Advanced Biology With Vernier: Second Edition

SECOND EDITION
Advanced Biology with Vernier
Experiments for AP* and College Biology
Experiments contributed by
David Masterman
Kelly Redding
John Melville, Ph.D.
Jack Randall
Diana Gordon
Steven L. Gordon, M.D.
Michael Collins
Bio-Rad Laboratories, Inc.
WARD’S Natural Science
Vernier Software & Technology

13979 S.W. Millikan Way • Beaverton, OR 97005-2886
Toll Free (888) 837-6437 • (503) 277-2299 • FAX (503) 277-2440
info@vernier.com • www.vernier.com
SECOND EDITION
Experiments for AP and College Biology

Proper safety precautions must be taken to protect teachers and
students during experiments described herein. Neither the authors nor
the publisher assume responsibility or liability for the use of material
described in this publication. It cannot be assumed that all safety
warnings and precautions are included. Teachers must follow local
regulations concerning safe handling, use, and disposal of the materials
and chemicals associated with the experiments included in this manual.
Copyright © 2015 by Vernier Software & Technology. All rights reserved. Purchase
of this book and accompanying CD includes a site license entitling the teachers at
one school to modify and reproduce student experiments for use by students at that
one school only. No part of this book or its accompanying CD may be used or
reproduced in any other manner without written permission of the authors except in
the case of brief quotations embodied in critical articles or reviews.
Logger Pro, Vernier LabQuest, Vernier LabQuest Mini, Vernier LabPro, and other
marks shown are our registered trademarks in the United States. All other marks
not owned by us that appear herein are the property of their respective owners, who
may or may not be affiliated with, connected to, or sponsored by us.
*AP and Advanced Placement Program are registered trademarks of the College Entrance Examination Board,
which was not involved in the production of and does not endorse this product.
Published by
Vernier Software & Technology
13979 SW Millikan Way
Beaverton, OR 97005-2886
(888) 837-6437
(503) 277-2299
FAX (503) 277-2440
info@vernier.com
www.vernier.com
ISBN 978-1-929075-20-1
Second Edition
Second Printing
Printed in the United States of America
Contents
Sensors Used in Experiments............................................................................................ vii

Preface .............................................................................................................................. ix
Experiments
1 A. Diffusion through Membranes .................................................................................... 1A-1
B. Osmosis.....................................................................................................................1B-1
2 Enzyme Action: Testing Catalase Activity (Method 1–O2 Gas Sensor)....................... 2-1 (O2)
Enzyme Action: Testing Catalase Activity (Method 2–Gas Pressure) ........2-1 (Gas Pressure)
3 Mitosis and Meiosis .......................................................................................................... 3-1
4 A. Plant Pigment Chromatography ................................................................................. 4A-1
B. Photosynthesis ...........................................................................................................4B-1
5 Cell Respiration (Method 1–CO2 and O2) .................................................... 5-1 (CO2 and O2)
Cell Respiration (Method 2–CO2)............................................................................ 5-1 (CO2)
Cell Respiration (Method 3–O2) .................................................................................. 5-1(O2)
Cell Respiration (Method 4–Gas Pressure) ...............................................5-1 (Gas Pressure)
6 A. Easy Transformation of E.coli using the pGLOTM Bacterial Transformation Kit ........... 6A-1
B. Analysis of Precut Lambda DNA (Option 1) ................................................ 6B-1 (Lambda)
B. Forensic DNA Fingerprinting (Option 2)..................................................6B-1 (Fingerprint)
7 Genetics of Drosophila ..................................................................................................... 7-1
8 Population Genetics and Evolution ................................................................................... 8-1
9 Transpiration .................................................................................................................... 9-1
10 A. Blood Pressure as a Vital Sign ................................................................................. 10A-1
B. Heart Rate and Physical Fitness .............................................................................. 10B-1
11 Animal Behavior ............................................................................................................. 11-1
12 A. Dissolved Oxygen in Water ...................................................................................... 12A-1
B. Primary Productivity ................................................................................................. 12B-1
13 The Visible Spectra of Plant Pigments............................................................................ 13-1
14 Determination of Chlorophyll in Olive Oil ........................................................................ 14-1
15 Enzyme Analysis using Tyrosinase ................................................................................. 15-1
16 Introduction to Neurotransmitters using AChE ................................................................ 16-1
17 Macromolecules: Experiments with Protein .................................................................... 17-1
v
Appendixes
A Using the CD .....................................................................................................................A-1
B Using TI Connect: Loading EasyData and Capturing Calculator Screen Images.................B-1
C Using Logger Pro to Transfer Data to a Computer ............................................................ C-1
D Safety Information ........................................................................................................... D-1
E Vernier Products for Advanced Biology..............................................................................E-1
F Equipment and Supplies .................................................................................................... F-1
G Bio-Rad Products for Advanced Biology ........................................................................... G-1
H Correlations to AP Biology Curriculum .............................................................................. H-1
Index ................................................................................................................................ Index-1
vi
Sensors Used in Experiments
White or Blue Digital

Bioimaging System
Dissolved Oxygen
Blood Pressure
Colorimeter or
Gas Pressure
Spectrometer
Conductivity
Temperature
Heart Rate
ProScope
CO2
O2
1A Diffusion through Membranes 1
1B Osmosis 1
Enzyme Action (Method 1) 1
2
Enzyme Action (Method 2) 1
3 Mitosis and Meiosis No sensor
4A Plant Pigment Chromatography No sensor
4B Photosynthesis 1
Cell Respiration (Method 1) 1 1
Cell Respiration (Method 2) 1
5
TM
6A pGLO Bacterial Transformation No sensor
Analysis of Precut Lambda DNA (Option 1) 1 1*
6B
Forensic DNA Fingerprinting (Option 2) 1 1*
7 Genetics of Drosophila No sensor
8 Population Genetics and Evolution No sensor
9 Transpiration 1 O
10A Blood Pressure as a Vital Sign 1
10B Heart Rate and Physical Fitness 1
11 Animal Behavior No sensor
12A Dissolved Oxygen in Water 1 1
12B Primary Productivity 1
+
13 The Visible Spectra of Plant Pigments 1
+
14 Determination of Chlorophyll in Olive Oil 1
15 Enzyme Analysis using Tyrosinase 1
16 Introduction to Neurotransmitters using ACHe 1
17 Macromolecules: Experiments with Protein 1
* - Included in the Blue or White Digital Bioimaging System

O - Optional
+ - Spectrometer only
vii
viii
Preface
This book contains experiments designed to help instructors incorporate technology into their
AP † Biology, Advanced Biology, and college General Biology courses. The first twelve
experiments have a direct one-to-one correlation with the twelve labs prescribed in the
2001 College Board’s AP Biology Lab Manual. Most of these experiments use Vernier sensors to
collect, display, print, graph, and analyze the data. Several others use Bio-Rad kits in conjunction
with Vernier technology such as our Digital Bioimaging Systems or one of our Spectrometers.
Others use no sensors at all, but are included to provide the convenience of a complete set of
experiments for those teaching the AP Biology curriculum.
There are three different versions of each experiment in this book:

• Computer (printed and on CD) These versions are written with directions for computer-
based data collection using Vernier Logger Pro software on a Windows or Macintosh
computer.
• LabQuest (on CD) These version are written with directions for LabQuest-based data
collection using the LabQuest App on a LabQuest interface.
• Calculator (on CD) These versions are written with directions for TI graphing calculator-
based data collection using the EasyData App (for use with Vernier LabPro, TI CBL 2, or
Vernier EasyLink).
The files for each experiment included on the CD in the back-inside cover of this book are
Microsoft Word® files. See Appendix A for details about using this CD. In the case of computer
users, the Word files on the CD are identical to the printed versions of the student experiments in
this book. Users of Vernier LabQuests and TI graphing calculators can print their respective
versions of the experiments from the CD Word files.
In all cases, purchase of this book entitles you to use these word-processing files in any way you
wish in your courses. This includes making edits to our files to match your own teaching style or
lab investigation pedagogy. We also give you permission to include these files or edited versions
in your course lab manual, as long as this manual is not distributed beyond your school
boundaries.
Following the student instructions for each experiment in the printed version of this book is a
Teacher Information section containing sample results, answers to questions, directions for
preparing solutions, and other helpful hints regarding the planning and implementation of a
particular experiment. The Teacher Information sections are applicable to all three data-collection
platforms and are therefore not included on the CD.
The appendices include valuable information that can help make you more comfortable with your
initial use of this equipment. Here is a short summary of the information available in each
appendix:
• Appendix A tells you how to use the word-processing files found on the CD.
• Appendix B tells you how to use TI Connect to load the EasyData App onto your calculator
and capture calculator screen images.
• Appendix C tells you how to use Logger Pro 3 software to display or print graphs and data
tables after importing data from LabQuest or a TI graphing calculator.
• Appendix D provides details about safety information.
• Appendix E provides information on Vernier products for advanced biology.
†
AP and Advanced Placement Program are registered trademarks of the College Entrance Examination Board,
which was not involved in the production of and does not endorse this product.
ix
• Appendix F provides a comprehensive list of all the equipment and supplies used in these
experiments.
• Appendix G provides information on purchasing Bio-Rad equipment and supplies.
• Appendix H provides correlations to the 2001 College Board’s AP Biology Curriculum.
We would like to thank Bio-Rad Laboratories Inc. and WARD’S Natural Science for their
invaluable contributions to this book. Thanks to Alex Plank, Gretchen Stahmer DeMoss, and
Robyn Johnson for their hard work that made this book a reality. And finally, thank you to the all
teachers who practically begged us to create this book for them. We are grateful to have such
dedicated and passionate professionals in the classroom.
Vernier Software & Technology Biology Department
x
Computer
Diffusion through Membranes

