Biol 1010U – Fall 2023

Laboratory # 3:
Membrane structure and function

Objectives:
• Determine the effect of temperature on membrane damage
• Learn how to use a spectrophotometer and prepare a blank solution
• Prepare and use a standard curve to determine the concentration of a substance
• Review the proper use of glass pipettes
• Review the concept of dilution
• Practice collecting and presenting data graphically

Please note that both the Introduction/Protocol and Assignment files need to be
printed out and brought to the lab in order for you to do lab 3. Make sure to
complete the pre-lab questions in your assignment before coming to the lab.

Introduction

Membranes perform several functions in a cell, including:

- Separating the contents inside the cell
from the external environment
- Organizing specific chemicals and
reactions into certain compartments,
known as organelles
- Regulating the transport of molecules
and ions in and out of organelles and
the cell itself

One way to study the function of a cellular

structure is to disrupt it and examine the effect
of this action on the cell. In this lab, you will
investigate the effect of temperatures on the
functioning of membranes of living beet cells
(Beta vulgaris). Beet cells contain a red
pigment called betacyanin that is located in
the large central vacuole of the cell (see
Figure 1). These vacuoles are surrounded by
a membrane called tonoplast and are located
in the cytosol of the cell. The cell itself is
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surrounded by another membrane called the plasma membrane. As long as both of these
membranes remain intact, the betacyanin remains in the cell. However, if these membranes
are damaged, the betacyanin ends up leaking out of the vacuole and out of the cell,
producing a red coloured water in which the beet is immersed. You will subject discs of beet
root to various temperatures, and assess the amount of damage to the cells by measuring
the amount of betacyanin that leaked from the cells.

As the damage to the membrane increases, greater amounts of betacyanin will leak from the
cells, causing the water surrounding the disc to change colour. Therefore, the intensity of the
colour in the water will depend on the amount of damage done to the cells. While your eyes
can distinguish relative variation in colour intensities, it cannot be used for quantitative
measurements these variations. Instead, you will use a spectrophotometer (see below) to
measure the amount of betacyanin that has leaked from the beet tissues by measuring the
absorbance value of the betacyanin in solution.

General Principles of Spectrophotometry

The presence of coloured

compounds, such as the
betacyanin pigment, can be
quantified using a visible light
spectrophotometer.
Visible light is an electromagnetic
energy made of various
wavelengths ranging from 380 nm
to 750 nm. We perceive these
wavelengths as different colours.

As light meets matter, it may be reflected, transmitted or absorbed. Substances that

absorb visible light are called pigments. A molecule absorbs wavelengths of light in a
characteristic pattern. In general, you can make a good guess about a solution's absorbance
pattern by looking at the colour of the solution: the colour of a solution is the colour that is the
most reflected or transmitted and therefore is the colour NOT absorbed. For example, a
solution of chlorophyll is green because it reflects and transmits green light, while absorbing
the other colours (see Figure 2). However, this does not mean that all greens are identical, or
that the absorbance of other colours is necessarily complete.

A spectrophotometer allows us to determine the transmission/absorbance of light by solutes

in a solution in order to determine their concentrations. A spectrophotometer is made of the
following components (Figure 2):

- Light source: tungsten filament lamp that produces white light.

- Prism: separates the white light beam into the different wavelengths or colours of light.
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 - Narrow slit: used to select one particular wavelength of light that passes through the
sample (incident light, IL).
- Sample holder: keeps the sample you are measuring in the proper orientation.
- Light detector: photosensitive cell that measures the light that passes through the
sample (transmitted light, TL). The energy received by the photocells is converted
into a voltage fluctuation, which is then displayed on a meter scale.

When light passes through a solution, some of it will strike molecules and be absorbed, thus
the amount of light coming out on the other side is less than the amount that entered. Note
that there are two ways of looking at this: you can either think about how much light makes it
through the solution (transmittance) or how much light gets absorbed by the molecules
(absorbance). Clearly the two are related, however they are measured differently.

- Transmittance is expressed as the percentage of light that passes through (%

Transmittance or %T):
TL
% T = T x 100 = x 100
IL

- Absorbance (A) is expressed as: A = - Log (T) = 2 - Log (%T)

So, if transmittance is 100%, then A=0. If transmittance is 1%, then A=2.

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Suppose you dissolve different amounts of a yellow dye and measure the amount of blue
light that is transmitted or absorbed. Typical results are shown below in Figure 3.

Absorbance values are usually used more than transmittance because they show a linear
relationship with the concentration of the solute in a solution; absorbance rises linearly with
concentration (black line in Figure 3), while transmittance decreases in an exponential
fashion compared to concentration (blue line in Figure 3).

