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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 is primarily composed of phospholipids,
cholesterol and proteins that move freely in the
Penetration of different substances into plane of the membrane. The phospholipid is
the cell is one of the most difficult problems generally composed of a hydrophobic tail and a
that a cell can encounter. As an answer to this, hydrophilic head (Khan Academy, 2015). The
a cellular structure, which is the plasma protein component of the membrane aids for
membrane, controls the entry and exit of the transport some nutrients against the
certain chemicals and substances into the cell. concentration gradient, which requires
The plasma membrane not only defines the additional energy. Lastly, cholesterol is tucked
border of the cell but also interacts with its between the hydrophobic tails of the membrane
environment in a controlled way. One of the phospholipids providing additional flexibility
major roles of the plasma membrane is to of the membrane. All of these work together
regulate materials into the cell and from the for the membrane to keep the inside and
cell to its environment. In order to carry out outside of the cell regulated. The permeability
this responsibility, the membrane is composed of the plasma membrane allows the cell to
of phospholipid bilayer. The main model in reach equilibrium and is also one of the reasons
understanding the structure of the plasma why difference in concentration occurs, which
membrane is called the fluid mosaic model in turn produces hemolysis. Osmosis in a
(Khan Academy, 2015). hypotonic solution causes the hemolysis of
cells. Also, the blood tonicity of the difference
in the concentration of substances inside and Penetration of alkali simply refers to the
outside the cell determines whether the cell entry of dissociated alkali metals, and/or
will shrink, swell or remain its normal size. In ammonium into the cell by diffusion (Brooks
addition, another factor that affects the rate of 1923). Alkali penetration is dependent on their
diffusion is the temperature. Higher degree of dissociation, since only ionized form
temperature increases the rate of diffusion of the alkali are transported through the
across a membrane. The mass or the size of the membrane, may it be passive or active
diffusing particle also affects rate of diffusion (Burggren 2002). Ionization degrees are higher
in the sense that as the mass becomes larger, with strong acids and bases and lower with the
the diffusion rate becomes slower. For a weak ones (Campbell & Farrell 2013).
smaller particle can move faster than a larger Penetration of alkali is commonly observed
particle. Furthermore, larger membrane surface with the use of dyes, like the neutral red dye,
area available for diffusion facilitates a faster which stains the golgi apparatus red, to stain
diffusion rate and a greater distance over which cells, and subjecting the set-ups to alkali
diffusion must take place results in a slower compounds, then observing the color changes
diffusion rate. before and after dissociation by the liquid.
There are several factors affecting the In the experiment alkali metals such as
permeability of the plasma membrane such as sodium and potassium were present in the form
the temperature, chemicals, size of the of NaCl and KCl. Sodium Chloride (NaCl) and
molecule passing across it and its nature. The Potassium Chloride (KCl) are both solutions
cell membrane is known to be selective in that are classified as non-penetrating
terms of allowing substances to pass through it, electrolytes. Both solutions cause hemolysis at
but with certain chemicals, it is possible that lower molar concentrations than the non-
the membrane will be destroyed together with penetrating nonelectrolyte, which are glucose
its selectivity. This also applies to temperature. and sucrose, to name a few, that cause
The size of the molecule passing through the hemolysis at the same molar concentrations.
membrane is also a factor that affects its This is because an electrolyte can dissociate
permeability. The larger the molecule the into two ions, and every ion in the dissociated
harder will it pass through the membrane, solution exerts the same osmotic pressure as is
unless it undergoes the process of endocytosis. produced by the entire molecule. Thus, at the
The nature of the molecule is also a big factor same molar concentrations, there would be
that affects the permeability of the cell more molecules per liter in the electrolyte
membrane. Water soluble and fat-soluble solution than in the nonelectrolyte solution and
substances can easily pass through the the solutions would exhibit different osmotic
membrane but substances, which are pressures. The osmotic pressure exerted by
electrolytes in nature, would be slower than the particles in a solution, whether they are
non-electrolytes due to the fact that electrolytes molecules or ions, is determined by the number
with high valency cannot penetrate the of particles per unit volume of fluid, not by the
membrane easily (Wong, nd). mass of the particles. This is because each
particle in a solution exerts the same amount of
pressure against the membrane (Guyton & The last part of the hemolysis experiment
Hall, 2006). In addition, depending on the established the hemolyzing concentration of
degree of dissociation of the particular glucose, NaCl, and KCl. 5 ml of each test
electrolyte in the solvent, different electrolyte solution or reagents having different
solutions can differ in the osmotic pressure concentrations were transferred to different test
they exert (Abramoff and Thomson, 1982). tubes. Each test tube was added with five drops
A coefficient called isotonic coefficient of the prepared red blood cell suspension.
