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Page 1: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)

(January - April 2018)

Assigned reading, problems, quizzes, graphing exercises and schedule.


(Lab classes #1-4)
(1) Lab Notes p. 1-9
(2) Lab Notes p. 16 Overview
(3) Resource Manual p. 28-32 Graphs for print publication.
(4) Resource Manual p. 35-42 Ionic product of water, pH and buffers
(solve problems)*
(5) Resource Manual p. 42-44 Light
(6) Resource Manual p. 50-51 Pipetting
(7) Resource Manual p. 58-61 Spectrophotometry (try calculations on p.61)*
(8) Resource Manual p. 92-94 Lab Notebook and Protocols
(9) Resource Manual p. 6 – 10 Philosophy and ground rules
(10) Resource Manual p. 67-68 Units of Measure & Exponents (solve problems)*
(11) Resource Manual p. 44-46 Light reactions & Light quantity
(12) Resource Manual p. 21 Chlorophyll
(13) Resource Manual p. 61-64 Suspensions, solutions, concentrations
(solve problems on pp. 63-64)*
(Lab classes #5-6)
(14) Lab Notes p. 10 - 15
(15) Lab Notes p. 17 Overview
(16) Resource Manual p. 18-20 Cell counting
(17) Resource Manual p. 20-21 Centrifugation
(18) Resource Manual p. 46-49 Microscopy
(19) Resource Manual p. 64-67 Dilutions (solve problems on pp.66 - 67)*
* Solutions to problems in the Resource Manual are on p.54-57 of the Resource Manual.

Lab class #1 Lab class #3 Lab class #5

Graph assignment #1 due Graph assignment #2 due

Lab class #2 Lab class #4 Lab class #6


Lab Quiz #1: 4 questions Hand in lab notebook at the
Graph assignment #1 set on (7), (10) and (13). start of class.
Graph assignment #2 set Lab Quiz #2: 5 questions
on (4) & (19)

Grading for this unit.


A total of 14% of the course mark will be available for the following:
Graph assignment #1 1%
Graph assignment #2 2%
Lab Quiz #1 4%
Lab Quiz #2 5%
Lab Book 2%
Total 14%
Page 2: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)

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 = Ecl 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.

Lab Class #1: Introduction to spectrophotometry (Resource Manual, p. 58-61).

(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:

Transmittance (T) %T Log %T Absorbance (A)

1.0 100 2.0 0


0.9 90 1.95 0.046
etc. etc. etc. etc.

(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)

(a) Use the data in the table to make a graph of A vs. T.


(b) Use the data in the table to make a graph of A vs. Log % T. (All graphs must have a descriptive
title). [GRAPHS REQUIRED].

(3) Absorption Spectra. Spectrophotometry can be used to identify or characterize compounds in


solution by producing an absorption spectrum. An absorption spectrum is a graph of the absorbance
values of a chemical compound in solution measured at a series of different wavelengths. We will make
an absorption spectrum for a food colouring dye and a solution of 2, 6- dichlorophenol indophenol
(DCPIP).
Protocol.
Set up the spectrophotometer by following the instructions on page 60 in the Resource Manual.
Solutions of a red food dye at a concentration of 0.03% (v/v) (Resource Manual, p. 61) and DCPIP at a
concentration of 2 x 10-5 moles/litre will be provided. Record the absorbance value for each solution at
20 nm increments over the range of 360 to 600 nm. YOU MUST ZERO THE INSTRUMENT EACH
TIME THAT YOU CHANGE THE WAVELENGTH.

Plot all of the data on one graph and note the wavelength at which absorbance is maximal (the max) for
each solution. [GRAPH REQUIRED].

(4) Standard Curve.


Protocol.
Use the stock DCPIP solution (1 x 10-4 mol/L) to make a dilution series according to the following table:

Tube # 1 x 10-4 M Deionised DCPIP Absorbance


DCPIP (mL) water (mL) concentration at max (= nm)
1 2.0 3.0 4 x 10-5 M
2 1.5 3.5
3 1.0 4.0
4 0.5 4.5
5 0 5.0
Read the absorbance (A) of each solution the wavelength of maximum (max ) absorbance for DCPIP.