1A
Diffusion is a process that allows ions or molecules to move from where they are more
concentrated to where they are less concentrated. This process accounts for the movement of
many small molecules across a cell membrane. Diffusion is the process by which cells acquire
food and exchange waste products. Oxygen, for instance, might diffuse in pond water for use by
fish and other aquatic animals. When animals use oxygen, more oxygen will diffuse to replace it
from the neighboring environment. Waste products released by aquatic animals are diluted by
diffusion and dispersed throughout the pond.
It is important to consider how the rate of diffusion of particles might be affected or altered.
• Diffusion may be affected by the steepness of the concentration gradient (the difference
between the number of ions or molecules in one region of a substance and that in an
adjoining region). The direction that a diffusing molecule or ion might travel in any
particular direction is random. While the particles are diffusing, is there a net movement
from where they are concentrated to where they are less concentrated?
• Diffusion might be affected by other different, neighboring particles. For instance, if
oxygen diffuses towards a single-celled pond organism at a certain rate, will that rate be
altered if some other molecule suddenly surrounded the organism? Would the presence of
other molecules block or enhance the diffusion of a molecule, or would the molecule’s rate
be independent of particles that do not alter the concentration gradient?
One way to measure the rate of diffusion of ions is to monitor their concentration in solution over
a period of time. Since ions are electrically charged, water solutions containing ions will conduct
electricity. A Conductivity Probe is capable of monitoring ions in solution. This probe however,
will not measure the amount of electrically neutral molecules dissolved in water. Salts, such as
sodium chloride, produce ions when they dissolve in water. If you place a salt solution in a
container such as dialysis tubing, the salt can travel through the very small holes in the tubing.
When dialysis tubing containing a solution of salt ions is placed into a beaker of water, the ions
can diffuse out of the tubing and into the surrounding water. In this way, you will be able to
measure the diffusion of salts in a solution of water and determine how concentration gradients
and the presence of other particles affect the diffusion of the salt across a membrane.
OBJECTIVES
In this experiment, you will
• Use a Conductivity Probe to measure the ionic concentration of various solutions.
• Study the effect of concentration gradients on the rate of diffusion.
• Determine if the diffusion rate for a molecule is affected by the presence of a second molecule.
MATERIALS
computer dialysis tubing, 2.5 cm × 12 cm
Vernier computer interface dropper pipet or Beral pipet
Logger Pro scissors
Vernier Conductivity Probe stirring rod
three 18 × 150 mm test tubes with rack 5% sucrose (table sugar) solution
1%, 5%, and 10% salt water dental floss or clamp
400 mL beaker ring stand and utility clamp
Advanced Biology with Vernier 1A - 1

Computer 1A
Figure 1
PROCEDURE
1. Connect the Conductivity Probe to the computer interface. Check to be sure the Conductivity
Probe is set to the intermediate setting, 2000 µS/cm (equivalent to a concentration of
1000 mg/L).
2. Prepare the computer for data collection by opening the file “01A Membrane Diffusion” from
the Advanced Biology with Vernier folder of Logger Pro.
Part I Concentration gradients

3. Test whether different concentration gradients affect the rate of diffusion. You will place
three solutions of differing salt concentrations (1%, 5%, and 10%) in distilled water. Each
salt solution will be placed in a dialysis tube and allowed to diffuse into the surrounding
water. When salt diffuses, the conductivity of water in the beaker will increase.
4. In Table 1, predict what you believe will happen in this set of experiments. How will the rate
of diffusion change when a 10% salt solution is placed in contact with pure water compared
to when a 1% salt solution is placed in contact with pure water?
5. Prepare the dialysis tubing. Obtain a piece of wet dialysis tube and a dialysis tubing clamp or
a short (approximately 10 cm) length of dental floss. Using the clamp or floss, tie one end of
the tube closed about 1 cm from the end, as shown in Figure 2.
1A - 2 Advanced Biology with Vernier

6. Place a 1% salt solution into a section of dialysis

tubing. To do this,
a. Obtain about 15 mL of a 1% salt water solution in
a test tube.
b. Using a funnel or Beral pipet, transfer about 10 mL
of the 1% salt water into the dialysis tube, as in
Figure 2. Note: To open the tube, you may need to
rub the tubing between your fingers a bit.
c. Tie off the top of the dialysis tube with a clamp or
a new length of dental floss. Try not to allow any
air into the dialysis tube. The tube should be very
firm after it is tied or clamped. Trim off any excess
dental floss extending more than 1 cm from either
knot.
d. Wash the outside of the tubing with tap water
thoroughly, so that there is no salt water adhering
to the tubing. Figure 2
7. Place 300 mL of water into a 400 mL beaker. If the conductivity of the tap water is low
(50 mg/L or less), use tap water to fill the beaker. Otherwise, use distilled water.
8. Position the Conductivity Probe into the water as shown in Figure 1. Place the dialysis tube
into the water. Be sure the tubing is submerged completely under the water. Important: Be
sure to position the Conductivity Probe and dialysis tubing the same distance apart in each
trial.
9. After stirring the solution for 30 seconds, begin data collection by clicking . Stir the
solution slowly and continuously throughout the two-minute data collection period. Data
collection will automatically end after two minutes have passed.
10. Determine the rate of diffusion. To do this:

a. Move the mouse pointer to the point where the data values begin to increase. Hold down
the mouse button. Drag the pointer to the end of the data and release the mouse button.
b. Click the Linear Fit button, , to perform a linear regression. A floating box will appear
with the formula for a best fit line.
c. Record the slope of the line, m, as the rate of diffusion in Table 2.
d. Close the linear regression floating box.
11. Remove one of the clamps. If the dialysis tubing is tied off with floss, use a pair of scissors
and carefully cut one of the dental floss knots and discard the floss. If you accidentally make
a cut in the tubing, replace it.
12. Empty all of the liquid out of the dialysis tube. Squeeze the excess liquid out with your
fingers.
13. Obtain 15 mL of a 5% salt solution in a test tube. Repeat Steps 6–12, substituting this 5% salt
solution for the 1% solution.
14. Obtain 15 mL of a 10% salt solution in a test tube. Repeat Steps 6–12, substituting this 10%
salt solution for the 1% solution.

Computer 1A
15. Examine your data closely and make a conclusion. Record your conclusion in Table 1.
Part II Effect of other molecules

16. Measure the rate of diffusion of salt while it is in the presence of another, non-conducting
solution. Since sugar does not form ions in solution, it should not conduct electricity. Sugar
will be added to the water to determine whether it interferes with the diffusion of salt.
17. In Table 1, predict what you believe will happen in this set of experiments. Will the non-
conducting sugar in the water block or reduce the diffusion rate of salt? Why?
Test to determine if water or a sugar solution conducts electricity.

18. Place some water in a clean 400 mL beaker.
19. Test the total dissolved solids concentration of the water by placing a clean Conductivity
Probe into it. Record the total dissolved solid concentration value in Table 3. The total
dissolved solids value should be displayed in the meter at the right of the screen.
20. Obtain 300 mL of a 5% sugar solution in a clean 400 mL beaker.
21. Test the total dissolved solids concentration of the 5% sugar solution by placing a clean
Conductivity Probe into it. Record the total dissolved solids value in Table 3. The total
dissolved solids value should be displayed in the meter on the right of the screen.
Test if 5% sugar interferes with the diffusion of a 5% salt solution.

22. Repeat Steps 6–12, with the following changes:
• Use 300 mL of sugar water in place of the water in Step 7.
• Substitute a 5% salt solution for the 1% solution.
• Record the rate of diffusion in Table 4.
23. Examine your data closely and make a conclusion. Record your conclusion in Table 1.
DATA
Table 1
Prediction Conclusion
Part I
Part II

Part I
Table 2
Salt concentration Rate of diffusion

(%) (mg/L/s)
10
Part II
Table 3 Table 4: Summary of Data
Solution Concentration Solution Rate of diffusion

(mg/L) (mg/L/s)
Distilled water 5% salt
Sugar water 5% salt / 5% sugar
QUESTIONS
1. What conclusion can you draw from the data in Table 2?
2. How did your conclusion compare to your prediction for Part I? Can you account for any
differences?
3. If the rates in any of the three experiments varied in Part I, calculate how much faster each
rate was compared to that for the 1% salt solution. For instance, if the rate of the 1% solution
was 1 μS/s and the rate of the 10% solution was 5 μS/s, then the rate of diffusion for the 10%
solution would be (5/1) five times the rate of the 1% salt solution.
4. Compare the ionic concentration of pure water with a sugar water solution. How do you
account for this?
5. What conclusion can you draw from the data in Tables 3 and 4?
EXTENSIONS
1. Make a plot of the rate of diffusion vs. the salt concentration in the dialysis bag. Using your
plot, estimate the rate of diffusion of a 3% salt solution.
2. If the results of the experiments in Part I can be extrapolated to diffusion in living systems,
how would a single-celled organism respond in an oxygen rich pond compared to an oxygen
poor pond? Explain.
3. Design an experiment to determine the effect of temperature on the diffusion of salt. Perform
the experiment you designed.