The linear relationship between absorption and concentration is described mathematically by

the Lambert-Beer law:
A = εcl
With:
- A: absorbance
- ε: extinction coefficient (in L mol-1 cm-1); ε is a measure of the amount of light absorbed per
unit of concentration and is the inverse of the slope of the line.
- c: concentration (mol L-1)
- l: light path, in cm (usually = 1 cm)

It is the linear relationship between absorbance and concentration that

makes absorbance useful in biology experiments;
If a solution shows twice the absorbance of another, then its
concentration is also twice as high.

Note that the Law is not obeyed at high concentrations of solute. The linear absorbance
range of most spectrometers is between 0.1 and 1. At high solute absorbance (> 1),
absorbance versus concentration is not linear anymore and therefore the law is no longer
valid. In this case, the solution will have to be diluted before taking the absorbance reading
of the solution.

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Measuring concentration using a spectrophotometer
When light passes through a solution, there are three sources of absorbance:

➢ the container (usually a clear glass test-tube or plastic cuvette)

➢ the solvent, which could be water or a buffer
➢ dissolved molecules

Because you only want to measure the concentration of the dissolved molecules, you need to
ensure that the container and solvent do not interfere with your reading. To do so, the
spectrophotometer must first be set to zero absorbance with a 'blank' made of a tube which
only contains the solvent (without the dissolved molecules you want to measure). This blank
is used as a reference point; by defining the amount of light that passes through the blank as
100% transmission or Abs = 0. Any reduction in the transmission or any increase in
absorbance after the light passes through the sample tube, will be due to the molecules of
interest in the solution.

How is the concentration of a substance determined?

To be able to determine the concentration of a substance using spectrophotometry, a
calibration curve or standard curve must first be obtained. A standard curve is a plot of the
measured responses (e.g. absorbance in the case of spectrophotometry) versus known
concentration or amount of the substance of interest. Because the relationship between
absorbance and substrate concentration is linear, the line of best fit with intercept at 0 (as
shown in the picture below) can be used to determine the concentration of a substrate in
unknown solutions from their absorbance values.

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Using Figures to present data
Two major types of figures, used to present scientific data in biology, include line graphs and
bar graphs. Both graphs have very specific requirement so make sure you follow the
guidelines mentioned below.

Please note that figure number and title (when required) is always located below the graphs.
The figure title should be relevant to the data presented and should not re-state the axis
labels. e.g. “Absorbance versus concentration” is not a proper title.
Graphs must have properly labeled axis: the axis’ titles must be descriptive of the data and
include the units used. Use the space of the Figure properly; adjust the scale so you don’t
have empty spaces. Keys to the symbols should also be present towards the top area of the
figure. When drawing a line graph, indicate on the graph both the individual points and the
best fit line.

Line graph

Bar graph:

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Protocol

Exercise 1: Using a spectrophotometer (Genesys 20)

1. Turn the power switch (1) at the back of the machine ON (I)
and let the spectrophotometer warm up for at least 15 min.
Warming up the spectrophotometer is necessary to
stabilize the light source and detector.
2. Press the A/T/C bottom on keyboard (4) to select for
absorbance (A).
3. Set the spectrophotometer to the desired wavelength using
the wavelength keys nm ▲ or nm▼ on keyboard (4).
You can change the wavelength quickly by holding the key.
4. To calibrate the spectrophotometer to 0.0 absorbance/100% transmittance, insert the “blank”
cuvette into the cuvette holder (3), close the lid and press the 0 ABS/100%T key on keyboard (4).
Make sure the cuvette is clean by wiping it with a tissue to remove liquid droplets, dust and
finger prints.
The “blank” tube contains everything except the molecule you want to measure.
Position the cuvette so that the light path (indicated by the arrow) passes through the clear
walls of the cuvette.
Finger prints on the clear walls of the cuvette will affect the absorbance readings. Always hold
the cuvette on the frosted sides.
5. Remove the blank from the sample compartment. The spectrophotometer is now ready to
measure the absorbance of your samples. To do so, insert the cuvette containing your sample and
read the absorbance from the digital display (2).
6. Remove the cuvette from the sample compartment and repeat step 5 with any remaining sample
solutions.
7. When all measurements are completed, turn off the spectrophotometer by switching to OFF (○)
at the back of the machine.

Exercise 2: Preparation of the standard curve for betacyanin

1. Please wear gloves for this experiment to avoid beet root stains!
2. Using a waterproof marker label seven tubes 1 to 7.