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 = Ec 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)

Lab Class #2: DICHLOROPHENOL INDOPHENOL (DCPIP)

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

Protocol. Room temperature = C


Prepare 7 tubes according to the following table:

Tube # 1 x 10-4 M Deionized Deionized 505 mM sodium


______ DCPIP (mL) water (mL) water (L) hydrosulphite (L)

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)

Lab Class #3: Experimental determination of rates of DCPIP photoreduction by a suspension of


chloroplasts isolated from spinach leaves.

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).

(i) Weigh out 25 grams of deribbed spinach leaves.


(ii) Chop leaves into small pieces with scissors and place them in a chilled Waring blender with 100 mL
cold isolation buffer (the recipe for this buffer is given in qu. 9 on p. 64, Resource Manual) to which
0.2L/mL of -mercaptoethanol has been added.
(iii) Blend the leaves with 3 x 5 second bursts at full speed in the blender.
(iv) Filter the resulting suspension through 4 layers of cheesecloth into a chilled beaker
(Volume = approximately 75 mL).
(v) Centrifuge the filtrate at 1300 x g. (see Resource Manual, p. 20-21) for 5 minutes.
(vi) Discard the supernatant and add isolation buffer to the pellet to a total volume equal to ~0.5 mL/g
spinach leaves used. Resuspend the pellet with a paint brush. This suspension is the chloroplast
preparation.

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/mgcm = _________mg/mL

= _________ g/L

the volume of chloroplast suspension that contain 20 g of chlorophyll:

= 20 g  ________ g/L = __________ L of the chloroplast suspension.


Page 7: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)

(2) Determination of the rate of photreduction of DCPIP by an isolated chloroplast suspension.

Protocol.
(1) Prepare 10 tubes according to the following table but do not add the chloroplast suspension yet:

Tube # 1 x 10-4 M Reaction Deionized Chloroplast


____________ DCPIP (mL) buffer (mL) water (mL) suspension (L*)
1 (blank) 0 1.0 4.0 *
2 1.5 1.0 2.5 *
3 1.5 1.0 2.5 *
4 1.5 1.0 2.5 *
5 1.5 1.0 2.5 *
6 1.5 1.0 2.5 *
7 1.5 1.0 2.5 *
8 1.5 1.0 2.5 *
9 1.5 1.0 2.5 *
10 (control) 1.5 1.0 2.5 *
* a volume of chloroplast suspension containing 20 g of chlorophyll.

(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.
Page 8: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)

(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:

Ac/minute x 5.0? mL = moles of DCPIP photoreduced/minute.


1000 mL/L
(e) Divide by the number of g of chlorophyll in the volume of chloroplast suspension used (typically
20 g) = moles of DCPIP photoreduced/minute/g of chlorophyll.

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]

(3) Measuring the photon fluence rate with a quantum sensor.

(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]

Tube # 1 x 10-4M DCPIP 5x reaction buffer Water [DCPIP] A600


(mL) (mL) (mL)
1 2.0 1.0 2.0 4 x 10-5M
2 1.5 1.0 2.5
3 1.0 1.0 3.0
4 0.5 1.0 3.5
5 Blank 0 1.0 4.0 0
Page 9: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)

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.

Tube # 1 x 10-4M 5 x reaction Deionized Methanol 30.3 M


DCPIP(mL) Buffer(mL) Water (mL) (L) DCMU(L)
1 Blank 0 1.0 4.0 50 0
2 Control-dark 1.5 1.0 2.5 50 0
3 Control-no DCMU 1.5 1.0 2.5 50 0
4 1.5 1.0 2.5 40 10
5 1.5 1.0 2.5 30 20
6 1.5 1.0 2.5 20 30
7 1.5 1.0 2.5 10 40
8 1.5 1.0 2.5 0 50

[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.

Cells and cell culture medium.


Each group will be provided with 5 mL of a suspension of yeast cells (Saccharomyces cerevisiae). The
cell cultures will have been incubated for 48 hours at room temperature, on a rotary shaker, in a sterile
culture medium called yeast carbon base (YCB). YCB contains all of the nutrients needed to sustain the
cells except for a source of nitrogen. To provide the necessary nitrogen source, the cultures are
supplemented with 0.05% (w/v) of the amino acid asparagine.
Because YCB absorbs light to some extent, it is useful to separate the cells from the culture medium and
resuspend them in deionized water. This allows us to use deionized water as the reference blank when
working with the spectrophotometer. We can separate the cells from the growth medium using a
centrifuge (Resource Manual, p. 20 – 21).

