Membrane Permeability in Red Beets

Fern Lawson

Lab Partner: Rachel Lusk

BIO 140 Lab, Sec. 001

Date Performed: February 10, 2009

Date Due: February 17, 2009

Introduction
In this experiment, the purpose was to test the permeability of the plasma

membranes of beet root cells under several different conditions. The first part of the

experiment involved testing the loss of the selective permeability of the plasma

membrane due to increasing temperature. Selective permeability occurs in the plasma

membrane because of the structure of the phospholipid bilayer with its hydrophobic

interactions. Hydrophobic interactions indicate the relationship of fatty acids and water,

where fatty acids act “scared” of water but in reality the two do not interact because fatty

acids are non-polar and water is polar. Another property of the plasma membrane

includes the channel and transport proteins that are incorporated both integrally and

peripherally on the cellular membrane. To test the selective permeability of the plasma

membrane, the heat stability of several cellular membranes of a beet were tested at

different temperatures. With increasing temperature, the structural integrity of the

cellular membrane is compromised because the proteins that make up the plasma

membrane are denatured and lose their shape. Secondly, the effects of temperature

extremes like extreme cold and heat were tested on the permeability of the beet root

membrane. Hypothetically, the extreme temperatures that the beet cell membranes were

put through should have caused denaturation of the proteins that make up the membrane

and allow the purple-red pigment bound within the plasma membrane to leak out of the

cell. Finally, the last portion of the experiment involved testing the effects of organic

solvents on the permeability of beet root membranes. An instability in the acidity or

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alkalinity as well as the concentration of the solutes in the external environment of the

beet cells can cause the breakdown of the cellular membrane components and thus the

release of the purple-red pigment from the cells. For each part of the experiment, the

ocular density of each sample of pigment that has leaked out of the beet root sample will

be quantified in a spectrophotometer.

Materials and Methods

The materials used in the experiment include a spectrophotometer, large red beet

roots, cork borer, test tubes, test tube rack, 1000-mL beaker, hot plate, thermometer, ring

stand and clamp, large forceps or dissection needle, colorimeter tubes, acetone, methanol,

toluene, single-edged razor blade, ruler, cutting board, and glass rod. The beet root

cylinders were prepared using the procedure in the Biology 140 lab manual (Schwarz 54).

When the beet root cylinders were washed, they were rinsed in cheese cloth until all of

the water ran clear and there was no red color. In Part I of the experiment (heat stability

of membranes), the procedure of steps one through nine in the lab manual were followed

(Schwarz 54). The temperature was noted, because at every ten degree decrease three

pieces of beet cylinder were placed in the water for one minute. Also, each beet cylinder

started incubation and the time was written down, so that their ocular density was

measured on time. The ocular densities or amount of pigment lost from the beet tissues

were recorded in the table provided and the averages of the ocular densities found versus

the heat treatment were graphed. In part two of the experiment, the procedural steps on

page 56 in the Schwarz lab manual were followed, and the times of the start of the

freezing of the beet cylinder and boiling were noted. Also, the times of their last

incubation time were noted so that the ocular densities were read on time. The ocular

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densities were recorded in the table provided. The last section of the lab involved using

several organic solvents so the fume hood was worked and gloves were worn when

handling the solvents. For this part of the experiment, the procedure in the lab manual

was followed (Schwarz 57). Also, the beet cylinder incubating in toluene was treated

differently then the other solvents, as noted in the lab manual. The data was recorded on

the table provided on page 57 in the lab manual.

Results
In this part of the experiment be sure to note that the 20 degrees Celsius is the control

temperature for this section.

Part I: Heat Stability of Membranes

Sample
Heat Treatment Sample #1 Sample #2 Sample #3 Average
in degree C
20 0.05 0.02 0.06 0.04
80 0.85 0.75 0.6 0.73
70 0.5 1 0.3 0.6
60 0.28 0.35 0.42 0.35
50 0.25 0.17 0.08 0.17
40 0.03 0.05 0.05 0.04

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 Ocular Density vs. Temperature

0.8

0.6

0.4
O.D. (475 nm)

0.2

0
20 30 40 50 60 70 80 90
Temperature (degree C)

Part II: Temperature Extremes

O.D. at 475
Treatment nm
Frozen and thawed 0.45
0.5
0.49
Treatment Average 0.48

Boiled 0.85
0.5
0.26
Treatment Average 0.54

Untreated Control 0.05

0.02
0.06
Treatment Average 0.04

Part III: Organic Solvent

Effects
Solvent O.D. at 475
Treatment nm
Distilled Water 0.05
0.07

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 0.11
Treatment
Average 0.08

Acetone 0.13
0.52
0.52
Treatment
Average 0.39

Toluene 1
0.5
0.32
Treatment
Average 0.61

Methanol 0.8
0.8
0.5
Treatment
Average 0.7

The first table and graph demonstrate the results obtained from part one of the

experiment. The graph plots the average amount of pigment lost from the beet tissue as

read by the spectrophotometer versus the temperature at which the beet cylinders were

submerged. The second table contains the data collected from the ocular density readings

involving membrane permeability and extreme temperatures. Also, the averages of these

ocular densities were recorded. Lastly, the third table includes the ocular density

readings collected and the averages of these when the beet cylinders were submerged and

incubated in several organic solvents.

