Gary Salonga

LSG15

ASMS
Stage 2 Biology

THE EFFECT OF
TEMPERATURE ON
MEMBRANE PERMEABILITY
IN BEETROOT CELLS
Gary Salonga
SACE No. 768456R
Biology | Penny Collins

Gary Salonga

LSG15

Abstract
The purpose of this experiment was to investigate the effects of temperature on
the plant cell membrane from beetroot. This was done by using beetroot and
placing them into different temperatures of water 30, 40, 50, 60, 70 and 80 oC.
The excess pigment was washed off before hand and discs of beetroot were
impaled onto skewers. The skewers were placed into separate test tubes of
varying temperatures of water and letting them soak. The water sample of the
test tubes were then taken out, placed into cuvettes and then placed into the
spectrophotometer. The absorbency reading was then recorded. It was supported
by the results that the water from higher temperatures would have a higher
absorbency reading.
Aim
To investigate the effects of temperature on the plant cell membrane from
beetroot.
Introduction
The cell membrane is composed primarily of phospholipids and proteins, these
are typically described as phospholipid bi-layers. Lysis refers to the breaking
down of the membrane of a cell. When the membrane is destroyed, the cellular
contents leak out and release any liquid held inside. There are several factors
that can denature the membrane, for example - heat denaturation, electric
shocks, pH factors and salinity. By cutting the cells, the cell membranes are
mechanically torn. The higher temperatures used in this experiment disrupt the
structure of proteins and certain chemicals like fat solvents dissolve the
phospholipids, leading to damage of the membrane.
Beetroots release a strong red pigment when the membrane is denatured. In this
experiment, the beetroot membrane was denatured using different temperatures
of water. The red pigment in beetroot is called betacyanin, which is located in the
large central vacuole of the beetroot cells. The betacyanin will leak out when the
membranes are damaged. In this case, the beetroot produces a red or dark pink
colour. When the cell membrane is damaged, the diffusion of betacyanin occurs
through the semi-permeable membrane by the act of osmosis. The betacyanin
molecules move from a higher concentrated area to a lower concentration
(123helpme.com, 2015).
Hypothesis
If the beetroot slices are placed into a higher temperature water, then it will leak
more colour and produce a higher absorbance.
Independent Variable
The independent variable is the different temperatures of water.
Dependent Variable
The dependent variable is the figure of absorbance that is produced from the
water where the beetroot has been.

Gary Salonga

LSG15

Materials
Beetroot

Measuring cylinder

Plastic pipette

6 test tubes

Mounted needle

Sieve

Test tube rack

Large beakers

Stopwatch

Marker pen

Ruler

2 Spectrophotometer cuvettes

Cork borer (1cm diameter)

Thermometer (alcohol)

Spectrophotometer

White tile

Hot plate

Sharp knife

Small beaker

Heat proof mat

Glass stirring rod

Method
1. Using a 1cm cork borer, cut cylinders from the flesh of the beetroot. Place the
cylinders of beetroot onto the tile and cut using the knife. Cut into 3mm wide
discs.
(Remember to keep the discs the same size and also in the same region of flesh.
By not doing this it may cause another random error).
2. Place all beetroot discs into a beaker and fill with enough water to submerge all
of them. Remove any excess pigment by swirling the beaker for at least 3
minutes and then draining the water. Repeat this step 3 times (9 minutes in
total) until the water runs clear.
3. Take five of the beetroot discs and slide onto the skewer. Discs should be spread
evenly (as shown in the diagram to the right) and 6 skewers like this in total.
4. You will need to label the top of six test tubes with different temperatures (30C,
40C, 50C, 60C, 70C, and 80C).
5. Add 10mL of distilled water to each of the test tubes. Make sure to label the top
of the test tube so you know which is yours. Place the tubes in the separate
water baths with different temperatures. When the temperature of your water
bath reaches desired temperature remove that test tube from the water bath and
place in a rack.
6. Record the temperature of the water in the test tube (desired temperature
3C). Immediately place the skewers of beetroot into the test tubes for one
minute. Place the test tubes into a room temperature water bath for 30 minutes.
7. Repeat the process described in Step 7 for each temperature.
8. Remove the beetroot stacks from the test tube after 30 minutes and throw away
skewers of beetroot.
Part B: Using the spectrophotometer.
1. Pipette 3mL of the water solution from the test tube into a cuvette.
2. Before and after placing the cuvette into the spectrophotometer, remember to
zero the machine. Also remember to have the absorbance of the
spectrophotometer at 530nm.
3. Rinse the cuvette using distilled water and repeat for each of the solutions. Read
the absorbance and record for each sample.