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3. Prepare the tubes according to Table 1 in your lab assignment. For each solution, determine the
dilution factor and the concentration of the diluted solution in Table 1. Show your calculations
below the Table. The calculations are part of the pre-lab assignment.
• Note that the stock concentration of betacyanin is 9 µM
4. Vortex each tube for a few seconds to completely mix the contents of the tubes. Label 7 plastic
cuvettes 1 to 7 on the top part of the cuvette. Pour ~ 3 mL of solution from each tube into the
corresponding cuvette.
• Make sure that your label will not interfere with the light path once the cuvette is inserted
in the cuvette holder of the spectrophotometer.
5. Set the spectrophotometer to a wavelength of 525 nm.
6. To calibrate your spectrophotometer, insert the cuvette containing the blank solution. Which
tube contains the blank solution? Record the information in your lab assignment (bottom of p. 2)
and make sure you double check with your TA before going forward with the experiment.
• Make sure to clean the clear sides of the cuvette with a kimwipe to get rid of any droplet of
liquid or finger prints before placing it in the spectrophotometer.
7. Press the 0 ABS/100%T to set the spectrophotometer to zero (0) absorbance. Your
spectrophotometer is now ready to measure the absorbance of the 6 other tubes. To do so,
simply remove the blank cuvette from the cuvette holder. and add instead the cuvette you want
to measure, close the lid and read the absorbance from the screen display. Record these values in
Table 1.
• Keep your blank cuvette as you will need it again in Exercise 3
8. Draw the standard curve on the graph paper provided (see below).

Exercise 3: Testing the effect of temperature on membrane structure

• Preparation of beet root discs
1. Using a cork borer, obtain two beet cores of ~ 5 cm in length. Cut the ends of the beet core (~ 0.2
cm at each ends) and discard in the garbage bag.
2. Starting from a freshly cut end of the beet root core, use a ruler and a razor blade to cut three
disks 1 cm in length.
3. Rinse the three discs by placing them in a beaker containing cool tap water. Gently shake. Discard
the water through a sieve. Continue rinsing the discs with water until the water in the beaker is
colorless.
4. Keep the discs in the water until needed.

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• Temperature stress effect
You will be testing the effect of three temperatures on cell membrane structure: -20oC, room
temperature (22oC) and 60oC. The discs will be exposed to each temperature for 3 minutes. The tubes
containing the discs are then placed in a beaker with cold tap water for 5 min. to stop the reaction.
The tubes are then vortexed to help with the release of betacyanin from the damaged cells. The
amount of betacyanin released is then quantified using the standard curve prepared in Exercise 2.
Please read all the instructions before starting and ask your TA if you have any questions.
1. Start by labelling 3 large test tubes A to C and your initials. Place your label on the side of the tube
of the tube. Add 5 mL of water to each of the tubes.
2. Leave Tubes A and B on the bench and place tube C in the 60oC water bath. Let the temperature
stabilize in the tube for 5 min or more:
o Tube A will be used for the disc placed in a -20oC freezer
o Tube B is testing the effect at 22oC (room temperature)
o Tube C is placed in the 60oC water bath
3. Label 3 cuvettes A to C (at the top of the cuvette) and place them on the cuvette holder.
4. Fill in a 500 mL beaker ¾ with cold tap water
5. Using forceps, place one of the beet discs on a petri dish and place the petri dish in the -20oC
freezer. Immediately start the timer counting up (0 min on the timer). Do not stop the timer until
the whole experiment is over.
6. Using long forceps, add another beet disc to tube B on the bench at the time indicated in column
3 of Table 2 (at 1 min on the timer). Make sure the beet disc is fully submerged in the water.
7. Using long forceps, add the last beet disc to test tube C that is in the 60 oC water bath at the time
indicated in column 3 of Table 2 (at 2 min on the timer). Make sure the beet disc is fully
submerged in the water.

8. After exactly 3 min (on the timer), remove the beet disk from the freezer and using long forceps
place the disc in tube A. Vortex for 30 sec and place tube A in the beaker containing cold water for
5 min.
9. At exactly 4 min (on the timer), vortex tube B for 30 sec and place it in the beaker containing cold
water for 5 min.
10. At exactly 5 min (on the timer), vortex tube C for 30 sec and place it in the beaker containing cold
water for 5 min.
11. Once the 5 min are up (following the times mentioned in column 5 of Table 2), take the tubes one
at a time from the beaker of water, dry the outside of the tubes with paper towel, vortex one
more time for 30 sec and immediately pour ~ 3mL of the surrounding water in the corresponding
sample cuvette labelled A, B and C.
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Once all 3 cuvettes are ready:
5. Make sure that the wavelength of your spectrophotometer is still set at 525 nm, and calibrate it
again with the same blank cuvette you used in Exercise 2.
6. Measure the absorbance of the water in the sample cuvettes (A to C) and record the value in
column 6 of Table 2. Use the standard curve prepared in Exercise 2 to determine the
concentration of betacyanin in the water.
7. Answer the questions.