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.

Care of the haemacytometer.


After each use, wash the haemacytometer chamber and its cover with warm water containing a small
quantity of detergent. Rinse with deionized water and place the chamber and cover on a paper towel on a
flat surface. Gently pat them dry with a Kimwipe™.

Protocol: Determination of the number of cells/mL using a haemacytometer and preparation of a


standard curve of the apparent absorbance at 400 nm (‘A400’) vs. the number of cells/mL (#cells/mL)
(1) Pellet cells in centrifuge (see Centrifugation – above), discard supernatant (growth medium), resuspend cells up
to 5 ml in deionized water.
(2) Make a 10-fold dilution of the resuspended cells (1.5 mL of cell suspension + 13.5 mL of D.I. water).
(3) Load a sample of the 10-fold diluted cell suspension on to the haemacytometer.
(4) Count the number of cells you see in 10 of the 0.04 mm2 squares (as explained in class):
First grid #cells/square Second grid #cells/square

Top left ___ Top left ___

Top right ___ Top right ___

Middle ___ Middle ___

Bottom left ___ Bottom left ___

Bottom right ___ Bottom right ___

(5) Calculate the average # cells in the 10 squares = cells/square.


Page 11: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)

(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 =

10-fold diluted Deionized


Tube # suspension (mL) water (mL) # cells/mL ‘A400’
1 5.0 0 *
2 3.0 2.0
3 2.0 3.0
4 1.0 4.0
5 Blank 0 5.0

* # cells/ml from your first haemacytometer count.


Experiment to determine the yield of Saccharomyces cerevisiae cells grown for 48 hours in
media containing different concentrations of nitrogen (i.e.asparagine).
Protocol:
Three duplicate pairs of cultures containing the following media will each be inoculated with 3.0
ml of a two day old culture of S. cerevisiae cells on the first day of the experiment.

(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)
(January - April 2018)

(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.

(b) In turn, read the “A400” for 1A, 1B and 1C:

“A400” for 1A =  slope of standard curve = cells/mL

“A400” for 1B =  slope of standard curve = cells/mL

“A400” for 1C =  slope of standard curve = cells/mL

(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:

Counts for 1A.


First grid #cells/square Second grid #cells/square

Top left ___ Top left ___

Top right ___ Top right ___

Middle ___ Middle ___

Bottom left ___ Bottom left ___

Bottom right ___ Bottom right ___

Calculate the average # cells in the 10 squares = cells/square.

Multiply the average #cells/square x 250,000 = original #cells/mL for 1A.

Counts for 1B.


First grid #cells/square Second grid #cells/square

Top left ___ Top left ___

Top right ___ Top right ___

Middle ___ Middle ___

Bottom left ___ Bottom left ___

Bottom right ___ Bottom right ___

Calculate the average # cells in the 10 squares = cells/square.

Multiply the average #cells/square x 250,000 = original #cells/mL for 1B.


Page 13: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)

Counts for 1C.


First grid #cells/square Second grid #cells/square

Top left ___ Top left ___

Top right ___ Top right ___

Middle ___ Middle ___

Bottom left ___ Bottom left ___

Bottom right ___ Bottom right ___

Calculate the average # cells in the 10 squares = cells/square.

Multiply the average #cells/square x 250,000 = original #cells/mL for 1C.

Day 0: Summary: original # cells/mL

# 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.

Resolution in microscopy.(Resource Manual, p.48 - 49)

Calculation of the theoretical resolution of your microscope.

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)

Protocol to complete the experimental work.

(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:

Dilution factor for 2A = -fold.

Dilution factor for 2B = -fold.

Dilution factor for 2C = -fold.

(c) In turn, read the “A400” for undiluted 2A, -fold diluted 2B and -fold diluted 2C:

“A400” for 2A =  slope of standard curve = x (d.f.) = cells/mL

“A400” for 2B =  slope of standard curve = x (d.f.) = cells/mL

“A400” for 2C =  slope of standard curve = x (d.f.) = cells/mL

(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:

Counts for 2A.