Computer 1A
4. Ectotherms are organisms whose body temperature varies with the surrounding environment.
On the basis of on your data from Extension Question 3, how do you expect the oxygen
consumption of ectotherms to vary as the temperature varies? Explain.
5. If waste products of a single celled organism were released by the organism into the pond,
how would that affect the organism’s ability to obtain oxygen as readily?

Experiment
TEACHER INFORMATION 1A
1. This experiment correlates with Lab 1: Exercise 1A in the 2001 College Board’s AP Biology
Lab Manual.
2. The student pages with complete instructions for data collection using LabQuest App,
Logger Pro (computers), and EasyData (calculators) can be found on the CD that
accompanies this book. See Appendix A for more information.
3. If the water in your area is very soft, you may want to use tap water instead of distilled water.
Test to see if the conductivity of the tap water is less than about 50 mg/L salt.
4. Provide each group with pre-cut, hydrated dialysis tubing. The tubing must be soaked in
water for at least ten minutes prior to use. The tubing should be soft and flexible.
5. Use dialysis tubing clamps if at all possible, as this will speed things up greatly. If desired,
use dental floss or string to tie off the dialysis tubing. The floss works exceptionally well.
You may want to show students how to tie off the dialysis tubes.
6. Have students check their dialysis tubes for leakage. This should be done before each
experiment. Leaky tubes should be replaced.
7. Any sugar may be used in Part II. Table sugar is inexpensive and readily available.
8. To prepare 5% sugar solution, add 50 grams of sugar to make one liter of solution (300 mL
per group is needed).
9. To prepare 1% salt solution, add 10 grams of NaCl to make one liter of solution (15 mL per
group is needed).
10. To prepare 5% salt solution, add 50 grams of NaCl to make one liter of solution (30 mL per
group is needed).
11. To prepare 10% salt solution, add 100 grams of NaCl to make one liter of solution (15 mL
12. The stored conductivity calibration (for 0–2000 µS/cm) works well for this experiment. The
ionic concentration is approximately proportional to the conductivity of the solution.
Ideas for Inquiry

This experiment lends itself well to development into an inquiry investigation. Some possible
investigations include:
• Investigate the effect of pH on the rate of diffusion.
• Determine the effect of the composition of the salt solution in the tubing on the rate of
diffusion (i.e., KCl vs. NaCl).
Advanced Biology with Vernier 1A - 1 T

Experiment 1A
• Determine the effect of the total number of ions in a compound on the rate of diffusion
(i.e., AlCl3, CaCl2, KCl).
• Investigate the effect of changing the concentration of the solution in the beaker on the
diffusion rate.
SAMPLE RESULTS
The following data may be different from students’ results.
Part I
Table 2
Salt concentration Rate of diffusion

(%) (mg/L/s)
1 0.25
5 1.23
10 2.19
Part II
Table 3 Table 4: Summary of Data
Solution Conductivity Solution Rate of diffusion

(mg/L) (mg/L/s)
Distilled water 1.5 5% salt 1.23
Sugar water 1.9 5% salt / 5% sugar 1.21
Diffusion through dialysis tubing of differing salt concentrations
1A - 2 T Advanced Biology with Vernier

Teacher Information Diffusion through Membranes
ANSWERS TO QUESTIONS
1. Rate of diffusion should increase with increasing salt concentration.
2. The rate of diffusion should increase as the concentration gradient becomes steeper. The rate
of the 10% salt solution should be the greatest and the rate of the 1% salt solution should be
the lowest of the three.
3. The rate of the 10% salt solution should be approximately ten times that of the 1% solution,
while the rate of the 5% salt solution should be five times that of the 1% solution.
4. The conductivity should be the same, as neither will conduct appreciably. Neither molecule is
electrically charged.
5. Student answers will vary. The rates of diffusion should be the same.

Computer
Osmosis
1B
In order to survive, all organisms need to move molecules in and out of their cells. Molecules
such as gases (e.g., O2, CO2), water, food, and wastes pass across the cell membrane. There are
two ways that the molecules move through the membrane: passive transport and active transport.
While active transport requires that the cell uses chemical energy to move substances through the
cell membrane, passive transport does not require such energy expenditures. Passive transport
occurs spontaneously, using heat energy from the cell's environment.
Diffusion is the movement of molecules by passive transport from a region in which they are
highly concentrated to a region in which they are less concentrated. Diffusion continues until the
molecules are randomly distributed throughout the system. Osmosis, the movement of water
across a membrane, is a special case of diffusion. Water molecules are small and can easily pass
through the membrane. Other molecules, such as proteins, DNA, RNA, and sugars are too large
to diffuse through the cell membrane. The membrane is said to be semipermeable, since it allows
some molecules to diffuse through but not others.
If the concentration of water on one side of the membrane is different than on the other side,
water will move through the membrane seeking to equalize the concentration of water on both
sides. When water concentration outside a cell is greater than inside, the water moves into the cell
faster than it leaves, and the cell swells. The cell membrane acts somewhat like a balloon. If too
much water enters the cell, the cell can burst, killing the cell. Cells usually have some mechanism
for preventing too much water from entering, such as pumping excess water out of the cell or
making a tough outer coat that will not rupture. When the concentration of water inside of a cell
is greater than outside, water moves out of the cell faster than it enters, and the cell shrinks. If a
cell becomes too dehydrated, it may not be able to survive. Under ideal conditions, the water
concentration outside is nearly identical to that inside.
In this experiment, you will use a Gas Pressure Sensor to measure the
rate of pressure change as water moves in to or out of the cell
(dialysis tubes filled with various concentrations of sucrose solution).
The pressure generated is called osmotic pressure and is in response
to the overall movement of molecules, both water and sucrose, inside
the dialysis cell.
OBJECTIVES
• Use a Gas Pressure Sensor to investigate the relationship
between water movement and solute concentration.
• Determine the water potential of potato cells.
Figure 1
Advanced Biology with Vernier 1B - 1

Experiment 1B
MATERIALS
computer test tube rack
Vernier computer interface prepared sucrose solutions
Logger Pro four 16 × 100 mm test tubes with stoppers
Vernier Gas Pressure Sensor dialysis tubing, 2.5 cm × 15 cm
25 mL graduated cylinder dialysis tubing clamp
15 cm piece of plastic tubing plastic tubing clamp
ring stand plastic tubing with Luer connector
utility clamp 600 mL beaker
20 mL syringe Parafilm or Seran wrap
PROCEDURE
1. Place four test tubes in a rack and label them 1.0 M, 0.9 M, 0.8 M,
and 0.7 M.
2. Fill each test tube with 10 mL of the correct solution.
3. Connect the plastic tubing with the Luer connector to the valve of
the Gas Pressure Sensor.
Figure 2
4. Connect the Gas Pressure Sensor to the computer interface.
Prepare the computer for data collection by opening the file “01B Osmosis” from the
Advanced Biology with Vernier folder of Logger Pro.
5. Using a ring stand and clamp, mount the Gas Pressure Sensor above the beaker, as
shown in Figure 1.
6. Obtain a piece of wet dialysis tubing and a dialysis tubing clamp. Fold 2 cm of the
dialysis tubing back on itself and clamp as shown in Figure 2.
7. Prepare the dialysis tubing.
a. Connect the 15 cm segment of plastic tubing to the syringe and draw up 15 mL of
the contents of the 1.0 M solution. Figure 3
b. Open the remaining end of the dialysis tubing by rubbing the tubing between your
fingers.
c. Insert the end of the syringe tubing into the dialysis tube and slowly inject the
solution into the dialysis tubing as shown in Figure 3.
d. Slide the plastic tubing clamp onto the upper portion of the plastic tube from the
Gas Pressure Sensor.
e. Place the plastic tubing from the Gas Pressure Sensor into the solution in the
dialysis tubing and wrap the excess around the plastic tubing being sure that
minimal no air space remains.
f. Wrap a 1 cm × 3 cm piece of Parafilm or Seran wrap tightly over the portion
of the dialysis tubing that is wrapped around the Gas Pressure Sensor tube in
order to protect the dialysis tubing from the clamp.
g. Slide the plastic tubing clamp down over the Parafilm or Seran wrap and
clamp tightly as shown in Figure 4. There should be minimal airspace above
the liquid.
h. Rinse the outside of the dialysis tubing with distilled water. Figure 4
1B - 2 Advanced Biology with Vernier

