Investigating the effects of certain detergents and soaps on denaturing of cell

membrane in beetroot cells

Research Question: What is the effect of soap VS hand sanitizer in the destruction of cell
membranes?
Aim: The aim of this experiment is to discover which types of soap and hand sanitizers are more
harmful to the cell membrane of bacteria, by using a beetroot as a model, and the intensity of
the beetroot pigment, measured with a spectrophotometer, when in contact with the detergents
used.
Personal Engagement: The topic chosen was very interesting to me as I use hand sanitizers
every day to make sure my hand is free of any bacteria. Often, people dont have the time to go
and wash their hands in the bathroom with soap and water, and instead use hand sanitizers as
an equally effective measure 1 to keep their hands clean. Soaps are often less accessible to
normal people who wish to clean their hands quickly and efficiently. I found it interesting to find
out how exactly detergents kill germs, and encountered that one of the main factors was how
their properties are able to pierce cell membranes and destabilize pathogenic microorganisms as
well as resident bacterial flora. Trying to see how I could prove if hand sanitizers had the same
effect as soaps in this endeavor, it occurred to me that beetroot cells could be used as models to
measure the effect done on the membrane, because they contain the Betalain pigment.
Furthermore, after finding this topic of interest, I delved more into it by adding a detergent as
one of my variables (in addition to the brands of hand sanitizers and soaps), because I assumed
that it would further damage the cell membrane and prove that my results hold a correlation.
Because I had the spectrophotometer available at my school, I realized that I would be able to
accurately measure the intensity of the pigment, and thus measure how much the cell
membrane was pierced once it was in contact with the detergents.
Background Information:
Cell membranes are complex, selectively permeable structures, which have the function of
controlling the movement of various substances in and out of the cell. Cell membranes are
composed of various lipids, proteins and ions. Moreover, they contain phospholipid bilayer that
makes the membrane both stable and flexible.
These phospholipid molecules have a
hydrophilic (attracted to water) head and a hydrophobic (repels water) tail, as shown in the
diagram below.

1 The Center for Disease Control states that the most important way to avoid diseases is to
frequently wash hands with soap and/or hand sanitizers. http://www.cdc.gov/handwashing/

There are numerous studies on the interactions of cell membranes and detergents, most are
based on the purification, isolation and solubilisation of membrane proteins. 2 Among properties
that affect interactions between membranes and surfactants, are surfactant concentration,
location of lipids, and interactions between proteins and lipids. 3

The term surfactant comes from the words surface active agent. 4 Like cell membranes,
surfactant molecules are amphipathic, meaning they have both hydrophilic and hydrophobic
parts.5 Specifically, they are composed of long chain hydrocarbons (fat soluble) and charged ions
(water soluble). Thus, surfactant molecules are able to concentrate at the meeting between the
phospholipids of the membranes in bacterial cells, and the surrounding solution. These properties
result in a leakage of the inside of the cell membrane. Soaps and detergents are classified as
surfactants. Although their properties work in practice against bacterial cells, these surfactants
are also active against vegetative cells like Beetroot.
Hand sanitizers and soap are marketed as equally effective measures to kill pathogenic
microorganisms as well as resident bacterial flora. 6 However, various brands of soaps and
sanitizers have different components, which have distinct effects in the cell membrane of the
pathogens they are attempting to eliminate. This experiment will test the following different
soaps and hand sanitizers:

1.

Antibacterial liquid soap - CONTAINING TRICOSLAN

Effect of Tricoslan on fatty acid synthesis:

Antibacterial soap effect: Molecule attacks multiple targets within the bacterial cellular
membrane and also inhibits the fatty acid synthesis during bacterial metabolism.

2.Regular liquid soap

3. PURELL (alcohol based) hand sanitizer Developed with 70% ethyl alcohol,
Alcohol based hand sanitizer effect: Denatures bacterial cellular components
essential to function by fracturing the physical structure of the proteins that make up the
major framework of the bacterial cell
4. SAPOLIO microbial detergent

2 http://shodhganga.inflibnet.ac.in/bitstream/10603/2913/9/09_chapter%203.pdf
3 (Lichtenberg, 1983; Higgins, 1987)
4 http://www.essentialchemicalindustry.org/materials-and-applications/surfactants.html
5 http://www.cemag.us/articles/2008/06/how-surface-active-agents-work
6 http://www.cdc.gov/handwashing/

The Peruvian brand, Sapolio has a microbial detergent which is categorized as an

anionic surfactant, composed mainly of alkyl aryl sulfonate. 7 Anionic surfactants lower
the surface tension of solvents, which means lower the cohesive forces exhibited by
molecules of a liquid.89 Thus, all anionic surfactants are able to bind to pathogens
residing in liquids, and this means that they are highly effective cleaners when mixed
with water.
5.Tricoslan hand sanitizer - DEVOSAN