Discussion
Permeability of the cellular membranes are regulated through several factors that

help maintain the homeostasis of the cell. Concentration and electrochemical gradients

of ions inside and outside of the cell, the temperature of the solution, the size and

structure of molecules trying to enter the cell, and the pH of the internal and external

environment of the cell all determine what enters and exits the cell membrane (Sadava

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105-108). The purpose of this experiment was to examine the permeability of the plasma

membranes of beet root cells by testing the durability of the membrane at different

temperature variables, in extreme temperatures, and when exposed to organic solvents.

Results from part one of the experiment reveal that at a higher more stressful temperature

of 80°C, the membrane of the beet root cells allows more of the purple-red pigment,

called betacyanin, to leak out of the cells into the water medium surrounding it. At a

control temperature of about 20°C the amount of pigment that leaked out of the cell was

minimal to none. The amount of betacyanin that leaked out of the cell membranes was

calculated by finding the ocular density of the solution in which the beet roots were

incubated using a spectrophotmeter. This ocular density depicted the concentration of the

pigment in the solution, which relates to the amount of cellular membrane breakdown in

the beet root cells. More membrane breakdown occurs at higher temperatures because

the proteins and lipids that make up the membrane are damaged by the extreme heat.

This damage compromises the membrane’s ability to selectively retain, absorb, and

excrete molecules.

In part two of the experiment, the stability of the beet root membranes at extreme

temperatures was tested by placing beet cylinders in extreme cold and extreme heat. The

beet cylinder that was placed in the freezer and then thawed, leaked out a substantial

amount of betacyanin pigment into the water solution in which it was incubated. The

beet cylinder placed in the boiling water for ten minutes also leaked a large amount of

pigment into the water solution. When a cell membrane is frozen, ice crystals form in the

cell cytoplasm and its membrane. Water expands when it freezes, so when the internal

water of the cell freezes it can harm the internal organelles of the cell and puts pressure

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on the plasma membrane. This pressure can cause the plasma membrane to burst or be

damaged, so that when the cells are thawed and put into water, betacyanin pigment is

released from the cell into the water. This increase in pigment concentration, increases

the reading of the ocular density. When the beet root is subjected to the extreme heat of

boiling water, the plasma membranes of the cells are damaged. The heat can denature the

proteins that make up the membrane and the dye of the beet root diffuses out of the cell

into the water.

Lastly, the effect of organic solvents on the plasma membranes of beet root cells

was tested using distilled water, acetone, toluene, and methanol. Water is a polar

molecule and because the membrane of a cell is made up of mainly nonpolar

phospholipids, water can’t penetrate the cell as well as a nonpolar substance. The

nonpolar hydrophobic tails repel the water and thus less pigment is leaked out of the cells

into the water. This is the reason for the low ocular density reading of 0.08. The next

solvent used was acetone, which is a substance that contains both polar and nonpolar

characteristics. With its nonpolar characteristics of the two methyl side groups that make

up acetone, it can some what permeate the cell membrane pulling out some pigment.

However, because acetone is also polar, it will not leak out as much pigment as a uniform

nonpolar molecule like toluene. Toluene is a nonpolar molecule and can easily penetrate

the membrane of the beet root cell, which releases a high amount of pigment into the

solution. This high concentration of pigment causes an increase in the ocular density

measurement of 0.61. Methanol was the last solvent used and is a highly polar molecule.

The membrane of the cell was disrupted by using the polar solvent, which acts almost

like a detergent by causing the pigment of the beet to leak out into the solution due to a

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damaged cell membrane giving it a high ocular density reading of 0.70.

In performing the experiment, it was seen that extreme cold and heat, different

organic solvents, and rising temperatures compromised the plasma membrane’s ability to

selectively allow certain materials to enter and exit its surface.

References
Schwarz, Otto J. Introduction to Basic Laboratory Skills for the Biological Sciences-An
Experimental Approach for Biology 140 Organization and Function of the Cell.
3rd Ed. Oak Ridge: Performance Press, 2000. p. 50-58.

Sadava, David. Life the Science of Biology. 8th Ed. Sunderland: Sinauer Associates,
Inc., 2008. p. 105-108.