Osmosis
8. Fill the beaker with 400 mL of distilled water and place the clamped dialysis tube in the
beaker. Check that the tubing bulb is completely submerged and that there are no kinks in the
tubing. Allow it to sit in the solution for five minute before starting data collection.
9. Click to begin data collection.
10. When data collection has finished, carefully release the tubing by unfastening the clamp.
Important: Do not disconnect the tubing from the sensor prior to releasing the tubing clamp.
There is enough pressure within the dialysis tubing to create a mess.
11. Rinse out the dialysis tubing and Styrofoam cup.
12. Determine the rate of change in osmotic pressure.

a. Highlight the data from the beginning of the region of increasing values to the end of the
data.
b. Click the Linear Fit button, , to perform a linear regression.
c. Record the slope of the line, m, as the rate of change in osmotic pressure for a 1.0 M
solution.
d. Close the linear regression box.
13. Store the data by choosing Store Latest Run from the Experiment menu.
14. Repeat Steps 6–13 for the remaining solutions.
DATA
Table 1
Sucrose solution Rate of pressure

concentration change
(M) (kPa/min)
1.0
0.9
0.8
0.7

Experiment 1B
PROCESSING THE DATA

1. On Page 2 of the Logger Pro experiment file, create a graph of the class average rates of
pressure change on the y-axis and sucrose solution concentration on the x-axis.
QUESTIONS
1. Which solutions, if any, produced a positive slope? Was water moving in or out of the cell
(dialysis tubing) under these circumstances? Explain.
2. Which solutions, if any, produced a negative slope? Was water moving in or out of the cell
under these circumstances? Explain.
3. Does sucrose move in or out of the cell? Explain.
4. Examine the graph of the rate of pressure change vs. the sucrose concentration. Describe any
pattern in the data.
5. Use the graph to estimate the concentration of sucrose that would yield no change in pressure.
Why is this biologically significant?
6. When wilted plants are watered, they tend to become rigid. Explain how this might happen.
7. Explain the strengths and weakness of this dialysis model with respect to an animal and plant
cell.
8. Discuss and explain potential reasons to account for variation in class average pressure change
at specific sucrose concentrations.
9. Predict the effect of increased and decreased initial temperature on rates of pressure change
for each of these different solution concentrations.
10. Predict the rates of pressure change if the dialysis tubing is placed in an insulated cup holding
1000 mL of 37°C water. Explain your reasoning.
11. Predict the rate of pressure change if the dialysis tubing were filled with 10 mL of 0.8 M
sucrose solution and 5 mL of 1.0 M sodium chloride solution. Explain your reasoning.

Osmosis
EXTENSION – WATER POTENTIAL

Water potential is a term used when predicting the movement of water into or out of plant cells.
Water always moves from an area of higher water potential to an area of lower water potential.
The symbol for water potential is the Greek letter Psi, Ψ. Water potential consists of a physical
pressure component called pressure potential Ψp, and the effects of solutes called solute potential,
Ψs.
Ψ = Ψp + ΨS
Water = Pressure + Solute
potential potential potential
Distilled water in an open beaker has a water potential of zero. The addition of solute decreases
water potential while the addition of pressure increases water potential. A water potential value
can be positive, negative, or zero. Water potential is usually measured in bars, a metric measure of
pressure. (1 kPa = .1 bar)
In this experiment, you will measure the percent change in mass of potato cores after they have
soaked in various concentrations of sugar solutions for a 24 hour period. You will use this data to
calculate the water potential of the potato cells.
MATERIALS
computer 250 mL beaker
Logger Pro plastic wrap
four potato cores balance
0 M, 0.33 M, 0.67 M, or paper towels
1.0 M sugar solution
PROCEDURE
You will be assigned one or more of the sugar solutions in which to soak your potato cores.
1. Pour 100 mL of the assigned sugar solution into a 250 mL beaker.
2. Measure and record the mass of the four potato cores together.
3. Put the four cores into the beaker of sugar solution.
4. Cover the beaker with plastic wrap and allow it to stand for a 24 hour period.
5. Remove the cores from the beaker, blot with a paper towel, and determine the mass of the
four cores together after soaking.
6. Calculate the percent change in mass and record your data for the sugar concentration tested
in Table 2 as well as on the class data sheet.
7. Repeat Steps 1–6 for any additional assigned sugar solutions.

Experiment 1B
DATA
Table 2
Sugar solution Initial mass Final mass Percent change in

concentration (g) (g) mass
0.00 M
0.33 M
0.67 M
1.0 M
Table 3
Class data of percent change in mass
Sugar solution Group Group Group Group Group Group Group Group Total Class
concentration 1 2 3 4 5 6 7 8 average
0.00 M
0.33 M
0.67 M
1.00 M
Table 4
Sugar molar concentration
Solute potential
Water potential
PROCESSING THE DATA

1. On Page 3 of the Logger Pro experiment file, create a graph with the percent change in mass
on the y-axis and concentration of sugar on the x-axis.
2. Perform a linear regression to determine the molar concentration of sugar solution at which
the mass of the potato cores does not change.
a. Click the Linear Fit button, , to perform a linear regression. A floating box will appear
b. Choose Interpolate from the Analyze menu. Move the cursor along the regression line to
the point where the line crosses the x axis. This point represents the molar concentration of
sugar with a water potential equal to the potato core water potential. Record this
concentration in Table 4.

Osmosis
3. Use the sugar molar concentration from the previous step to calculate the solute potential of
the sugar solution.
a. Calculate the solute potential of the sugar solution using the equation
Ψs = −iCRT
where i = ionization constant ( 1.0 for sugar since it doesn’t ionize in water)
C = sugar molar concentration (determined from the graph)
R = pressure constant (R = 0.0831 liter bars/mol-K)
T = temperature (K)
b. Record this value with units in Table 4.
4. Calculate the water potential of the solution using the equation
Ψ = Ψp + Ψs
The pressure potential of the solution in this case is zero because the solution is at equilibrium.
Record the water potential value with units in Table 4.
QUESTIONS
1. What may happen to an animal cell if water moves into it? How does this differ from what
would happen in a plant cell?
2. What factors affect water potential?
3. If a plant cell has a higher water potential than its surrounding environment and the pressure is
equal to zero, will water move in or out of the cell? Explain why.

Experiment
TEACHER INFORMATION 1B
Osmosis
1. This experiment correlates with Lab 1: Exercise 1B in the 2001 College Board’s AP Biology
Lab Manual.
3. The Extension on water potential has been added to this experiment to further address the
concept of water movement in and out of cells.
4. Soak 15 cm segments of dialysis tubing in distilled water prior to the lab.
5. Prepare a stock 1.0 M sucrose solution by combining 342.0 grams of sucrose (table sugar)
with 1.5 L of boiling distilled water. Stir into solution and gently bring volume up to 2.0 L
with distilled water. Table 1 presents directions for preparing 250 mL aliquots of each of the
four solutions. This will produce enough of each solution to accommodate sixteen runs.
Note: These solutions offer a rich medium for bacterial and fungal growth and can become
contaminated upon extended exposure to air. Autoclaving or bringing solutions to an initial
boil in a microwave may extend solution life.
Table 1
Amount of 1.0 M
Tube composition Amount of water
sucrose
1.0 M 250.0 mL 0.0 mL
0.9 M 225.0 mL 25.0 mL
0.8 M 200.0 mL 50.0 mL
0.7 M 175.0 mL 75.0 mL
6. If more than one sensor is available to each group, prepare and connect additional dialysis
tubing/sucrose solution setups for each additional Gas Pressure Sensor. Use a separate beaker
for each setup.
Ideas for Inquiry

• Determine the effect of adding salt to the water in the beaker on the rate of osmotic
pressure change.
• Investigate how other types of sugars or mixtures inside the dialysis tube affect the rate of
osmotic pressure change.
• Determine the effect of changing the thickness of the dialysis tube.
• Determine the effect of surface area on the rate of osmotic pressure change.
Advanced Biology with Vernier 1B - 1 T

Experiment 1B
SAMPLE RESULTS
Table 1
Concentration Rate of pressure

change (kPa/min)
1.0 M 0.3
0.9 M 0.27
0.8 M 0.22
0.7 M 0.16
EXTENSION SAMPLE DATA

Average percent change in mass - class data
0.00 M 18.5
0.33 M 1.6
0.67 M –15.0
1.0 M –18.0
Sugar molar concentration 0.41 M
Solute potential –10.0 bars
Water potential –10.0 bars
1B - 2 T Advanced Biology with Vernier