Analysis of the results:

Standard curve:
- Plot the absorbance values versus [betacyanin] as a line graph on graph paper provided.
- Make sure you properly label the axis and include units when necessary.
- Draw the standard curve (best fit line that passes through 0)
- Label the figure, Figure 1, below the figure and add a descriptive title.

Effect of temperatures:
- Plot the [betacyanin] versus temperature as a bar graph on graph paper provided.
- Make sure you properly label the axis and include units when necessary. Note that you will have
two X axis labels: one for each of the bar’s treatment and a general one for what the axis is.
- Label the figure, Figure 2, below the figure and add a descriptive title.

Clean up:
- Discard the beet root in the regular garbage bag.
- Dump the content of the test tubes and cuvettes down the drain, rinse them with tap water
and place in the wash basin.
- Place the 5 mL pipettes, tip down, into the pipette wash bucket located at the end of your
bench.
- Turn the spectrophotometer off.

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 Appendix A:
Review: Performing dilutions expressed as fraction (check the BIOMath file
on Canvas for additional information)

The dilution factor is defined by the following fraction:

𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 =
(𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 + 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡)

The final concentration (Cf) of the diluted solution can be calculated by multiplying the dilution
factor (DF) by the concentration of the stock (Cs) solution:
Final concentration = dilution factor x stock concentration of solution
Cf = DF x Cs

Example 1: A solution of 95% ethanol is diluted 1/5. What is the concentration of the diluted
solution?
95% ethanol x (1/5) = 19% ethanol

Example 2: A solution has an original concentration of 20 mg/mL. The solution is diluted 4

times (note that this corresponds to a 1/4 dilution). The concentration of enzyme in the diluted
solution is:
20 mg 1 5mg
x = = 5mg / ml
1 mL 4 1 mL

Preparation of solutions using fractional dilutions

A 1/10 dilution means that you add 1 part of solute to 9 parts of solvent to bring the total
volume to 10 parts.

For example, adding 1 mL of solute to 9 mL of water (or other solvent) will give you 10 mL of
1/10 diluted solute.

The final volume does not have to always add up to 10, in this case multiply the final volume
with the dilution factor to determine the amount of solute needed and subtract this volume to
the final volume to determine the amount of solvent to add.

For example, you want to prepare a 1/5 dilution of a solute in a final volume of 2 mL.
- First determine the volume of solute needed:
(1/5) x 2 mL = 0.4 mL
- Then subtract from the final volume to determine the amount of solvent to add:
2 mL - 0.4 mL = 1.6 mL
So to prepare a 1/5 dilution of the solute in a final volume of 2 mL, you will need to mix 0.4
mL of the solute with 1.6 mL of water (or other solvent).
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 Appendix B
A review: Measuring volumes of liquid using a glass pipette and pipette bulb

In this lab you will use graduated serological pipettes to accurately transfer volumes of solutions
ranging from 1 to 10 mL.

The pipette bulb is a device that is used to provide suction to draw the liquid up into the pipette. The
nice thing about a pipette bulb is that you do not have to remove it at any point during the transfer of
liquid. There are two valves on the bulb and each serves a specific purpose (see Figure below). To
pipette solutions with a pipette bulb, follow the steps below:

1. Squeeze the bulb to expel the air from it.

2. Carefully place the bottom part of the bulb over
the mouth of the pipette. Push gently in a turning
motion to attach the bulb to the pipette.
3. Place the tip of the pipette below the surface of the
solution you want to pipette. Gradually squeeze the
suction valve (↑) to draw liquid into the pipette.
While drawing the solution in, make sure there is
enough solution in your beaker or flask to completely
fill the pipette and that the pipette tip remains below
the surface of the liquid; this will prevent air from
being drawn into the pipette.
4. Stop squeezing the suction valve (↑) when the
liquid is above the volume you want to pipet. Do not
remove the bulb from the pipette.

Gently press the empty valve (↓) until the bottom of the concave surface
(the meniscus) of the liquid in the pipette is at the mark corresponding to
the volume you want to measure (e.g. 5 mL in the picture). Touch the tip of
the pipette onto the wall of the vessel to remove any liquid that may be
present outside of the tip.
5. Transfer the pipette into the receiving vessel, and press the empty valve (↓)
to draw the liquid out of the pipette. To accurately transfer the desired
volume of solution, blow the last drop of liquid from the pipette by pressing
the “blow-out” ending of the bulb or pressing the small ball between the
two suction valves.

NEVER use your mouth to draw liquid into a pipette!

DO NOT allow liquid to enter the pipette bulb.
Be careful, the graduations on serological pipettes are up-side-down!

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