Fahim Khan

02: Cellular Effects of Hypertonic Saline Solutions

Purpose
Cells respond to their external environment - in fact cells live and die
according to their environment. This seems obvious, but it becomes
more difficult to imagine the specific relationships between the cell and
its environment. This lab explores simple osmotic interactions, and the
resulting cell damage, between beet cells and increasingly hypertonic
saline solutions. As we will see, the color intensity of the solutions is a
function of the rate of osmosis occurring since the water-soluble red
pigments in the beet cells diffuse accordingly.

Background
We all know from biology class that cells have plasma membranes that
are semi-permeable i.e. allow certain substances through while leaving
out others. For example, water (effectively) flows freely in and out of
cells, as do substances that dissolve in water. Now, we also all know
from chemistry class that osmosis is a natural phenomenon that
maintains the isotonicity (balance of solutes) of an environment
through diffusion of water until equilibrium is reached. Interpolating
these pieces of information, we can explain what will happen if a cell is
placed in a solution that contains a higher concentration of solutes
than the interior of the cell (or a tonoplast vacuole, as it may be and is
in this experiment). If the external solutes cannot diffuse into the cell
to achieve isotonicity, then osmosis will occur with the water in the
vacuole exiting until a solute concentration balance is reached
(hopefully before the cell dies of excessive dehydration). If we have
some way of observing this process, we can measure it – fortunately
we do have that ability, by the virtue of a visual indicator in the form of
a water-soluble pigment. The pigment is betacyanin (it’s red, but
shouldn’t it be blue?), and it is what gives beets their color. Being
water-soluble, it is transported out of the tonoplasts during osmosis in
a hypertonic environment and the amount that is transported out
logically coincides with the rate of osmosis – so, predictably, the
reddest solutions should be the most hypertonic ones. The reddest
solutions also indicate the most damaged cells, because these
solutions contain the highest osmotic pressures (pressure exerted upon
the cell structures by the flow of water from low to high solute
concentration areas), which damages the cellular water channels. To
exactly measure “redness” a colorimeter will be used. A colorimeter is
an instrument that passes light of a precise wavelength (470 nm blue
in this case) through a cuvette containing a solution (we will be going
through six different concentrations here) and strike a photocell that
can measure tiny changes in the color of the original light to determine
the amount of absorption by the solution. A clear solution results in
next to no absorption while a completely dark solution will fully or
almost fully absorb the light.

Procedure
Having six test tubes with differing solution concentrations of salt and
water (0%, 3%, 6%, 9%, 12%, 15% salt), twelve cleaned beet squares
(two per tube), and only fifteen minutes to mix the beets into the
solutions, we set about the experiment. While the time was counting
down, we set up the colorimeter per specifications (eventually). When
the time ran out, we poured the six solutions into six cuvettes and put
them all one by one into the colorimeter, writing down our
measurements. We then cleaned up.

Data
%
Beak sal Absorban
er t ce
1 0 0.475
2 3 0.467
3 6 0.475
4 9 0.503
5 12 0.405
6 15 0.5
 Absorbance vs. % salt

0.6

0.503 0.5
0.5 0.475 0.467 0.475

0.405
0.4

0.3

Absorbance
0.2

0.1

0
0 3 6 9 12 15
% salt

Results
We had predicted that there was a linear correlation between light
absorbance/osmosis rate and the degree of hypertonicity of the
solution. However, the results (more or less stable absorbance values)
did not fit the theory so we can’t really know what to make of the data.
While we could just replace our theory with one that works, this would
be a bit presumptuous as the likelihood of the experiment having
somehow been botched is higher than the likelihood of the theory of
electromagnetism and the laws of thermodynamics being wrong. So,
possible mistakes roughly in order of decreasing probability are (1) not
having properly cleaned beets or cuvettes or maybe miscalibrated
colorimeter, (2) having inconsistent solution volumes among test
tubes, (3) the beets having been already damaged, (4) instrument
malfunction and (5) incorrectly prepared solution concentrations (this
is least likely because I trust Mr. Randolph – and notice that I trust him
more than any mere machine, near-infallible though they are).

Conclusion
The experiment, with regards to proving the hypothesis, was
inconclusive. Despite that, I thought the experiment was educational,
as far as serving as a refresher of 10th grade Chemistry, and enjoyable,
as far as lab experiments go. For a moment, I wondered over the
results whether the connection between saline concentration and
osmosis rate was not simply linear, but instead made complicated by
interfering cellular reactions (like optimal operating concentrations for
the water channels or something). But a moment later, I realized it was
a stupid idea.

Questions
1. Which solution of salt produced the most intensely red solution? The
least?
Well, the 15% salt solution should have been the most red, and
the 0% the least red because higher solute concentration means
higher osmosis rate and pressure. The results did not show that, and
while the solutions seemed to all look the same to me, the colorimeter
showed minute differences – 12% showed lowest absorption and 9%
showed highest absorption at 0.405 and 0.503 respectively.
2. Which salt concentration(s) had the least effect on the beat
membrane? Why?
Using the data, I would say the answer is the 12% solution
because it was the least intense meaning the least amount of
betacyanin was released as a result of osmosis. However, if the reason
really was osmosis, then logically the 0% solution should show least
amount of betacyanin since no osmosis as a result of hypertonicity
would occur.
3. Did more damage occur at high or low salt concentrations? Explain
why this might be.
More damage occurred at low and high concentrations according
to the data. More damage should occur at high concentrations since
osmotic pressure and rate is highest in these solutions, with the most
amount of force being applied on the water channels and most amount
of dehydration.
4. An effective way to kill a plant is to pour salt onto the ground where
it grows. How might the salt prevent the plants growth? Is this
consistent with your data?
The osmosis explanation is that the roots absorb less water (and
possibly lose water) because of the osmotic potential between the
soil’s saline solution and the plant’s “pure” water. If the roots absorb
less water, then simply put there is less to go around for the metabolic
processes required for survival and growth. To beat a dead horse for a
fourth time, no, this explanation is not consistent with the data.