Just like biological membranes, detergents

This property enables soap to disrupt the cell membrane and thus stop the functioning of
pathogens. Detergent molecules permit the dispersion of hydrophobic compounds, thus they
extract and solubilize membrane proteins, seeping in between the fatty acids of the membrane
and pulling it apart, releasing the cell membrane contents.
This experiment will use beetroot to test the effect of these cleansers. Beetroot contains a red
pigment called Betalin, which is soluble in water. When the cleansers destroy the cell membrane
of the beetroot, the red pigment will leak out, down the concentration gradient and die the
solution of water. The deepness of the red pigment will depend on the amount of damage the
cleanser did to the cell membrane. Thus, with the visible spectrometer that is able to measure
wavelengths and absorbance, one could notice which cleanser affected the cell membrane the
most.
Hypothesis:
I predict that the antimicrobial detergent will have the highest impact on the cell membrane,
and release the most pigment into the solution, because it has a higher ability to destruct
barriers of skin compared to the alcohol solutions in hand sanitizers and soaps. 10

Variables

Units

Uncertai
nty

What?

Why?

How?

7http://www.silsa.com.pe/intranet/InformacionProductosLimpieza/HOJASEGURIDAD/HOJA%20DE
%20SEGURIDAD%20DETERGENTE%20SAPOLIO.pdf
8 http://www.wisegeek.com/what-is-an-anionic-surfactant.htm
9 http://www.wisegeek.com/what-is-surface-tension.htm
10

Anti-bacterial
liquid soap

To see and quantify

the differences and
effectiveness of
Regular
liquid each cleanser and
soap
its components in
destroying the cell
Antimicrobial
membrane of
Detergent
beetroot cells.
(ARIEL)

Independe
nt
Variables

Dishwashing
detergent
(SAPOLIO)

By getting
each cleanser
and mixing it
with the same
volume of
water and
inserting the
beetroot
inside for the
same amount
of time in
each trial.

Hand
sanitizer
made of 0.3%
Tricoslan.

Dependen
t Variable

Control
Variables

Au

Degrees
Celcius
C

+/0.0511
Au

+/- 0.05
C

Absorbance of
light

Temperature of
water and cleanser
mixture.

To measure how
much pigment
leaked out of the
beetroot cell and
thus the impact of
the cleanser in the
cell membrane

By using a
Vernier
Spectrophoto
meter that
graphs
absorbance of
light VS
wavelength
and selecting
the
wavelength of
550.5 nm in
all trials,
variables and
results.

To
ensure
that
temperature does not
affect the solubility
and piercing of the cell
membrane in beetroot
cells, because a higher
temperature
would
have the effect of
disintegrating
the
membrane
and
therefore disrupt the

Before each
variable, the
temperature
will be recorded
using a
thermometer to
ensure that the
solution is in
room
temperature.
Trials will also

11 Vernier Manual for Spectro Pro: http://www2.vernier.com/manuals/SpectroProManual.pdf

experiment that tests be carried out

membrane destruction the same day at
for cleansers.
approximately
the same time

Units

Uncertai
nty

What?

Control
Variables

Cm3

Cm

+/- 0.5
cm3

+/- 0.05
cm

Volum
e of
water
and
cleans
er

Size of
Beetro
ot
pieces

Why?

How?

The beetroots will be

mixed with the solution
of water + cleanser. It is
important that all trials
carry the exact same
concentration of water
and soap, because the
beetroot will be put
inside it, and a higher
concentration of soap
vs water would damage
the cell membrane
more if these volumes
were to vary.
If a beetroot has a
bigger size and a bigger
surface area to volume
ratio than another, then
it will naturally release
more pigment into the
solution.
Thus,
the
beetroots
must
be
exactly the same size in
length and diameter.
These conditions must
apply to all trials.

Using a measuring
cylinder, the cleansers
will be measured to 0.5
cm3 and the tap water
also to 0.5 cm3 before
inserting both into the
plastic cup and before
inserting the beetroot.
Once inside the plastic
cup, they will be
checked with the
measurements there to
ensure that no mistake
has been made
A cork borer with a
diameter of 1cm and a
15 cm ruler will be used
to measure in total 25,
2 cm strands of
beetroot for every trial.
The beetroots will be
placed in line to ensure
that they are the same
size.

Beetro
ot
types

Seco
nds

+/- 0.5 s

Time

Each different beetroot

might have different
weakness in cell
membranes, and to
ensure that no minimal
variances disturb the
results, the same
beetroot types must be
used.