Teacher Information Osmosis
1. All solutions produce a positive slope as water will seek to move from high concentration
into the sucrose solution with a lesser water concentration. As the water moves into the
“cell”, the osmotic pressure increases since the larger sugar molecules are not able to diffuse
the opposite direction across the membrane.
2. None of the solutions produced a negative slope as the solute inside the “cell” consisted of a
solution of sugar molecules that were too large to diffuse through the membrane into the
surrounding water. The only overall movement would be the water from outside the “cell”
moving into the cell attempting to dilute the inner solution to a state similar to the outer
region. Diffusion and the special case, osmosis, seek to establish equilibrium that would, if
attained, result in a zero slope with concentrations being similar on both sides of the
membrane.
3. Sucrose, due to its disaccharide molecular shape and the small pore size of the dialysis
membrane, remains inside this cell.
4. All trials seem to proceed to a pressure that ultimately results in the rupture of the cell, time
to reach this state appears to offer the only difference.
5. The sucrose concentration that would yield no change in pressure would be one with possibly
just a trace of sucrose. This solution would be near isotonic. The significance of this isotonic
situation is that the pressure maintained inside this cell is the same as that of the fluid
surrounding the cell with respect to sucrose. This maintains cell size and function at normal
osmotic pressure in this model.
6. Cells in wilted plants are in some state of dehydration. For those plants that have not
exceeded their critical wilting point, watering will rehydrate the cells and increase their turgor
allowing the plant to resume life in a state of healthy water balance. The turgor is brought
about by osmotic pressure within plant cells.
7. This dialysis model of a cell works in offering a descriptive and manipulative structure for
studying the relative nature of a cell’s plasma membrane. One can interact with this system
by adjusting the solute conditions and recording changes in pressure. The drawback to this
method is the matter of scale, membrane pore size, lack of control of ion movement, and
inability to detect changes in solutions resembling those found in inter and intra cellular
environments.
8. Some of the reason for variation would be technique differences between the groups, varying
volumes of solution and air space in the dialysis tubing, varying initial temperatures and
volumes of water surrounding the dialysis cell, and variations in the region of the graph
selected to find rate of osmotic change.
9. Increasing the temperature would seem to speed up the collisions of water molecules with the
membrane tubing thereby increasing the rate of osmosis while lowering the temperature
would slow the osmotic rate of change. In humans, temperature remains constant at 37°C so,
as in this example, change will be due to variations in solution concentration.
10. By using a less insulated container more heat energy will be lost to the environment. This
cooling of the water surrounding the dialysis cell will slow the rate of osmotic pressure
change. However, there may be a trade-off between the doubling of the volume of water
surrounding the cell and the cooling due to a material of lesser insulating ability.

Experiment 1B
ANSWERS TO EXTENSION QUESTIONS

1. An animal cell will swell and maybe burst when water moves into it. A plant cell has a cell
wall that prevents the cell from bursting.
2. The two factors that affect water potential are solute potential and pressure potential.
3. Water will move out of the plant cell because water always moves from an area of higher
water potential to an area of lower water potential.

Computer
Enzyme Action:
2
Testing Catalase Activity
(Method 1–O2 Gas Sensor)
Many organisms can decompose hydrogen peroxide (H2O2) enzymatically. Enzymes are globular
proteins, responsible for most of the chemical activities of living organisms. They act as catalysts,
substances that speed up chemical reactions without being destroyed or altered during the
process. Enzymes are extremely efficient and may be used over and over again. One enzyme may
catalyze thousands of reactions every second. Both the temperature and the pH at which enzymes
function are extremely important. Most organisms have a preferred temperature range in which
they survive, and their enzymes most likely function best within that temperature range. If the
environment of the enzyme is too acidic or too basic, the enzyme may irreversibly denature, or
unravel, until it no longer has the shape necessary for proper functioning.
H2O2 is toxic to most living organisms. Many organisms are capable of enzymatically destroying
the H2O2 before it can do much damage. H2O2 can be converted to oxygen and water, as follows:
2 H2O2 → 2 H2O + O2
Although this reaction occurs spontaneously, enzymes increase the rate considerably. At least two
different enzymes are known to catalyze this reaction: catalase, found in animals and protists, and
peroxidase, found in plants. A great deal can be learned about enzymes by studying the rates of
enzyme-catalyzed reactions. The rate of a chemical reaction may be studied in a number of ways
including:
• measuring the rate of appearance of a product (in this case, O2, which is given off as a gas)
• measuring the rate of disappearance of substrate (in this case, H2O2)
• measuring the pressure of the product as it appears (in this case, O2).
In this experiment, you will measure the rate of

enzyme activity under various conditions, such as
different enzyme concentrations, pH values, and
temperatures. It is possible to measure the
concentration of oxygen gas formed as H2O2 is
destroyed using an O2 Gas Sensor. If a plot is made,
it may appear similar to the graph shown.
At the start of the reaction, there is no product, and
the concentration is the same as the atmosphere.
After a short time, oxygen accumulates at a rather
constant rate. The slope of the curve at this initial
time is constant and is called the initial rate. As the
peroxide is destroyed, less of it is available to react
and the O2 is produced at lower rates. When no
more peroxide is left, O2 is no longer produced.
Advanced Biology with Vernier 2 - 1 (O2)

Computer 2
OBJECTIVES
• Use a computer and an Oxygen Gas Sensor to measure the production of oxygen gas as
hydrogen peroxide is destroyed by the enzyme catalase or peroxidase at various enzyme
concentrations.
• Measure and compare the initial rates of reaction for this enzyme when different
concentrations of enzyme react with H2O2.
• Measure the production of oxygen gas as hydrogen peroxide is destroyed by the enzyme
catalase or peroxidase at various temperatures.
• Measure and compare the initial rates of reaction for the enzyme at each temperature.
catalase or peroxidase at various pH values.
• Measure and compare the initial rates of reaction for the enzyme at each pH value.
MATERIALS
Vernier computer interface pH buffers
computer enzyme suspension
Logger Pro three 18 × 150 mm test tubes
Vernier O2 Gas Sensor test tube rack
10 mL graduated cylinder ice
400 mL beaker thermometer
250 mL Nalgene bottle three dropper pipettes
3.0% H2O2
PROCEDURE
1. Obtain and wear goggles.
2. Connect the Oxygen Gas Sensor to the computer interface. Prepare the computer for data
collection by opening the file “02 (O2) Enzyme” from the Advanced Biology with Vernier
folder of Logger Pro.
Part I Testing the Effect of Enzyme Concentration

3. Place three test tubes in a rack and label them 1, 2, and 3. Fill each test tube with 5 mL of
3.0% H2O2 and 5 mL of water.
4. Initiate the enzyme catalyzed reaction.

a. Using a clean dropper pipette, add 5 drops of enzyme suspension to test tube 1.
b. Begin timing with a stopwatch or clock.
c. Cover the opening of the test tube with a finger and gently invert the test tube two times.
d. Pour the contents of the test tube into a clean 250 mL Nalgene bottle.
e. Place the O2 Gas Sensor into the bottle as shown in Figure 1. Gently push the sensor down
into the bottle until it stops. The sensor is designed to seal the bottle without the need for
unnecessary force.
f. When 30 seconds has passed, click to begin data collection.
2 - 2 (O2) Advanced Biology with Vernier

Enzyme Action: Testing Catalase Activity (O2)
Figure 1
5. When data collection has finished, remove the O2 gas sensor from the Nalgene bottle. Rinse
the bottle with water and dry with a paper towel.
6. Move your data to a stored run. To do this, choose Store Latest Run from the Experiment
menu.
7. Collect data for test tubes 2 and 3:

• Add 10 drops of the enzyme solution to test tube 2. Repeat Steps 4–6.
8. Using the mouse, select the initial linear region of your data on the graph. Click on the Linear
Fit button, . Click and a best-fit linear regression line will be shown for each run
selected. In your data table, record the value of the slope, m, for each of the three solutions.
(The linear regression statistics are displayed in a floating box for each of the data sets.)
9. To print a graph of concentration vs. volume showing all three data runs:
a. Label all three curves by choosing Text Annotation from the Insert menu, and typing
5 Drops (or 10 Drops, or 20 Drops) in the edit box. Then drag each box to a position near
its respective curve. Adjust the position of the arrow head.
b. Print a copy of the graph, with all three data sets and the regression lines displayed. Enter
your name(s) and the number of copies of the graph you want.
10. Determine the rate of reaction for each of the time intervals listed in Table 3 using the
procedure outlined in Step 8. Record the rates for all three data runs in the Table 3.
Part II Testing the Effect of Temperature

Your teacher will assign a temperature range for your lab group to test. Depending on your
assigned temperature range, set up your water bath as described below. Place a thermometer in
your water bath to assist in maintaining the proper temperature.
• 0–5°C: 400 mL beaker filled with ice and water.
• 20–25°C: No water bath needed to maintain room temperature.
• 30–35°C: 400 mL beaker filled with very warm water.
• 50–55°C: 400 mL beaker filled with hot water.

