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 er 1 2 3 4 5 6 % sal t 0 3 6 9 12 15 Absorban ce 0.475 0.467 0.475 0.503 0.405 0.5

Absorbance vs. % salt
0.6 0.5 0.4 0.3 Absorbance 0.2 0.1 0 0 3 6 % salt 9 12 15 0.475 0.467 0.475 0.503 0.405 0.5

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.

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