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The Effect of Temperature and Solvent (Acetone and Methanol) on Beet Tissue

Sarah Dover

BIOL 202 - A02: Principles of Cell and Molecular Biology

Dr. Sarah Emel

October 26, 2021

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Abstract

A lab was conducted, in which the effect of temperature and solvent type and

concentration (1%, 25%, and 50% acetone or methanol) on the cell membranes of beet tissue

was examined. This entailed exposing beet tissue to various temperatures (-20℃, 1℃, 25℃,

38℃, 52℃, and 72℃) or solvent solutions for 20 minutes and obtaining the absorbance of the

solution the beet tissue was held in to examine the intensity of the disruption. It was found that

temperature deviating from room temperature (25℃) caused disruption, but warmer

temperatures appeared to cause more damage. Additionally, the methanol and acetone solutions

both indicated more beet tissue damage as their concentration increased. However, out of the

three examinations, only the acetone solution was shown to be statistically significant.

Introduction

Every cell on this planet has one goal in mind: to attain equilibrium. All of these cells

enjoy being at this stage by remaining at a certain temperature, pH, etc. Deviating from such

conditions can lead to poor outcomes, such as cell damage or death. As Ebrahimi and colleagues

(2018) found in their research article, small shocks of heat have been shown to affect the cell

membrane of samples of ​Salmonella enterica serovar Typhimurium, leading to the leakage of

intracellular components found within the cytoplasm. Once a certain intensity of the heat applied

to the cell is reached, the cells will die, due (in part) to weakened interactions among lipids

within the envelope of the cell (Ebrahimi et al., 2018).

Joo et al. (2012) found that methanol can cause quite an uproar when interacting with the

cell membrane, as they noticed that methanol interacts with the lipids in the membrane, altering

its fluidity and causing certain proteins associated with the membrane to change. Another study

found that acetone will also affect the fluidity of the membrane (Posokhov & Kyrychenko,
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2013). It will do so by creating a gap between the phospholipid heads and reducing the density of

the tail region (Posokhov & Kyrychenko, 2013). With all of the articles in mind, it is

hypothesized that the most extreme of the temperatures (72℃) and solvent treatments (50%

acetone and 50% methanol) will elicit the greatest disruption of the beet tissue.

Methods

The steps taken to obtain the data were derived from the lab manual provided to all of the

students in the Biology 202 lab. This lab was conducted within two lab periods, which were one

week apart. During the first week, six 4 centimeter long cylinders were cut from a beet root with

a cork borer. Although the manual instructed to, the beet cylinders were not rinsed with sucrose

solution, but they were dried with a paper towel. Each cylinder was then placed into a test tube,

along with 10 milliliters of 0.5 M sucrose solution. This solution was created by combining 20.5

g of sugar with 120 milliliters of distilled water.

Then, each test tube was placed in one of six different liquid temperature environments

for 20 minutes: -20℃, 1℃, 25℃, 38℃, 52℃ (which measured 55℃ upon retrieval), and 72℃

(measured 71℃ upon retrieval). While the test tube held at -20℃ was placed in an antifreeze

bath, all of the other test tubes were placed in a water bath. Once the time ended, the test tubes

were collected, the beet cylinders were removed, and approximately 2 mL of each of the

solutions left in the test tubes were placed into cuvettes to be measured in the mass

spectrophotometer at 550 nm. It was important to blank the machine before the absorbance

values were obtained. In this case, the solution was the same, so only one blank was required.

The temperatures were considered the independent variables, while the milligrams of disrupted

beet tissue were considered the dependent variable.

During the second week of the experiment, another seven cylinders were created from a
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beet root via a cork borer. They were cut to be 7 cm long and were rolled along a paper towel to

be dried. Each beet cylinder was placed into a test tube that contained a different type of solvent

solution for 20 minutes. One contained an isotonic solution of 0.5 M sucrose (which was made

by adding 68.5 grams of sugar to 400 mL of distilled water). This solution could be considered

the control of the experiment, as it does not contain either of the solvents. The remaining test

tubes contained some percentage of acetone or methanol combined with the sucrose solution.

Two test tubes contained 1% solvent solution, which was created by combining 1 mL of the

solvent (one had acetone, while the other methanol) and 99 mL of the sucrose solution. Another

two test tubes contained 25% solvent solution. This was created by combining 12.5 mL of the

solvent (acetone or methanol) with 37.5 mL of the sucrose solution. Additionally, the last two

test tubes were made to be 50% solvent solutions, with 10 mL of the solvent (acetone or

methanol) and 10 mL of the sucrose solution. While the type/concentration of solvent solution is

considered the independent variable, the amount of disrupted beet tissue is considered the

dependent variable.

Upon completion of the 20 minutes, the beet tissue was removed and about 2 mL of each

of the solutions from the test tubes was placed into a cuvette. Before the absorbance was

obtained at 550 nm, a blank cuvette needed to be placed into the mass spectrophotometer. The

cuvette contained the solvent treatment without any beet particles in it. Once the blank was

placed, the absorbance value for all of the test tubes was able to be obtained and recorded.

Any obtained absorbance values that measured beyond 1.0 within any section of the

experiment needed to be diluted to be within the range of 0.1-1.0. Only one of the samples

exhibited an initial absorbance value that was greater than 1.0 (the sample from the first part of

the lab, which was stored at 70℃). In order to make the absorbance value fall back within the
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desired range, the sample was diluted by a dilution factor of 10.