Computer 2
11. Rinse the three numbered test tubes used for Part I. Fill each test tube with 3 mL of 3.0%
H2O2 and 3 mL of water. Place the test tubes in the water bath. The test tubes should be in the
water bath for 5 minutes before proceeding to Step 12. Record the temperature of the water
bath, as indicated on the thermometer, in the space provided in Table 4.
12. Find the rate of enzyme activity for test tubes 1, 2, and 3:
• Add 10 Drops of the enzyme solution to test tube 1. Repeat Steps 4–6.
13. Repeat Step 8 and record the reaction rate for each data set in Table 4. Calculate and record
the average rate in Table 4.
14. Record the average rate and the temperature of your water bath from Table 4 on the class
data table. When the entire class has reported their data, record the class data in Table 5.
Part III Testing the Effect of pH
15. Place three clean test tubes in a rack and label them pH 4, pH 7, and pH 10.
16. Add 5 mL of 3% H2O2 and 5 mL of a pH buffer to each test tube, as in Table 1.
Table 1
Volume of 3% H2O2 Volume of buffer
pH of buffer
(mL) (mL)
pH 4 5 5
pH 7 5 5
pH 10 5 5
17. Using the test tube labeled pH 4, add 10 drops of enzyme solution and repeat Steps 4–6.
20. Repeat Steps 8 and 9 to calculate the rate of reaction and print your graph. Record the
reaction rate for each pH value in Table 6.
DATA
Part I Effect of Enzyme Concentration
Table 2
Slope or rate
Test tube label
(%/min)
5 Drops
10 Drops
20 Drops

Enzyme Action: Testing Catalase Activity (O2)
Table 3
Time intervals (minutes)
Rates 0–0.5 min 0.5–1.0 min 1.0–1.5 min 1.5–2.0 min 2.0–3.0 min
5 Drops
10 Drops
20 Drops
Part II Effect of Temperature
Table 4 Table 5 (Class Data)
Test tube label Slope, or rate Temperature tested Average rate

(%/min)
Trial 1
Trial 2
Trial 3
Average
Temperature range:____°C
Part III Effect of pH
Table 6
Test tube label Slope or rate

(%/min)
pH 4
pH 7
pH 10
PROCESSING THE DATA

1. On Page 2 of this experiment file, create a graph of the rate of enzyme activity vs.
temperature. Plot the rate values for the class data in Table 5 on the y-axis, and the
temperature on the x-axis. Use this graph to answer the questions for Part II.

Computer 2
QUESTIONS
1. How does changing the concentration of enzyme affect the rate of decomposition of H2O2?
2. What do you think will happen to the rate of reaction if one increases the concentration of
enzyme to twenty-five drops? Predict what the rate would be for 30 drops.

3. At what temperature is the rate of enzyme activity the highest? Lowest? Explain.
4. How does changing the temperature affect the rate of enzyme activity? Does this follow a
pattern you anticipated?
5. Why might the enzyme activity decrease at very high temperatures?

6. At what pH is the rate of enzyme activity the highest? Lowest?
7. How does changing the pH affect the rate of enzyme activity?
EXTENSIONS
1. Different organisms often live in very different habitats. Design a series of experiments to
investigate how different types of organisms might affect the rate of enzyme activity. Consider
testing a plant, an animal, and a protist.
2. Presumably, at higher concentrations of H2O2, there is a greater chance that an enzyme

molecule might collide with H2O2. If so, the concentration of H2O2 might alter the rate of
oxygen production. Design a series of experiments to investigate how differing concentrations
of the substrate hydrogen peroxide might affect the rate of enzyme activity.
3. Design an experiment to determine the effect of boiling the catalase on the rate of reaction.
4. Explain how environmental factors affect the rate of enzyme-catalyzed reactions.

Experiment
TEACHER INFORMATION 2
Enzyme Action:
1. This experiment correlates with Lab 2 in the 2001 College Board’s AP Biology Lab Manual.
3. This experiment may take a single group several lab periods to complete. A good breaking
point is after the completion of Step 10, when students have tested the effect of different
enzyme concentrations. Alternatively, if time is limited, different groups can be assigned one
of the three tests and the data can be shared.
4. Your hot tap water may be in the range of 50–55°C for the hot-water bath. If not, you may
want to supply pre-warmed temperature baths for Part II, where students need to maintain
very warm water. Warn students not to touch the hot water.
5. Many different organisms may be used as a source of catalase in this experiment. If enzymes
from an animal, a protist, and a plant are used by different teams in the same class, it will be
possible to compare the similarities and differences among those organisms. Often, either beef
liver, beef blood, or living yeast are used.
6. To prepare the yeast suspension, dissolve 7 g (1 package) of dried yeast per 100 mL of 2%
glucose solution. A 2% glucose is made by adding 20 g of glucose to enough distilled water to
make 1 L of suspension. Incubate the suspension in 37–40°C water for at least 10 minutes to
activate the yeast. Test the experiment before the students begin. The yeast may need to be
diluted if the reaction occurs too rapidly. The reaction in Step 4, with 5 mL of 3.0% hydrogen
peroxide, and 5 drops of suspension produces enough oxygen to exceed a measured
concentration of 22% in 40 to 60 seconds.
To ensure uniform yeast suspension concentration, make the suspension available on a
magnetic stirrer and instruct your students to withdraw their portions from the center of the
mixture as it is being stirred.
7. To prepare a liver suspension, homogenize 0.5 to 1.5 g of beef liver in 100 mL of cold water.
You will need to test the suspension before use, as its activity varies greatly depending on its
freshness. Dilute the suspension until the reaction in Step 4, with 5 mL of 3.0% hydrogen
peroxide, and 5 drops of suspension produces enough oxygen to exceed a measured
concentration of 22 % in 40 to 60 seconds. The color of the suspension will be a faint pink.
Keep the suspension on ice until used in an experiment.
8. The 3% H2O2 may be purchased from any supermarket. If refrigerated, bring it to room
temperature before starting the experiment.
9. To extend the life of the O2 Gas Sensor, always store the sensor upright in the box in which it
was shipped.
Advanced Biology with Vernier 2 - 1 T (O2)

Experiment 2
10. Vernier Software sells a pH buffer package for preparing buffer solutions with pH values of 4,
7, and 10 (order code PHB). Simply add the capsule contents to 100 mL of distilled water.
11. You can also prepare pH buffers using the following recipes:
• pH 4: Add 2.0 mL of 0.1 M HCl to 1000 mL of 0.1 M potassium hydrogen phthalate.
• pH 7: Add 582 mL of 0.1 M NaOH to 1000 mL of 0.1 M potassium dihydrogen phosphate.
• pH 10: Add 214 mL of 0.1 M NaOH to 1000 mL of 0.05 M sodium bicarbonate.
12. You may need to let students know that at pH values above 10, enzymes will become
denatured and the rate of activity will drop. If you have pH buffers higher than 10, have
students perform an experimental run using them.
Ideas for Inquiry

• Determine the effect of the type of catalase on the rate of enzyme activity.
• Determine the effect of changing the substrate (H2O2) concentration on enzyme activity.
• Determine the effect of adding salt to the reaction.
• Investigate the optimal pH for enzyme activity of catalase.
SAMPLE RESULTS
Sample class data
Test tube label Slope, or rate (%/min)
5 Drops 0.27
10 Drops 0.73
20 Drops 1.59
0–5 °C range: 4°C 0.58
20–25 °C range: 21 °C 0.82
30–35 °C range: 34°C 1.43
50–55 °C range: 51°C 0.36
pH 4 0.36
pH 7 0.89
pH 10 0.97
2 - 2 T (O2) Advanced Biology with Vernier

Teacher Information Enzyme Action: Testing Catalase Activity (02)
The effect of H2O2 concentration on the rate of The effect of pH on the rate
enzyme activity of enzyme activity
The effect of temperature on the rate of enzyme activity
Sample data: Effect of enzyme concentration on the rate of activity
1. The rate should be highest when the concentration of enzyme is highest. With higher
concentration of enzyme, there is a greater chance of an effective collision between the
enzyme and H2O2 molecule.
2. Roughly, the rate doubles when the concentration of enzyme doubles. Since the data are

Experiment 2
somewhat linear, the rate is proportional to the concentration. At a concentration of 30 drops,

the rate would be about 2.90%/min.
3. The temperature at which the rate of enzyme activity is the highest should be close to 30°C.
The lowest rate of enzyme activity should be at 60°C.
4. The rate increases as the temperature increases, until the temperature reaches about 50°C.
Above this temperature, the rate decreases.
5. At high temperatures, enzymes lose activity as they are denatured.
6. Student answers may vary. Activity is usually highest at pH 10 and lowest at pH 4.
7. Student answers may vary. Usually, the enzyme activity increases from pH 4 to 10. At low pH
values, the protein may denature or change its structure. This may affect the enzyme’s ability
to recognize a substrate or it may alter its polarity within a cell.

