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General Introduction.
Read “The Laboratory Notebook” in the Resource Manual (p. 92-94). Follow these guidelines for
keeping your lab notebook. It should be clear what each graph, table or piece of information represents.
Information should be clearly titled and recorded on numbered and dated pages.
Experimental Spectrophotometry.
OBJECTIVES: At the end of the 6 lab classes in this unit you should be able to:
(1) draw a diagram of the optical system of the Spectronic 20 and explain how the instrument works.
(2) describe what is meant by transmittance (T) and absorbance (A) and be able to convert transmittance
values to absorbance values. Use the equation A = Ecl to calculate A, c or E.
(3) use the spectrophotometer with confidence.
(4) state Lambert’s Law and Beer’s Law in words.
(5) make an appropriate reference blank to zero the spectrophotometer in an experimental situation.
(6) give an example of each of the principal uses of the spectrophotometer (Lab Notes, p.16).
(7) calculate reaction rates using spectrophotometric data (A/time).
(8) describe the meaning and units of photosynthetic photon flux (Resource Manual, p. 45).
(9) explain the meaning of resolution in microscopy and the theoretical limit to resolution by reference
to Abbe’s Equation (Resource Manual, p. 48-49).
(10) use S.I. units of measure and exponents (scientific notation) with confidence.
(11) calculate molar concentrations and dilutions.
(12) remember and use the following equations: [H+][OH-] = 1 x 10-14, pH = - log[H+],
[H+] = 10-pH, pOH = - log[OH-], [OH-] = 10-pOH, pH + pOH = 14, pH = pKa + log
base .
acid
(13) make a buffer solution and explain how it minimizes changes in pH.
(14) be aware of factors to consider when selecting a buffer solution for a specific experimental
context (Resource Manual, p. 41-42).
(15) know how to determine the number of cells/mL using a haemacytometer and a spectrophotometer.
(16) design an experiment, with appropriate controls, to investigate the rate of electron flow from
photosystem II in isolated, illuminated chloroplasts.
(1) An image of the optical system of the Spectronic 20 (Resource Manual. P. 59) will be displayed and
a dismantled model is available for inspection.
(2) In your lab book, make a table with the following headings:
(Suggested values for T = 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, and 0.1)
1
Use the equation A = log to calculate the values for A.
T
Page 3: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)
Plot all of the data on one graph and note the wavelength at which absorbance is maximal (the max) for
each solution. [GRAPH REQUIRED].
Make a graph of absorbance vs. DCPIP concentration. The line should pass through the origin because
at zero DCPIP concentration the absorbance will be zero. This graph, a standard curve, exemplifies
Beer’s Law (i.e. up to a concentration limit, absorption is linearly related to the concentration of the
absorbing compound in solution). [GRAPH REQUIRED].
The spectrophotometer can therefore be used to determine an unknown solution concentration if you
first prepare a standard curve of known concentrations of that substance vs. absorbance.
The absorption coefficient (E) for DCPIP in deionized water can be calculated from the slope of the
standard curve:
If l = I cm, then A = Ec E = A/c
Calculate the value for E (the absorption coefficient). You will use this derived E value to analyse data
obtained in the next lab class.
Page 4: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)
We will be using the dye 2,6-dichlorophenolindophenol (DCPIP) as an artificial electron acceptor in our
experimental work on the rate of electron flow from photosystem II in isolated chloroplasts. In the
oxidized state, DCPIP is blue. When it is reduced by gaining electrons, DCPIP becomes colourless. The
reduction of DCPIP can therefore be monitored with a spectrophotometer by measuring the decrease in
absorbance as it is decolourized. To optimize the data obtained from using DCPIP as an electron
acceptor, we need to determine two things:-
(i) The wavelength of light that is maximally absorbed by DCPIP. (You determined the max for
DCPIP from its absorption spectrum in lab class #1).
(ii) The range of DCPIP concentration over which a plot of concentration vs. absorbance is linear. This
is determined by producing a standard curve of DCPIP concentration vs. absorbance at the wavelength
at which DCPIP shows maximum absorbance (λmax). (also done in lab class #1).