If a beetroot is left in
the solution more time
than the rest, the cell
membrane would be
more affected as
exposure time
increases. It is
important that each
beetroot spends the
same amount of time in
contact with the
solution in order to be
able to measure the
results when changing
the independent
variable

For each cleanser, one

strand of beetroot from
one specific beetroot
which is going to be
labeled is going to be
used. As there are 5
trials and 5 beetroots, 5
different
beetroot
strands taken from 5
different beetroots of
the same type, will be
used for the same
cleanser in each trial.
A chronometer will be
used in each trial for
each beetroot to stay
exactly 5 minutes in
contact with the
solution. A tweezer will
be used to take out the
beetroot from the
solution when the time
is out and discard it.

Safety
1. Be cautious with the knife and scalpel.
2. Wear glasses to ensure no red pigment enters eye or no cleanser enters eye.
3. Handle technological equipment and water with care for no collision to occur.

Materials
o
o
o
o
o
o
o
o
o

Cork Borer with 1 cm diameter

Scalpel
20 cm Ruler
Pipette x 5
Measuring cylinder x2
Plastic cups x 10
Beetroot x 5
Chronometer
Thermometer

o
o
o
o
o
o
o
o
o
o
o
o
o
o

Knife
Spectrometer
Logger Pro 3.7 Software
Computer
Blank sheet of paper
Cuvette
Cloth
Distilled Water
Anti-bacterial liquid soap
Regular liquid soap
Ariel antimicrobial Detergent
Hand sanitizer made of quaternary ammonium compounds
Hand sanitizer made of Tricoslan.
Tweezer

Five trials per cleanser

Method:
1.
2.
3.
4.
5.
6.

Start by measuring and recording room temperature.

Using a knife cut the top vegetative potion and the base of the five beetroots
Place a blank sheet of paper on the table and the five beetroots that will be used on top.
Label each beetroot on the bottom of the paper (Beetroot 1, Beetroot 2, etc.).
Take Beetroot 1 and using a scalpel cut it approximately in half.
Using the 1cm diameter cork-borer, extract the first strand of beetroot. Measure the strand
with a ruler to 2cm, and cut this length with the scalpel. Make sure to leave space in the
beetroot to collect more strands.
7. Take four more 2cm strands of the same beetroot with the cork borer.
8. Place the five strands of beetroot extracted below the label of Beetroot 1. And discard the
remains of the first beetroot.
9. Repeat steps 3 through 6, four more times, with the four beetroots remaining.
10.Once there are a total of 25 2cm strands of beetroot, label 5 plastic cups (beetroot 1,
beetroot 2, etc)
11.Place each group of strands in their respective plastic cups.
12.Measure 50 cm3 of distilled water in the measuring cylinder and add to plastic cup 1.
13.Repeat the last step with the 4 more plastic cups.
14.Measuring with a chronometer, keep the beetroot strands in the distilled water solution for
5 whole minutes.
15.After 5 minutes have passed, discard the distilled water from each of the plastic cups and
leave the beetroot strands inside.
16.Pour 30 cm3 of anti-bacterial liquid soap in the measuring cylinder and transfer it to a
clean plastic cup. Measure 30 cm3 of tap water and mix with the soap in the plastic cup.
17.Take the first strand of beetroot from plastic cup beetroot 1 and place it in the plastic
cup with the solution of soap and water. Momentarily start the chronometer. When the
clock reaches 5 minutes stop the chronometer, extract the strand of beetroot from the
plastic cup and discard it.
18.Open the Spectrophotometer Software in your computer.
19.Connect the visible spectrophotometer
20.Calibrate using distilled water.
21.Clean the cuvette and pour the beetroot pigmented solution taken from step 16 inside.
Place the cuvette inside the spectrophotometer and save the results.
22.Repeat steps 15 through 20, using one beetroot strand from each plastic cup labeled
beetroot 1, beetroot 2 etc. And, using the same cleanser. This would amount to a total of
5 trials per cleanser once all the trials are finished.

23.After, finishing recording the first batch of results, save the file in the Spectrophotometer
Software as Antibacterial Soap and open a new one.
24.Repeat steps 15 through 21. This time, taking one strand of beetroot from each plastic cup
but with Regular Liquid Soap instead of antibacterial soap.
25.After finishing recording the second batch of results, save the file in the
Spectrophotometer Software as Regular Liquid Soap.
26.Repeat steps 15 through 21. This time, taking one strand of beetroot from each plastic cup
but with hand sanitizer made of quaternary ammonium compounds.
27. After finishing the third batch of results, save the file in the Spectrophotometer Software
as Hand sanitizer made of QAC.
28.Repeat steps 15 through 21. This time, taking one strand of beetroot from each plastic cup
but with hand sanitizer made of tricoslan.
29.After finishing the fourth batch of results, save the file in the Spectrophotometer Software
as hand sanitizer made of tricoslan.
30.Repeat steps 15 through 21. This time taking one strand of beetroot from each plastic cup
but with Ariel antimicrobial Detergent.
31.After finishing the fifth batch of results, save the file in the Spectrophotometer Software as
Ariel antimicrobial Detergent.
32.End by measuring and recording room temperature for changes in environmental
conditions.