Computer
Enzyme Action:
2
(Method 2–Gas Pressure)
Many organisms can decompose hydrogen peroxide (H2O2) enzymatically. Enzymes are globular
proteins, responsible for most of the chemical activities of living organisms. They act as
catalysts, substances that speed up chemical reactions without being destroyed or altered during
the process. Enzymes are extremely efficient and may be used over and over again. One enzyme
may catalyze thousands of reactions every second. Both the temperature and the pH at which
enzymes function are extremely important. Most organisms have a preferred temperature range in
which they survive, and their enzymes most likely function best within that temperature range. If
the environment of the enzyme is too acidic or too basic, the enzyme may irreversibly denature,
or unravel, until it no longer has the shape necessary for proper functioning.
H2O2 is toxic to most living organisms. Many organisms are capable of enzymatically destroying
the H2O2 before it can do much damage. H2O2 can be converted to oxygen and water, as follows:
2 H2O2 → 2 H2O + O2
Although this reaction occurs spontaneously, enzymes increase the rate considerably. At least
two different enzymes are known to catalyze this reaction: catalase, found in animals and
protists, and peroxidase, found in plants. A great deal can be learned about enzymes by studying
the rates of enzyme-catalyzed reactions. The rate of a chemical reaction may be studied in a
number of ways including:
• measuring the rate of appearance of a product (in this case, O2, which is given off as a gas)
• measuring the rate of disappearance of substrate (in this case, H2O2)
• measuring the pressure of the product as it appears (in this case, O2).
In this experiment, you will measure the rate of

enzyme activity under various conditions, such as
different enzyme concentrations, pH values, and
temperatures. It is possible to measure the
concentration of oxygen gas formed as H2O2 is
destroyed using an O2 Gas Sensor. If a plot is
made, it may appear similar to the graph shown.
At the start of the reaction, there is no product,
and the concentration is the same as the
atmosphere. After a short time, oxygen
accumulates at a rather constant rate. The slope of
the curve at this initial time is constant and is
called the initial rate. As the peroxide is
destroyed, less of it is available to react and the O2
is produced at lower rates. When no more
peroxide is left, O2 is no longer produced.
Advanced Biology with Vernier 2 - 1 (Gas Pressure)

Computer 2
OBJECTIVES
• Use a computer and a Gas Pressure Sensor to measure the production of oxygen gas as
hydrogen peroxide is destroyed by the enzyme catalase or peroxidase at various enzyme
concentrations.
• Measure and compare the initial rates of reaction for this enzyme when different
concentrations of enzyme react with H2O2.
catalase or peroxidase at various temperatures.
• Measure and compare the initial rates of reaction for the enzyme at each temperature.
catalase or peroxidase at various pH values.
• Measure and compare the initial rates of reaction for the enzyme at each pH value.
MATERIALS
Vernier computer interface enzyme suspension
Logger Pro four 18 X 150 mm test tubes
Vernier Gas Pressure Sensor ice
1-hole rubber stopper assembly pH buffers
10 mL graduated cylinder test tube rack
250 mL beaker of water thermometer
3% H2O2 three dropper pipettes
METHOD 2 PROCEDURE
2. Connect the Gas Pressure Sensor to the computer interface. Prepare the computer for data
collection by opening the file “02 (Pressure) Enzyme” from the Advanced Biology with
Vernier folder of Logger Pro.
3. Connect the plastic tubing to the valve on the Gas Pressure Sensor.
1 2 3 4
Figure 1
Part I Testing the Effect of Enzyme Concentration
4. Place four test tubes in a rack and label them 1, 2, 3, and 4. Partially fill a beaker with tap
water for use in Step 5.
2 - 2 (Gas Pressure) Advanced Biology with Vernier

Enzyme Action: Testing Catalase Activity (Gas Pressure)
5. Add 3 mL of water and 3 mL of 3% H2O2 to each test tube.
6. Using a clean dropper pipette, add 1 drop of enzyme suspension to Test Tube 1. Note: Be
sure not to let the enzyme fall against the side of the test tube.
Table 1
Test tube label Volume of 3% H2O2 (mL) Volume of water (mL)
1 3 3
2 3 3
3 3 3
4 3 3
7. Stopper the test tube and gently swirl to thoroughly mix the contents. The reaction should
begin. The next step should be completed as rapidly as possible.
8. Connect the free-end of the plastic tubing to the connector in the

rubber stopper as shown in Figure 2. Click to begin data
collection. Data collection will end after 3 minutes.
9. If the pressure exceeds 130 kPa, the pressure inside the tube will be too
great and the rubber stopper is likely to pop off. Disconnect the plastic
tubing from the Gas Pressure Sensor if the pressure exceeds 130 kPa.
10. When data collection has finished, disconnect the plastic tubing
connector from the rubber stopper. Remove the rubber stopper from
the test tube and discard the contents in a waste beaker.
11. Find the rate of enzyme activity:

Figure 2
a. Move the mouse pointer to the point where the data values begin to
increase. Hold down the mouse button. Drag the mouse pointer to the point where the
pressure values no longer increase and release the mouse button.
with the formula for a best-fit line.
c. Record the slope of the line, m, as the rate of enzyme activity in Table 4.
12. Find the rate of enzyme activity for test tubes 2–4:
a. Add 2 drops of the enzyme solution to test tube 2. Repeat Steps 7–11.
b. Add 3 drops of the enzyme solution to test tube 3. Repeat Steps 7–11.
c. Add 4 drops of the enzyme solution to test tube 4. Repeat Steps 7–11.
Part II Testing the Effect of Temperature
13. Place four clean test tubes in a rack and label them T 0 – 5, T 20 – 25, T 30 – 35, and T 50 – 55.

Computer 2
14. Add 3 mL of 3% H2O2 and 3 mL of water to each test tube, as shown in Table 2.
Table 2
Test tube label Volume of 3% H2O2 (mL) Volume of water
T 0–5 3 3
T 20–25 (room temp) 3 3
T 30–35 3 3
T 50–55 3 3
15. Measure the enzyme activity at 0–5°C:

a. Prepare a water bath at a temperature in the range of 0–5°C by placing ice and water in a
600 mL beaker. Check that the temperature remains in this range throughout this test.
b. Place Test Tube T 0–5 in the cold water bath until the temperature of the mixture reaches
a temperature in the 0–5°C range. Record the actual temperature of the test-tube contents
in the blank in Table 4.
c. Add 2 drops of the enzyme solution to Test Tube T 0–5. Repeat Steps 7–11.
a. Prepare a water bath at a temperature in the range of 30–35°C by placing warm water in a
600 mL beaker. Check that the temperature remains in this range throughout this test.
b. Place Test Tube T 30–35 in the warm water bath until the temperature of the mixture
reaches a temperature in the 30–35°C range. Record the actual temperature of the test-tube
contents in the blank in Table 4.
a. Prepare a water bath at a temperature in the range of 50–55°C by placing hot water in a
600 mL beaker (hot tap water will probably work fine). Check that the temperature
remains in this range throughout this test.
b. Place Test Tube T 50–55 in the warm water bath until the temperature of the mixture
reaches a temperature in the 50–55°C range. Record the actual temperature of the test-tube
contents in the blank in Table 4.
18. Measure the enzyme activity at 20–25°C (room temperature):
a. Record the temperature of Test Tube T 20–25 in Table 4.
b. In the tube labeled T 20–25, add 2 drops of the enzyme solution. Repeat Steps 7–11.

Enzyme Action: Testing Catalase Activity (Gas Pressure)
Part III Testing the Effect of pH

19. Place three clean test tubes in a rack and label them pH 4, pH 7, and pH 10.
20. Add 3 mL of 3% H2O2 and 3 mL of each pH buffer to each test tube, as in Table 3.
Table 3
pH of buffer Volume of 3% H2O2 (mL) Volume of buffer (mL)
pH 4 3 3
pH 7 3 3
pH 10 3 3
21. In the tube labeled pH 4, add 2 drops of the enzyme solution. Repeat Steps 7–11.
DATA
Table 4
Test tube label Slope, or rate (kPa/min)
1 Drop
2 Drops
3 Drops
4 Drops
0 – 5°C range: °C
20 – 25°C range: °C
30 – 35°C range: °C
50 – 55°C range: °C
pH 4
pH 7
pH 10
PROCESSING THE DATA

Enzyme concentration plot
1. On Page 2 of this experiment file, create a graph of the rate of enzyme activity vs. enzyme
concentration. The rate values should be plotted on the y-axis, and the number of drops of
enzyme on the x-axis. The rate values are the same as the slope values in Table 4.

Computer 2
Temperature plot
2. On Page 3 of this experiment file, create a graph of the rate of enzyme activity vs.
temperature. The rate values should be plotted on the y-axis, and the temperature on the x-
axis. The rate values are the same as the slope values in Table 4.
pH plot
3. On Page 4 of this experiment file, create a graph of rate of enzyme activity vs. pH. The rate
values should be plotted on the y-axis, and the pH on the x-axis. The rate values are the same
as the slope values in Table 4.
QUESTIONS
1. How does changing the concentration of enzyme affect the rate of decomposition of H2O2?
2. What do you think will happen to the rate of reaction if the concentration of enzyme is
increased to five drops? Predict what the rate would be for 5 drops.

3. At what temperature is the rate of enzyme activity the highest? Lowest? Explain.
4. How does changing the temperature affect the rate of enzyme activity? Does this follow a
pattern you anticipated?
5. Why might the enzyme activity decrease at very high temperatures?