So far you have used the spectrophotometer to (i) produce characteristic absorption spectra for
compounds in solution and (ii) to determine an unknown concentration for a specific compound in
solution by preparing a standard curve of solute concentration vs. absorbance. Another major use of this
instrument (Lab Notes, p. 16) is to determine the rate of a (bio)chemical reaction.
1. Experiment to determine the rate of reduction of DCPIP by different concentrations of the reducing
agent, sodium hydrosulphite
1 1.5 3.5 0 50
2 1.5 3.5 10 40
3 1.5 3.5 20 30
4 1.5 3.5 30 20
5 1.5 3.5 40 10
6 CONTROL 1.5 3.5 - *
7 BLANK 0 5.0 - -
[Best practice: Instead of adding 1.5 mL of dye and 3.5 mL of D.I. water individually to each of tubes
1 to 6, make a bulk solution by mixing 10.5 mL of stock DCPIP and 24.5 mL of D.I. water in a beaker.
Then transfer 5 mL aliquots to each of tubes 1 – 6. This is more efficient and all of the components in
tubes 1 – 6 will be at exactly the same concentration].
Read the initial absorbance (zero time) of the solution in tube #1 tube just before you add the reducing
agent. Then start timing when you add the 50 L of 5.05 mM sodium hydrosulphite and invert the tube
three times to mix the contents. When 30 seconds have elapsed from the time that you added the
reducing agent, take the absorbance reading. Then take absorbance readings at 60, 90 and 120 seconds
from the time you added the reducing agent. Reactions in tubes 2 – 5 are started by adding 40, 30, 20
and 10 L of reducing agent respectively. * The control is started by adding 50 L of deionized water.
Page 5: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)
Draw a single graph to show the A (change in absorbance) vs. time for reactions in tubes 1 – 5 to
illustrate the kinetics of these reactions. [GRAPH REQUIRED]. Make a second graph to the rate of
DCPIP reduction (moles/minute) vs. the initial concentration of the reducing agent present in each
reaction. [GRAPH REQUIRED].
2. Calculating the average rate of reaction during the first minute of each reaction using A values and
the absorption coefficient (E) for DCPIP derived from the slope of the standard curve.
In the example standard curve for DCPIP shown below, the slope of the line (the absorption coefficient
or E) = 16,400 litres/mole/cm.
As an example, if the absorbance of a reaction mixture is 0.500 at the start of the reaction and declines to
0.336 in the first minute of the reaction, then the change in absorbance during the first minute (the
A/min) is 0.500 – 0.336 = 0.164/min.
0.45
0.4
0.35
0.3
0.25
A600
0.2
0.15
0.1
0.05
0
0 0.5 1 1.5 2 2.5
DCPIP concentration (m oles/L x 10-5)
The change in the DCPIP concentration during the first minute (c/min) can be calculated using the
absorption coefficient (E).
A / min 0.164
c/min = = = 1 x 10-5 moles/litre of DCPIP reduced/minute
E 16,400
Since you are using 5.0 mL of DCPIP solution plus 50 L of reducing agent, the actual amount of
5.05ml
DCPIP reduced in 1 minute = x (1 x 10-5 M) = 5.05 x 10-8 moles of DCPIP
1000ml / L
reduced/minute (average rate).
Preparation of a buffer solution. In the next class you will be measuring the rate of reduction of DCPIP
by illuminated chloroplasts isolated from spinach leaves. Like most biological reactions, this reaction is
very sensitive to changes in pH. Consequently, the reactions will be run in the presence of a buffer
solution to minimize changes in pH during the course of the reactions. In preparation for the next class,
an instructor will show your group how to make this buffer solution. Instructions for preparing this
buffer are on pages 40 – 41 in the Resource Manual.
Page 6: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)
The experience you have gained in using the Spectronic 20 to measure reaction rates will be used to
study a biological phenomenon: namely, the rate at which electrons flow from photosystem II in
illuminated, isolated spinach chloroplasts. A brief description of this phenomenon will be given in class
(you should already have reviewed the notes on light harvesting and electron transport in
photosynthesis, p. 43-45, Resource Manual). In summary, photons of light are absorbed by pigment
molecules in the antenna complexes surrounding photosystem II in the thylakoid membranes. Some of
the absorbed energy is funneled towards a pair of chlorophyll a molecules (the reaction centre) in
photosystem II which respond by emitting electrons that sequentially pass through other electron
acceptor molecules to photosystem I. The electrons lost from photosystem II are replaced by electrons
released in the oxidation of water. (See Resource Manual, p. 44-45).