First grid #cells/square Second grid #cells/square

Top left ___ Top left ___

Top right ___ Top right ___

Middle ___ Middle ___

Bottom left ___ Bottom left ___

Bottom right ___ Bottom right ___

Calculate the average # cells in the 10 squares = cells/square.

Multiply the average #cells/square x 250,000 = final #cells/mL for 2A.


Page 15: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)

Counts for 2B.


First grid #cells/square Second grid #cells/square

Top left ___ Top left ___

Top right ___ Top right ___

Middle ___ Middle ___

Bottom left ___ Bottom left ___

Bottom right ___ Bottom right ___

Calculate the average # cells in the 10 squares = cells/square.

Multiply the average #cells/square x 250,000 = x d.f. = final #cells/mL for 2B.

Counts for 2C.


First grid #cells/square Second grid #cells/square

Top left ___ Top left ___

Top right ___ Top right ___

Middle ___ Middle ___

Bottom left ___ Bottom left ___

Bottom right ___ Bottom right ___

Calculate the average # cells in the 10 squares = cells/square.

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)

OVERVIEW – EXPERIMENTAL SPECTROPHOTOMETRY

Introduction – Direct observation is not always possible in biological research: the


biologist frequently has to use specialized equipment to follow the
course of a biological process. An understanding of how the
equipment works, and what it can and cannot do, is often important
for producing reliable, reproducible information. In other words,
many machines can generate numbers but these will only be
meaningful if the instrument is correctly set up, properly operated and
measuring in the range for which it is designed.

Spectrophotometry – In this rotation, you will ultimately use a


spectrophotometer to study the rate of electron flow from
photosystem II in isolated chloroplasts.

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:

(1) to produce absorption spectra that can be used to


characterize or identify compounds in solution.

(2) to make quantitative determinations of the


concentration of materials in solution by reference to
your own standard curves.

(3) to study the rate at which (bio)chemical reactions


proceed.

Having worked through the exercises, you should have the


experience and skill required to use this technology in a more
independent investigation.

With the assistance of instructors and colleagues, you will


carry out experiments to investigate factors affecting the
rate of electron flow in isolated chloroplasts.

Important technical and background information can be found


in the Spectrophotometry section of the Resource Manual and
your first year biology textbook.

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)

Overview: Estimation of cell numbers.

Introduction When working with cells in culture media, it is frequently necessary to


know how many cells are present. Direct counts of the number of cells in
a sample of culture medium can be made using a particular kind of
microscope slide, the haemacytometer. On the upper mirrored surface of
the haemacytometer are precisely etched lines that intersect to form
squares of known area. With an appropriate type of cover in place, a
known volume of fluid is enclosed above each square. Therefore, if the
number of cells contained in the liquid above each square is counted, the
number of cells in a given volume of the culture from which the sample
was taken can be calculated.

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.

In this class you will participate in an experiment to determine the


yield of cells over a given time period as a function of the nutritional
quality of the medium or other experimental variables.
Page 18: Biology 2290G Lab Notes (Dean Unit, North Campus Building, Room 325)
(January - April 2018)

Appendix 1. Haemacytometer structure and the logic of its use.

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)

Rules for counting.


(a) There must be a minimum (on average) of 10 cells per square to be counted.
(b) Do not count cells that touch any of the three parallel lines on the top or left side of the
square.
(c) Count all cells that touch any of the three parallel lines on the bottom or right side of the
square.
Calculate the average number of cells per square. At this point you have determined the
average number of cells observed in a 0.2 mm x 0.2 mm square. We now need to know what
volume is occupied above a 0.2 mm x 0.2 mm square and how many times this volume fits
into a millilitre.
Area of a square = 0.2 mm x 0.2 mm = 0.04 mm2
When the glass cover is in place, the depth of the chamber above the grid is 0.1 mm.
∴ Volume above a square = 0.04 mm2 x 0.1 mm = 0.004 mm3
(1 mL = 1 cm3 = 1000 mm3)
∴ 1000 mm3 = 250,000 times
0.004 mm3

i.e. the volume above a 0.04 mm2 square is 1/250,000 mL.

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.