Ethical of 0.1 moles. To ensure that

Ethical considerations/Safety

This experiment does not run the
risk of harming individuals, the environment or the property of the lab in
which the experiment is conducted. All remaining or waste products produced in
this investigation, including ethanol, distilled water & beetroot pigment solutions
and remaining beetroot, will be disposed of appropriately in the chemical waste
bucket for further processing or in a designated bin. The chemical waste will
be treated so no harmful substances are released into the environment.

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To ensure the safety of the
experiment and those around, suitable clothing, lab coat and eyewear is
required in the collection of data. Surrounding objects must be out of harm’s
way and should not interfere will the experimentation as glass objects and flammable
material are used. Ethanol is poisonous and flammable so precautions should be
taken to ensure that there are no flames present during the experiment. The
methodology of this investigation was researched from “Brilliant Biology Student” and will provide a
fair means for testing this investigations research question.



Solutions, creating ethanol solutions
ranging from 0 to 0.5 moles at increments of 0.1 moles

The calculation and creation of different
concentrations of ethanol in distilled water needs to be established to form
the basis of this investigations independent variable: different
concentrations of ethanol ranging from 0 to 0.5 moles at increments of 0.1
moles. To ensure that
there is a large spread of data and sufficient evidence to carry out data
analysis through standard deviation, five increments of ethanol concentration
will be used. Although ethanol used in disinfectants
are “typically at a concentration of ?70 to 85% (vol/vol)”- Chatterjee1,
Indranil, et al. – by having concentrations
under 50%, the characteristics of the tonoplast
threshold introduced by the publication from the American Chemical Society can be determined.


1.     Prepare
50ml measuring cylinder, 1 mole ethanol, distilled water, 6x100ml beakers and

2.     Calculate
necessary volume of 1 molar ethanol and distilled water. Amount of ethanol
added is equal to the desired concentration of ethanol solution multiplied by

For example, for a 0.5 molar ethanol solution: 0.5x50ml = 25ml. To calculate
the necessary volume of distilled water simply take 50ml and subtract it by the
calculated volume of ethanol

3.     Measure
necessary volume of 1 molar ethanol and distilled water using a 50ml measuring
cylinder and pour in a 100ml beaker. For smaller volumes of ethanol use a 10ml
measuring cylinder

4.     Label
beaker with the correct ethanol concentration

5.     Repeat
so one ethanol solution at ethanol concentrations ranging from 0 to 0.5 moles at increments of 0.1 moles is achieved


beet, cutting cubes of beet

beet needs to be cut and prepared prior to its contact with ethanol solutions
to ensure all the beetroot is processed the same way and the alcohol can make
contact with its vacuole membrane (tonoplast).

1.     Prepare
knife, distilled water, stopwatch, 5 spot plates, forceps, beetroot and peeler

2.     Rinse
outside of beetroot to remove any impurities

3.     Peel
the beetroot to remove the skin and place in a designated bin, ensuring there
are no stray edges

4.     Place
the beetroot on a plate and cut the peeled beetroot into a large cube to make
sure the measurement and slicing of each cube is accurate

5.     Using
a ruler and knife mark the dimensions of 6 1onto
the large cube and proceed to cutting out the cubes

6.     Using
forceps, place the 6  cubes into a 50 beaker of distilled water to ensure that the betacyanin released during slicing is
not present

7.     After
10 minutes of rinsing, measured using a stopwatch, place the 6  cubes onto a spot plate



Interacting beetroot cubes with
ethanol solution

The interaction of beetroot
cubes in ethanol solution is essential in determining the validity of this
investigation’s hypothesis, namely whether ethanol has a deteriorative effect
on the vacuole membrane (tonoplast)
of beetroots (beta vulgaris).

1.     Collect
cling film and stirrer

2.     Using
both hands, forceps and a stopwatch, simultaneously place a  cube of beetroot into an ethanol solution and
start the stopwatch

3.     Cover
the solution with cling film

4.     Every
minute partially remove the cling film and gently stir the solution, recover
with the cling wrap

5.     After
10 minutes, remove the cling wrap and  cube of beetroot from the solution and place
in a designated bin

6.     Repeat
for the remaining 4 different ethanol solutions


damage to tonoplast, measuring transmittance level/% using a

transmittance level of the betacyanin pigment carried in ethanol
solution will be observed and recorded using a colorimeter and LoggerPro to
determine the values for this investigations’ dependent variable: transmittance
level of solution containing betacyanin from the vacuole membrane
(tonoplast) of a beetroot (beta vulgaris).


1.     Prepare
pipette, colorimeter, lint-free tissues, cotton swab and computer with

2.     Prepare a blank cuvette using distilled water to calibrate the
colorimeter. Connect the colorimeter to a computer and open LoggerPro. Place
the cuvette into the colorimeter and close the lid. Select 470 nm (Blue) and
press CAL button for the duration of the red light

3.     Using
a pipette and 10ml measuring cylinder measure 2ml of the solution with betacyanin into a blank cuvette

4.     Make sure when each sample is taken: position the cuvette on the
correct sides, tap the cuvette on a hard surface to get rid of any bubbles,
wipe the outside of the cuvette using lint-free tissues and only hold the
cuvette on the correct sides

5.     After 1 minute period of recording transmittance, stop recording and
remove the cuvette and its contents. Dry the insides with a cotton swab to
remove excess liquid

6.     Enter the data into a data collection table

7.     Repeat until every solution is tested


Repeat all four steps 5 times so a total of
30 data sets is collected; 5 results for each increment of ethanol
concentrations will produce more reliable results and enable error analysis
through the calculation of standard deviations. 


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