Oxidized DCPIP can be used to intercept the electrons somewhere between photosystem II and
photosystem I and as it does so, it becomes reduced and loses its blue colour. The rate at which DCPIP
loses colour, detected by a decrease in its A600 with time, is proportional to the number of electrons that
the dye has accepted from photosystem II.
(1) Protocol for Chloroplast Isolation. (The chloroplast suspension will be prepared prior to the class).
Determination of Chlorophyll concentration in the chloroplast suspension. (See Resource Manual p. 21).
Briefly stated, 50 L of the suspended chloroplast preparation is added to 5 mL of 80% (v/v) aqueous
acetone and the mixture is centrifuged to remove debris.
The A652 of the resulting solution = _______. The concentration of chlorophyll in the chloroplast
suspension is calculated as follows:
mg of chlorophyll/mL = A652 ___________ x 100 34.5 mL/mgcm = _________mg/mL
= _________ g/L
Protocol.
(1) Prepare 10 tubes according to the following table but do not add the chloroplast suspension yet:
(2) There is a piece of masking tape marked in 5 cm increments from 0 up to 40 cm on the bench. Place
the lamp over the 0 cm ruling. Reaction tubes are supported in a 100 mL beaker. The 100 mL beaker can
be placed astride any of the ruled lines on the masking tape to vary the amount of light falling on the
reaction mixtures (see diagram below)
(3) Add a volume of chloroplast suspension containing 20 g of chlorophyll to the blank (tube #1) and
invert three times to mix. Use the blank solution to set the spectrophotometer to zero absorbance.
(4) Working quickly, add a volume of chloroplast suspension containing 20 g of chlorophyll to tube
#2, invert to mix and record the absorbance of the reaction mixture at 600 nm (= A600 at 0 minutes).
(5) Place the reaction mixture in a beaker astride the line at 5 cm from the lamp and start timing when
you turn on the lamp.
(6) After 55 seconds have elapsed, put the tube into the spectrophotometer and record the 1 minute
absorbance reading (do not turn off the lamp and do not stop timing).
(7) Immediately return the tube to the beaker at 5 cm from the lamp and repeat the procedure at 55
seconds into the second minute and 55 seconds into the third minute.
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(8) Repeat steps 4 – 7 with the remaining reaction mixtures at 10, 15, 20, 25, 30 35 or 40 cm away
from the lamp respectively.
(9) The control (tube #10) is treated identically except that it is not illuminated and is wrapped in
aluminum foil between A600 readings.
Rate calculations.
(a) Subtract the A600/ 2 minutes for the control (tube #10) from the A600/ 2 minutes for each of the
other reactions to derive the corrected A600/ 2 minutes for each reaction.
(b) Divide each corrected A600/ 2 minutes by 2 to derive the corrected A600/ minute value for
each reaction.
(c) Find the c/min by dividing each corrected A600/minute by the slope (the absorption coefficient or
E) of the new standard curve at pH 7.5 (see 4, below) to determine rates in terms of moles/litre of
DCPIP photoreduced/minute.
(d) Correct for the volume of the reaction mixture by multiplying each c/minute
by the total volume of the reaction mixture (5.0 mL plus the volume of the chloroplast
suspension[?]) expressed as a fraction of a litre, i.e:
The rate of photoreduction in each tube should be plotted against the photon fluence rate measured at
each experimental distance from the lamp (see 3, below). [GRAPH REQUIRED]
(See Resource Manual, p. 45. Light Ouantity: fluence measurements). Use the Li-Cor quantum sensor to
measure the photon fluence rate, in moles photons/m2/sec, at each distance from the lamp that you ran
the reactions. How does the photon fluence rate change with distance from the lamp? Make a graph of
photon fluence rate vs. distance from the lamp. [GRAPH REQUIRED].