Gary Salonga

LSG15

Results
Table 1

Temperat
ure (oC)

30

40

50

60

70

80

Test Tube
Temperat
ure (oC)

27

38

48

57

67

74

Absorban
ce (abs)

0.402

0.481

0.550

0.553

0.801

0.860

The effect of temperature on membrane permeability in beetroot

1

0.8

0.6
Absorbance (abs)
0.4

0.2

0
20

30

40

50

60

70

80

90

Temperature (oC)

Graph 1

The graph above shows evidence that there is a higher absorbance when the
pigment of beetroot leaks into a higher temperature of water.
Discussion
Random errors
Another random error may be that students forgot to zero the
spectrophotometer after each use. Since the value does not go straight to zero
after removing the cuvette, it would have most likely added onto the number
after the first sample.

Gary Salonga

LSG15

Placing the skewers with the beetroot discs into the test tubes may also be a
random error. Initially when the discs were put onto the skewer the spacing
between them was quite large. When the skewers were to be placed into the test
tube, the top two discs were not submerged in the water. We then had to take
out the skewers, rearrange them and place them back in. However the very top
disc was barely in water. Since all of the discs were not submerged, it would not
have released the potential amount of pigment into the water.
Cutting the beetroot into similar size discs may have presented a random error.
Although it may not be an error that has a high chance of happening, it can still
present some irregularities in data. Referring back to the method, it says to use
the cork borer around the same area in the beetroot. Since our beetroot was very
oddly shaped, it was necessary to get flesh from any part of the vegetable. The
flesh was not the same the whole way through and may have affected the
quality of the discs we cut. The differing sizes of discs would affect the amount of
surface area on each slice. This would then directly affect the rate of diffusion.
Systematic errors
The temperature in the water baths would be classed as systematic errors. This
is an error because the temperature in the bath did not reach the desired
temperature of what it should have been. The method indicates that it should
have at most, 3 degrees Celsius above or below the stated temperature.
However looking at the test tubes, some water baths were still not in that range.
The water at 80 degrees Celsius did not reach the desired temperature and the
thermometer stated that it was only at 74 degrees Celsius. If all the water baths
were at the desired temperature, it would have supported in getting optimal
results.
For most of the groups, there was not enough time for the test tubes to be
submerged into the room temperature water bath for the whole 30 minutes. For
our group, it was only able to be submerged for 20 minutes. This would not have
given the potential amount for all the pigment to release into the water solution.
Although it is highly unlikely, there could have been a fault with the equipment
used, specifically the cuvettes. If the cuvettes were in any way marked or
scratched, it may have altered the results. If there were any scratches they
would not be visible to the naked eye. These flaws could restrict the light and
alter the amount of light that travels through the sample.
Evaluation
Our final results did produce some reliable data. The main issues are that at 60
degrees Celsius the absorbance did not raise as much as we had hoped, and also
at 80 degrees Celsius, the value was not high enough to give the right shape for
graph. For this experiment, the graph should produce an exponential growth with
accurate results. However with the results that we produced, the graph looks
more linear. Before the cuvette was placed into the spectrophotometer, the 50
and 60 degree Celsius samples looked very similar which we assumed would
have a very similar absorbency value. This proved correct as the difference
between those two values was miniscule. Since the absorbency of 80 degrees
Celsius was quite low compared to other groups, it is most likely that something
went wrong during the skewers sitting in the test tube for the one minute. It
could also have been that the test tube with 80 degree water cooled down very

Gary Salonga

LSG15

quickly as the environmental temperature was quite low. The sudden change of
temperature may have cooled the water down quicker than the other
temperatures.
The validity of this experiment showed to be very high. What we had planned to
measure was exactly what the results showed. Although the results did not
represent an accurate representation in a graph, it did not show any major
outliers.
The reliability of the experiment was also quite high. There were not many errors
that would have caused a major contribution to the results, and because of this it
is most likely that if the experiment was to be performed again, it would have
much more accurate data.
The equipment used was very reliable. Since the spectrophotometers were quite
new, they would have a very small margin of error. Since the instructions that
were to be done by hand were very precise, they also would not have given
much error. However, the water bath was the main concern of the equipment.
The desired temperatures were not able to be produced in any of the water baths
and this may have been a very important factor as to why many groups had an
outlier.
Conclusion
As the report shows, the hypothesis was supported by the results. As the
temperature in the samples increased where the beetroot was placed, there was
a higher value of absorbance in the water sample.

Gary Salonga

LSG15

Bibliography
123helpme.com, (2015). The Effect of Temperature on the Cell Membranes of
Beetroot
Cells
::
Papers.
[online]
Available
at:
http://www.123helpme.com/view.asp?id=123054 [Accessed 17 May 2015].
Wikipedia,
(2015).
Betalain.
[online]
Available
http://en.wikipedia.org/wiki/Betalain [Accessed 18 May 2015].

at:

Beetroot 2015 Cells Summative Prac and Rubric

Informational hand out
Penny Collins [Accessed 14 May 2015].
Khirwadkar, S. (2015). An Investigation to determine the effect of temperature on
cell membrane permeability. [online] Available at: https://biobrainstorm.wikispaces.com/file/view/cell_membrane_permeability.pdf [Accessed
19 May 2015].