(i) expresses the relationship of the electrolyte Hemolysis reading was done at 5-minute
and the nonelectrolyte. It can be computed intervals. The values obtained from the
using the following equation: hemolysis reading were used to calculate the
isotonic coefficients of the salts by using
(Equation 1) Equation 1.

Isotonic Coefficient
Hemolytic point of electrolyte (molar concentration)
For the penetration of alkali experiment,
= 10 ml of neutral red solution was added to 15
Hemolytic point of non electrolyte
ml of yeast suspension. Color changes of the
This experiment aims to help the mixture in a span of 5 min were noted. Each of
researchers understand osmosis, the selective the four labeled test tubes (A, B, C and D) was
permeability of the cell, and the factors that added with 2 ml of the prepared mixture of
determine it. neutral red solution and yeast suspension. 0.5
ml of 0.01 N NH4OH, 0.5 ml of 0.01N KOH,
Materials and Methods 0.5 ml of 0.01N NaOH and 0.5ml of distilled
water were added to test tubes A, B, C and D,
The red blood cell suspension used for respectively. Each mixture was filtered. The
the hemolysis experiment was prepared by color of yeast cells and the filtrate was then
mixing ten drops of blood from the fingertips observed under the microscope.
of the donor, and isotonic saline solution to a
test tube. Isotonic saline solution was Results and Discussion
continuously added to the suspension until the
color of the suspension became reddish pink. In the first part of the experiment,
For the first part of the hemolysis experimenters made red blood cell
experiment, one test tube was filled with 5 ml suspensions. Making RBC suspension is
of distilled water, while another test tube was especially important for this experiment to
filled with 5 ml of 0.2M NaCl. Each test tube make sure that the cells are purified; so that no
was added with five drops of the prepared red plasma is attached to the cells.
blood cell suspension. The test tubes were then
mixed gently by inverting the test tube from Hemolysis of RBC (Red Blood Cells)
side to side, several times. The test tubes were
placed against a proper background and were As RBC suspensions were placed in
observed for hemolysis. different solutions with varying
concentrations, the first setup to exhibit physiological salinity of plasma is 0.85%
hemolyzation was 0.02 M NaCl with a NaCl, which is quite close, and was suppose
duration of 4 minutes. The intracellular fluid to provide an isotonic environment. However,
of erythrocytes is a solution of salts, glucose, after 29 minutes the RBC in the solution
protein, and hemoglobin. According to hemolyzed. This might be due the RBC itself,
Koeppen & Stanton (2009), a saline solution since the blood suspensions added to the
that is isotonic to red blood cells has a solutions cleared up as soon as the test tubes
concentration of 0.85% NaCl. This greatly where it was placed were shaken. The
affected the rate of diffusion due to the duration of the hemolyzation of the cells as
difference of concentration gradient of the compared to other groups with different blood
external and internal environment. The source was unusually faster.