(4) Make a new standard curve for DCPIP at pH 7.5. (see table below). [GRAPH REQUIRED]
Lab class #4: The effect of different concentrations of dichlorophenyl dimethyl urea (DCMU) on
the rate of photoreduction of DCPIP by isolated chloroplasts.
In this class you will again measure the rate of photoreduction of DCPIP but, this time, in the presence
of different concentrations of dichlorophenyl dimethyl urea (DCMU). DCMU is an agricultural
herbicide marketed as Diuron™. When working with reagents that affect the rates of biological
reactions, two questions are commonly asked:
(i) Is the relationship between the reaction rate and the reagent concentration linear or is there another
relationship between these two variables?
(ii) If the reagent inhibits the reaction, what concentration of the reagent reduces the reaction rate to 50%
of the rate in the absence of the inhibitor (the control rate)?
Protocol.
It is not necessary to repeat the protocol for the basic method. Just refer to the lab notebook page number
on which the protocol from the previous class is recorded. You should, however, include the following
information and the table below:
Room temperature = C.
The photon fluence rate used = moles of photons/m2/second.
The volume of chloroplast suspension that contains 20 g of chlorophyll = L.
[Best practice: Instead of adding 1.5 mL of dye, 1.0 mL of buffer and 2.5 mL of D.I. water individually
to each of tubes 2 to 8, make a bulk solution by mixing 12.0 mL of stock DCPIP, 8.0 mL of buffer
solution and 20. mL of D.I. water in a beaker. Then transfer 5 mL aliquots to each of tubes 2 – 8].
[BAR GRAPH REQUIRED].
At the start of lab class #4, the instructor will describe the components of the microscopes and show you
how to set them up for optimal optical performance. The instructor will also explain the logic of using a
haemacytometer (Resource Manual, p. 18 – 20, Lab notes, p. 18-19) to determine the number of
cells/mL in liquid suspension cultures of cells.
Page 10: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)
Lab class #5: In this class you will use a microscope, a haemacytometer and a spectrophotometer to
determine the number of cell/mL in a liquid suspension cell culture. These methods will be used to start
a simple experiment that will be completed in lab class #6.
Centrifugation.
(a) Place tubes in the swinging buckets in the centrifuge. Make sure that the rotor is balanced.
Close the lid.
(b) Turn on the centrifuge at full speed (# 7) and wait for 30 seconds before turning off the power.
(c) Wait for the rotor to stop completely before removing the tubes.
(d) Carefully discard the supernatant (growth medium) and add enough deionized water to bring the
total volume to 5 mL.
(e) Thoroughly resuspend the cells in the deionized water by aspiration with a Pasteur pipette.
(6) Multiply the average #cells/square x 250,000 = cells/mL. (Place this value next to
the * in the following table and use this value to calculate the #cells/mL in tubes # 2, 3 and 4).
(7) Read the “A400” for tubes #1 – 4 and make a standard curve to show the relationship between the
“A400” and the #cells/mL. [GRAPH REQUIRED] Calculate the slope of the graph. Slope =
(a) 1A and 2A: 65.0 ml of 1.17% (w/v) yeast carbon base medium (no asparagine).
(b) 1B and 2B: 65.0 ml of 1.17% (w/v) yeast carbon base medium + 0.01% (w/v) of asparagine.
(c) 1C and 2C: 65.0 ml of 1.17% (w/v) yeast carbon base medium + 0.05% (w/v) of asparagine.
On day zero (lab class #5), the initial number of cells/ml in one of each pair of cultures will be
determined using both the haemacytometer and the spectrophotometer.
The remaining 3 duplicate cultures will be maintained at room temperature for 48 hours. In the
final lab you will estimate the number of cells/ml in these cultures using the haemacytometer and
the spectrophotometer to determine the increase in the number of cells/ml in each treatment
during the 24 hour incubation period.
Final #cells/ml
Yield = Original #cells/ml
At the end of the experiment, produce a bar chart for the yield of cells against asparagine
concentration. At each concentration, place two bars: one to show the yield as determined by
using the haemacytometer, and one to show the yield as determined by spectrophotometry.