Data collection
Data Overview
The data will be analyzed to check for the hand sanitizers, soaps and detergents that induced the
strongest effect in the cell membrane of the beetroot. The mean absorbance will be calculated,
as well as a standard deviation for each cleansers results. Then, the results will be checked for
anomalies by plotting the points in a graph and discarding the results that are completely out of
range. Once the data is analyzed, a simple plotted graph will be used, without a best fit line
because line graphs are solely used to show trends in data, and these results dont show a trend
but a comparison.
Table 1: Condensed raw data collected from spectrophotometer for Absorbance (+/- 0.5 Au) in a
550 nm wavelength for each cleanser and all trials
Absorbange of light (+/- 0.5 Au) in 550nm
wavelength
Trial 1

Trial 2

Trial 3

Trial 4

Trial 5

Sapolio
Detergent
Devosan
Hand
Sanitizer (0.3
% tricoslan)
Plain Liquid
Soap
Antibacterial
soap
(Tricoslan)
PURELL
alcohol
based hand
sanitizer

0.294

0.144

0.193

0.208

0.241

0.115

0.151

0.107

0.132

0.140

0.123

0.102

0.136

0.120

0.137

Qualitative Data: Color of solutions for each cleanser

FOTOS

Sample calculations
12

Mean

Example: For Sapolio Detergent

12 Image of Mean calculations webpage:
http://denninginstitute.com/modules/dau/stat/data/mean.gif

Mean = 0.294 + 0.144 + 0.193 + 0.208 + 0.241

5
Mean = 0.040 Au
Standard Deviation

Table 2: Mean values of Absorbance (+/- 0.5 Au) and standard deviation for each independent
variable.
Absorbange of light (+/- 0.5 Au) in 550nm wavelength
Standar
d
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
Mean
Deviatio
n
Sapolio
detergent
Devosan
hand
sanitizer
Plain
liquid
soap
Antibacte
rial soap

0.294

0.144

0.193

0.208

0.241

0.216

0.040

0.115

0.151

0.107

0.132

0.140

0.129

0.019

0.123

0.102

0.136

0.120

0.137

0.124

0.016

Ariel
antimicro
bial
detergent
Data presentation
Graph showing absorbance VS cleanser
Evaluation of results

Analysis of standard deviation: Was the data collected far from the mean?

Evaluation of variables
Even though water and cleansers were measured with a measuring cylinder, each type of
cleanser had different consistencies so it was difficult to tell whether each was measured
accurately. For further improvements, a more accurate instrument for measuring could have
been used such as a pipette, which has a lesser uncertainty of 0.03 cm 3. Also, the cleansers
chosen could have been all liquid, instead of powder and gel, which are harder to measure.
Additionally, a ruler was used to measure the 2cm strands of beetroot, but it was later noted that
a ruler is not the most accurate instrument. To fix this, a ruler with a higher precision than one of
a classroom one, could have been used.
Although the beetroots were washed with tap water before beginning to test the destruction of
cell membrane, it is unsure whether some pigment stayed in the surroundings of each beetroot,
and if it did, if all the beetroots had the same leakage before beginning the experiment.
Therefore, the beetroots should have been washed more thoroughly to ensure that no pigment
remained of the damage done to the cell membrane once it was cut.
The best effort was done to extract pieces of beetroot from the same part, so that each strand
had the same amount of pigment. However, it was very difficult to estimate the darkness of each
strand of beetroot, so some of them were effectively darker than others. This was a problem in
the experiment, because darker beetroots would release more pigment into the solutions of the
cleansers. Some beetroots even had dark spots that could not be washed with tap water. To
improve this, it would be helpful to select a strand of beetroot before beginning the experiment
and then compare each new strand color with that model. Although it is hard to have 25
beetroots of exactly red color, it could be done with more time to collect and analyze data.
For colorless cleansers, distilled water for the calibration of the spectrophotometer was used.
Nevertheless, it would have been better to calibrate the spectrophotometer with a mixture of the
cleanser and the water, as it might have increased the amount of light absorbance if the color of
the solution was slightly denser.
The cuvettes were cleaned and shaken before being inserted into the spectrophotometer.
However, it is possible that some pigment in the solution remained at the bottom of the plastic
cup, because it was hard to mix the solution without it pouring towards the sides of the plastic
cup. This must have affected some results, because the more mixed the solution is, the more the
pigment is distributed among it and the higher the absorbance value. Therefore next time, bigger
plastic cups should be used. Preferably with a lid, so they could be shaken evenly before testing.