates into Na+ and Cl-.
0.05 M
Isotonic Coefficient KCl = = 0.5
0.1 M
Which also suggests that in every 100
molecules of glucose, 50 molecules of KCl
should be present.
During the exercise, the hemolytic point
for glucose was not observed since the time
given was limited for the hemolysis to occur
on the cells. Which is why, the hemolytic
point for non-electrolyte was just theoretical
in this paper. The concept of concentration of
a solution in terms of numbers of particles
was considered.
Lastly, five milliliters of glucose with
varying concentration (0.1 M, 0.2 M, and 0.3
M) were also added with the red blood cell
suspension into three different test tubes. The
table above showed that none of the three set
up hemolysed with the given time. Glucose is
an example of a non-penetrating
nonelectrolyte and glucose causes hemolysis
or the influx of water by osmosis this means
that glucose will be absorbed by the cell
resulting to the swelling of the cell. In the
results gathered, none of the set ups
hemolysed in the given time. The primary
reason would be the amount of the glucose
concentration in the solution is much smaller
than the concentration of glucose inside the
cell, since cells uses and takes in glucose for
the rigidity of the membrane to increase. In
addition to this, even if different
concentrations of nonelectrolyte were used in
this set up, it would still have the same molar
concentration. Therefore, it will result to the
same osmotic pressure, which would allow
the cell to swell, burst and further release its
hemoglobin into the solution in the long run
(Upal, 2016).
Penetration of Alkali different reagents. A factor that affects the
permeability of the cell membrane would
Table 2. Color Change in Neutral Red and Yeast be electrolytes. This is because the entry of
Suspension electrolytes into cells is generally found to
be slower than that of non-electrolytes of
Neutral Red and Yeast Suspension
comparable size (Wong, n.d.). Weak
Initial Color Color after 5 min
red red electrolytes, which dissociate into ions,
enter more rapidly than strong ones, and as
Table 2 shows the initial color and mentioned a while ago, the higher valency
color change after 5 minutes of the neutral of the ions, the slower its penetration
red and yeast suspension. The initial color (Wong, n.d.). Strong bases are strong
should have been yellow-orange and would electrolytes while weak bases are weak
later turn to red. It is possible that either electrolytes (Patrick, 2013). KOH and
one or both reagents were contaminated or NaOH are strong bases while NH4OH and
expired. The yellow-orange initial color water are weak bases. Since KOH and
would turn to red because neutral red NaOH were strong electrolytes, it resulted
diffuses into yeast cells and turns red since to slow penetration or transport of KOH or
the intracellular fluid is acidic (Flinn NAOH to the yeast cells because the color
Scientific, Inc., 2007). of the yeast cells remained to the neutral
red stain. As for NH4OH and water, they
Table 3. Color of Filtered Yeast Cells and Filtrate of were weak electrolytes and it resulted to a
the Neutral Red and Yeast Suspension fast penetration or transport of NH4OH or
water to the yeast cells because the color of
the yeast cells changed to yellow.
Test Reagent Color Color of
Tube Added of Filtrate
Yeast References
Cells
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NH4OH brown Movement of materials through cell
B 0.01 N Red- Mauve to membranes. Pages 109–121, in
KOH brown brown Laboratory outlines in biology. W. H.
C 0.01 N Red- Brown
Freeman, New York, 529 pages.
NaOH brown
Distilled Yellow- Fuchsia Bettelheim, F. A., March, J., &
D
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*Refer to Appendix for photos of the and Colloids. In Introduction to
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Appendix Test Tube D - Distilled water

Color of Yeast Cells in Test Tube A to D

Test Tube A - 0.01 N NH4OH

Test Tube B - 0.01 N KOH

Test Tube C - 0.01 N NaOH