Page 12: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
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(a) Pellet the cells in 1A, 1B and 1C in the centrifuge, discard supernatant (growth medium), resuspend cells up
to 5 ml in deionized water.
(c) In turn, load samples of resuspended 1A, 1B and 1C on to the haemacytometer. In each sample,
count the number of cells you see in 10 of the 0.04 mm2 squares:
# of cells/mL in culture
Method 1a 1b 1c
Spectrophotometer
Haemacytometer
Lab class #6: Your laboratory notebook should be handed in at the beginning of lab class #6. It should be
complete up to and including the work for labs 1 – 5. After the second lab quiz, the instructor will explain the
meaning of resolution in microscopy and the use of Abbe’s equation to calculate the theoretical limit to resolution
in microscopy. We will then complete the experimental work started in lab class #5.
0.612
Use the equation d =
n sin
to calculate d, the theoretical resolution of your microscope for both the x10 and x 40 objective
lenses. Use a wavelength of 550 nm and give the answer in m. The numerical aperture (N.A.)
value (n sin ) is stamped on each objective lens. N.A. for x 10 objective lens = 0.25 and for the x
40 objective lens = 0.66.
Page 14: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)
(a) Pellet the cells in 2A, 2B and 2C in the centrifuge, discard supernatant (growth medium), resuspend cells up
to 5 ml in deionized water.
(b) Dilute cell suspensions as necessary:
(c) In turn, read the “A400” for undiluted 2A, -fold diluted 2B and -fold diluted 2C:
(d) In turn, load samples of resuspended undiluted 2A, -fold diluted 2B and -fold diluted 2C on to the
haemacytometer. In each sample, count the number of cells you see in 10 of the 0.04 mm2 squares:
Multiply the average #cells/square x 250,000 = x d.f. = final #cells/mL for 2B.
Multiply the average #cells/square x 250,000 = x d.f. = final #cells/mL for 2C.
Yield Calculations.
By haemacytometer By Spectrophotometer
(i) 2A (iv) 2A
1A 1A
(ii) 2B (v) 2B
1B 1B
(iii) 2C (vi) 2C
1C 1C
Page 16: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)
However, you will first spend some time on seeing how the
spectrophotometer is constructed and in understanding the
theoretical aspects that are important in its use. You will then
gain practical experience using the instrument in exercises
designed to demonstrate its major uses:
Review your first year biology notes and the relevant section in
the textbook to refresh your memory on the mechanism of the
light-dependent stage of photosynthesis.
Page 17: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)
Rapid estimates of cell numbers per unit volume of culture can be made
with a spectrophotometer. A beam of light passing through a suspension
of cells is scattered in proportion to the turbidity of the suspension. The
turbidity of the suspension, over a limited range of cell concentration, is
proportional to the number of cells per unit volume of suspension. To use
this method, it is first necessary to make a standard curve of absorbance at
a given wavelength versus the number of cells per millilitre. The number
of cells per ml must be determined by some other method such as
haemacytometry.
Figure 1. The haemacytometer is a thick glass slide with an H-shaped gutter etched on the
upper surface. A mirrored rectangle is located on either side of the cross bar of the H-shaped
gutter. A very small grid is located at the centre of each mirrored rectangle. Each grid looks
like the image below.
Figure 2. Each grid is divided into 9 squares that are 1 mm x 1 mm. The most complex of
these 9 squares is located at the centre of the grid. For counting yeast cells, we will use only
the centre square. The centre square is further subdivided into 25 smaller squares that are 0.2
mm x 0.2 mm. These 25 squares each contain 16 smaller squares surrounded by three
parallel lines. To use the haemacytometer, first put the glass cover on the chamber and then
load a sample of cells suspended in a liquid. Count the number of cells in a sample of ten 0.2
mm x 0.2 mm squares. Five counts are made on one grid using the squares indicated by X in
figure 2. The other five counts are made in the corresponding squares on the second grid.
Page 19: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)
so, make the 10 counts following the rules described above, divide by 10 to find the average number of
cells per square and multiply by 250,000 to find the number of cells/mL in the sample loaded on to the
chamber.