In a previous lab, the lab group obtained the absorbance values at varying beet

concentrations, which were then documented in a graph with a best-fit line. The absorbance

values from this lab were able to be compared to the graph that was generated to estimate the

concentrations of milligrams/milliliter (mg/mL) of beet tissue within the samples. Converting

from mg/mL to milligrams entailed multiplying the concentration by the dilution factor (if any

was needed) and then multiplied by 10.

Each lab group in both sections of the Biology 202 lab classes documented their own

calculations on a spreadsheet so the overall average could be obtained. ANOVA tests on these

average values were performed utilizing Excel in order to determine if temperature and/or the

type of solution (acetone or methanol) indicated a significant relationship within the experiment.

Results

The values within this section reflect the average values obtained from each of the

student lab groups' documented data from both sections of the lab. The ANOVA values for

temperature and methanol solvent solution displayed p-values of 0.19375 and 0.10475

respectively. Meanwhile, the ANOVA regarding the results from acetone solvent solution

showed a p-value of 0.039898.

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Figure 1. The effects of temperature on disruption of beet tissue. The green bars indicate the

resulting concentration of beet tissue disrupted by the temperature, while the thin black bars are

the error bars.

As observed in Figure 1, the temperature of 0℃ seemed to account for the least amount

of disrupted tissue. The amount of tissue disrupted appeared to increase the farther the

temperature extended from 0℃, with 70℃ exhibiting the largest concentration of beet tissue

disrupted.
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Figure 2. The effect of solvents on disruption of beet tissue. The green bars indicate the

milligrams of beet tissue disrupted due to the concentration of solvent (acetone or methanol),

while the thin black bars are the error bars.

In comparison to the isotonic solution, it appears as though the 1% acetone solution

yielded a smaller amount of disrupted beet tissue. The solutions with a greater percentage of

acetone resulted in a larger disruption in the beet tissue. Meanwhile, all of the methanol solutions

resulted in tissue disruption greater than the amount of disruption observed within the isotonic

solution.

Discussion

The more the beet cells were damaged, the more they released betacyanin, which further

intensified the reddish/pink color of the solution that the beet cylinders were in. Based on the

section of the experiment that examined the effect of temperature, it appeared as though both

extremes (hot and cold) exhibit beet tissue disruption, but greater temperatures caused a larger

effect on the disruption of beet tissue. This may likely be due to higher kinetic energy causing

damage at a faster rate, as the greater kinetic energy would lead to the cellular components

moving faster more imminently than it would take for ice to form and wreak havoc on the cell by

expanding, causing the cell to burst, or becoming jagged, puncturing the cell membranes.

However, the p-value from this section of the experiment was 0.19375, which is greater than

0.05, indicating that these results show no significant effect. As a result, the statistics do not

suggest such a relationship, so the hypothesis is unable to be supported based on the data that

was collected from this experiment.

Similarly, the second week’s experiment with the methanol solvent solution also resulted

in an insignificant effect on the disruption of beet tissue with a p-value of 0.10475. Meanwhile,
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part of the second part of this section of the experiment suggested a significant effect, as its

p-value was calculated to be 0.039898, which is less than 0.05. As observed in figure 2, it

appeared as though the concentration of solvent within the solution was directly related to the

amount of disrupted beet tissue. Based on the statistics, the hypothesis is only supported in

regards to the acetone solution being able to disrupt the beet tissue.

Although this experiment was simple within its scope, the concept of the lab can reach

further than just beets, as cell membranes are found on any living cell. The purpose of cell

denaturation by temperature or even solvent exposure could lead to interesting, and even

concerning findings. For instance, Sonmez and colleagues (2013) found that the addition of

alcohols, including methanol, can lead to severe disruptions in the various functions and the

structure of red blood cells.

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References

Ebrahimi, A., Csonka, L.N., & Alam, M.A. (2018). Analyzing thermal stability of cell membrane

of Salmonella using time-multiplexed impedance sensing. Biophysical Journal, 114,

609-618. https://doi.org/10.1016/j.bpj.2017.10.032

Joo, H.J., Ahn, S.H., Lee, H.R., Jung, S.W., Choi, C.W., Kim, M.S., Bae, M.K., Chung, I.K.,

Bae, S.K., Jang, J.O., & Yun, I. (2012). The effect of methanol on the structural

parameters of neuronal membrane lipid bilayers. The Korean Journal of Physiology &

Pharmacology, 16(4), 255-264. https://doi.org/10.4196/kjpp.2012.16.4.255

Posokhov, Y.O. & Kyrychenko, A. (2013). Effect of acetone accumulation on structure and

dynamics of lipid membranes studied by molecular dynamics simulations. Computational

Biology & Chemistry, 46, 23-31. http://dx.doi.org/10.1016/j.compbiolchem.2013.04.005

Sonmez, M., Ince, H.Y., Yalcin, O., Ajdžanović, V., Spasojević, I., Meiselman, H.J., & Baskurt,

O.K. (2013). The effect of alcohols on red blood cell mechanical properties and

membrane fluidity depends on their molecular size. Plos One, 8(9).

https://doi.org/10.1371/journal.pone.0076579