relationship of the steepness or the difference Low concentration of NaCl in the
in the concentration gradient to the rate of extracellular environment induces hypotonic
diffusion is that, the greater the difference condition to the RBCs. The 0.2 M NaCl
between concentrations of two sides the solution exhibited a turbid solution, which
higher the rate of diffusion (Tortora, 2009). suggests that the water is still flowing into the
Moreover, this explains why the duration of cell and might or might not hemolyze through
hemolysis of RBC also increases as the time. Suspension in distilled water, on the
concentration of NaCl increase (As seen in other hand, hemolyzed for the solution
table 1). became transparent as observed against a
white background. Since distilled water is
Table 1. Time of hemolyzation of RBC in different permeable, it does not contribute to osmotic
solutions with varying concentrations; (-) pressure (Guyton & Hall, 2006). Thus, a cell
RBC did not hemolyze after 30 minutes will swell in distilled water and the plasma
membrane may rupture due to an excessive
Reagent Duration of internal pressure that is much higher than that
Hemolyzation of the NaCl. The 0.1 M NaCl solution, as
0.9% NaCl 29 minutes expected hemolyzed but at a much slower
0.1 M NaCl 26 minutes rate for the isosmotic concentration for NaCl
0.02 M NaCl 4 minutes and plasma membrane is 0.154 M, which is
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 0.2, 0.1) and five milliliters of each of the
0.1 M KCl 25 minutes potassium chloride with varying
0.2 M KCl 22 minutes concentrations (0.05 M, 0.1 M, 0.2 M) were
placed into different test tubes and added with
In the 0.9% NaCl solution, it was five drops of the red blood cell suspension.
expected that the red blood cells will not The table above shows the results in which the
hemolyze, for as mentioned before, the blood hemolysed faster. Hypotonic solutions
are solutions in which their osmolarity is lower
than the suspended cells (Betteilheim, 1989). 0.05 M
Isotonic Coefficient KCl = = 0.5
In this set up, the Potassium chloride solution 0.1 M
is a hypotonic solution. Just like the sodium
chloride, potassium chloride is also an Which also suggests that in every 100
electrolyte, which dissociates into K+ and Cl- molecules of glucose, 50 molecules of KCl
exerting the same osmotic pressure inside and should be present.
outside the cell resulting now to swelling. This During the exercise, the hemolytic point
swelling results to the chloride interaction with for glucose was not observed since the time
the potassium channels which further leads to given was limited for the hemolysis to occur
the opening of the channels allowing the entry on the cells. Which is why, the hemolytic
of the potassium ions into the cell. Hence, the point for non-electrolyte was just theoretical
chloride anion can now be inferred to be the in this paper. The concept of concentration of
reason of the potassium ion influx into the cell a solution in terms of numbers of particles
which further swells, releasing hemoglobin was considered.
and dragging water into the cell leading to an Lastly, five milliliters of glucose with
early hemolysis (Nepal, 2012). Additionally, varying concentration (0.1 M, 0.2 M, and 0.3
the higher the concentration of the potassium M) were also added with the red blood cell
chloride the faster will be the action of suspension into three different test tubes. The
hemolysis. In the results gathered above, it can table above showed that none of the three set
be inferred that a minimal error has occurred up hemolysed with the given time. Glucose is
into the setup since the 0.1 M concentration of an example of a non-penetrating
potassium chloride was the first one to nonelectrolyte and glucose causes hemolysis
hemolyze instead of the solution with a 0.05 or the influx of water by osmosis this means
molar concentration. This may be due to the that glucose will be absorbed by the cell
chemicals used in the experiment and/or the resulting to the swelling of the cell. In the
measurement of the required chemicals used results gathered, none of the set ups
was not accurate. hemolysed in the given time. The primary
The isotonic coefficient for NaCl and KCl reason would be the amount of the glucose
were computed and were found to be both 0.5. concentration in the solution is much smaller
The solutions can be seen below: than the concentration of glucose inside the
cell, since cells uses and takes in glucose for
0.02 𝑀 the rigidity of the membrane to increase. In
Isotonic Coefficient of NaCl = = 0.5 addition to this, even if different
0.04 M
concentrations of nonelectrolyte were used in
So that in every 100 molecules of this set up, it would still have the same molar
glucose, 50 molecules of NaCl should be concentration. Therefore, it will result to the
present to maintain an isotonic environment same osmotic pressure, which would allow
and prevent the bursting of the cell. Since, the cell to swell, burst and further release its
NaCl will exert same osmotic pressure when hemoglobin into the solution in the long run
it dissociates into Na+ and Cl-. (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
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NH4OH brown Movement of materials through cell
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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