6. At what pH is the rate of enzyme activity the highest? Lowest?
7. How does changing the pH affect the rate of enzyme activity?
EXTENSIONS
1. Different organisms often live in very different habitats. Design a series of experiments to
investigate how different types of organisms might affect the rate of enzyme activity.
Consider testing a plant, an animal, and a protist.
2. Presumably, at higher concentrations of H2O2, there is a greater chance that an enzyme

molecule might collide with H2O2. If so, the concentration of H2O2 might alter the rate of
oxygen production. Design a series of experiments to investigate how differing
concentrations of the substrate hydrogen peroxide might affect the rate of enzyme activity.
3. Design an experiment to determine the effect of boiling the catalase on the rate of reaction.
4. Explain how environmental factors affect the rate of enzyme-catalyzed reactions.

Experiment
Enzyme Action:
3. This experiment may take a single group several lab periods to complete. A good breaking
point is after the completion of Step 12, when students have tested the effect of different
enzyme concentrations. Alternatively, if time is limited, different groups can be assigned one
of the three tests and the data can be shared.
4. Your hot tap water may be in the range of 50–55°C for the hot-water bath. If not, you may
want to supply pre-warmed temperature baths for Step 17, where students need to maintain
very warm water. Warn students not to touch the hot water.
5. Many different organisms may be used as a source of catalase in this experiment. If enzymes
from an animal, a protist, and a plant are used by different teams in the same class, it will be
possible to compare the similarities and differences among those organisms. Often, either beef
liver, beef blood, or living yeast are used.
6. To prepare the yeast suspension, dissolve 7 g (1 package) of dried yeast per 100 mL of 2%
glucose suspension. Incubate the suspension in 37–40°C water for at least 10 minutes to
activate the yeast. Test the experiment before the students begin. The yeast may need to be
diluted if the reaction occurs too rapidly. The reaction in Step 12, with 3 mL of 3% hydrogen
peroxide, 3 mL of water, and 2 drops of suspension should produce a pressure of
1.3 atmospheres in 40 to 60 seconds.
To prepare a 2% sugar solution, add 20 grams of sugar to make one liter of solution (100 mL
To ensure uniform yeast suspension concentration, make the suspension available on a

magnetic stirrer and instruct your students to withdraw their portions from the center of the
mixture as it is being stirred
7. To prepare a liver suspension, homogenize 0.5 to 1.5 g of beef liver in 100 mL of cold water.
You will need to test the suspension before use, as its activity varies greatly depending on its
freshness. Dilute the suspension until the reaction in Step 12, with 3 mL of 3% hydrogen
peroxide, 3 mL of water, and 2 drops of suspension produces a pressure of 130 kPa in 40 to
60 seconds. The color of the suspension will be a faint pink. Keep the suspension on ice until
used in an experiment.
8. You can purchase 3% H2O2 from any supermarket. If refrigerated, bring it to room
temperature before starting the experiment.
Advanced Biology with Vernier 2 - 1 T (Gas Pressure)

Experiment 2
9. Emphasize to your students the importance of providing an airtight fit with all plastic-tubing
connections and when closing valves or twisting the stopper into a test tube.
10. The accessory items used in this experiment are the #1 single hole stopper fitted with a
tapered valve connector and the section of plastic tubing fitted with Luer-lock connectors.
11. The length of plastic tubing connecting the rubber stopper assemblies to each gas pressure
sensor must be the same for all groups. It is best to keep the length of tubing reasonably small
to keep the volume of gas in the test tube low. Note: If pressure changes during data
collection are too small, you may need to decrease the total gas volume in the system.
Shortening the length of tubing used will help to decrease the volume.
12. If the Vernier Gas Pressure Sensor or Biology Gas Pressure Sensor is unavailable, the Vernier
Pressure Sensor may be used as an alternative.
13. Vernier Software sells a pH buffer package for preparing buffer solutions with pH values of 4,
7, and 10 (order code PHB). Simply add the capsule contents to 100 mL of distilled water.
14. You can also prepare pH buffers using the following recipes:
• pH 4: Add 2.0 mL of 0.1 M HCl to 1000 mL of 0.1 M potassium hydrogen phthalate.
• pH 7: Add 582 mL of 0.1 M NaOH to 1000 mL of 0.1 M potassium dihydrogen phosphate.
• pH 10: Add 214 mL of 0.1 M NaOH to 1000 mL of 0.05 M sodium bicarbonate.
15. You may need to let students know that at pH values above 10 enzymes will become
denatured and the rate of activity will drop. If you have pH buffers higher than 10, have
students perform an experimental run using them.
Ideas for Inquiry

• Determine the effect the type of catalase has on the rate of enzyme activity.
• Determine the effect changing the substrate (H2O2) concentration has on enzyme activity.
• Explore the effect of adding salt to the reaction.
• Determine is the optimal pH for enzyme activity of catalase.
2 - 2 T (Gas Pressure) Advanced Biology with Vernier

Teacher Information Enzyme Action: Testing Catalase Activity (Gas Pressure)
SAMPLE RESULTS
Table 5
Test tube label Slope, or rate (kPa/min)
1 Drop 10.23
2 Drops 44.98
3 Drops 59.36
4 Drops 98.26
0–5 °C range: 4°C 41.43
20–25 °C range: 21°C 48.02
30–35 °C range: 34°C 73.85
50–55 °C range: 51°C 27.55
pH 4 36.57
pH 7 66.86
pH 10 75.27
The effect of enzyme concentration on the rate of activity.
The effect of pH on the rate of enzyme activity

Experiment 2
The effect of temperature on the rate of enzyme activity
1. The rate should be highest when the concentration of enzyme is highest. With higher
concentration of enzyme, there is a greater chance of an effective collision between the
enzyme and H2O2 molecule.
2. Roughly, the rate doubles when the concentration of enzyme doubles. Since the data are
somewhat linear, the rate is proportional to the concentration. At a concentration of 5 drops,
the rate in the above experiment should be about 111 kPa/min.
3. The temperature at which the rate of enzyme activity is the highest should be close to 30°C.
The lowest rate of enzyme activity should be at 60°C.
4. The rate increases as the temperature increases, until the temperature reaches about 50°C.
Above this temperature, the rate decreases.
5. At high temperatures, enzymes lose activity as they are denatured.
6. Student answers may vary. Activity is usually highest at pH 10 and lowest at pH 4.

7. Student answers may vary. Usually, the enzyme activity increases from pH 4 to 10. At low pH
values, the protein may denature or change its structure. This may affect the enzyme’s ability
to recognize a substrate or it may alter its polarity within a cell.

Computer
Mitosis and Meiosis

3
Part I Mitosis
It was discovered in 1858, by Rudolf Virchow, that new cells can only arise from previously
existing cells. This is done in two ways: mitosis and meiosis. Somatic (body) cells divide
exclusively by mitosis followed by cytokinesis, while germ cells produce gametes by the process
of meiosis. Plant cells grow by enlargement, essentially by taking up water. When they reach a
certain size, they divide, forming two identical daughter cells. The various parts of the cell are
divided in such a way that the new daughter cell is identical to the parent cell.
Strictly speaking, mitosis implies only the division of the nucleus, and is therefore distinct from
cell division, in which the cytoplasm is divided. In most organisms, cells divide by ingrowth of
the cell wall, if present, and the contraction of the cell membrane, a process that cuts through the
spindle fibers. In land plants (bryophytes and vascular plants) and a few algae, cell division takes
place by the formation of a cell plate. Small droplets appear across the equatorial plate of the cell
and gradually fuse, forming a disc that grows outward until it reaches the wall of the dividing
cell, which completes the separation of the two daughter cells.
The DNA of prokaryotes is simply replicated before division. In eukaryotes, however, the
hereditary material is part of their complex chromosomes. Equal division of this material requires
a more complex method by which the chromosomes are replicated, separated, and apportioned
precisely between the daughter cells.
Mitosis, or nuclear division, ensures the equal division of the nuclear material between the
daughter cells in eukaryotic organisms. During mitosis the chromosomes condense, and move to
the center of the cell where they fully contract. They then split longitudinally into two identical
halves that appear to be pulled to opposite poles of the cell by a series of
microtubules. In these two genetically identical groups, the coiling of the
chromosomes relaxes again, and they are reconstituted into the nuclei of the
two daughter cells. It is a continuous process that can be divided into five
major phases: interphase, prophase, metaphase, anaphase, and telophase.
Interphase: The chromatin, if visible at all, can only be seen as small
grains or threads. Interphase is generally considered to be a “resting phase.”
However, the cell is replicating the genetic material, preparing for mitosis.
Prophase: The beginning of mitosis is illustrated by the chromosomes
gradually becoming visible. They start out as elongated threads that shorten
and thicken. Chromosomes become more condensed and undergo spiral
contractions, like a thin wire being turned into a coiled spring. This coiling
involves the entire DNA–protein complex. Each chromosome is composed
of two longitudinal halves, called chromatids, joined in a narrow area
known as the centromere, where the chromatids are not coiled. The
centromere, located on each chromosome, divides the chromosomes into
two arms of varying lengths. As prophase progresses, the nucleoli grow
smaller and finally disappear. Shortly after, in most cell types, the nuclear
envelope breaks down, putting the contracted chromosomes into direct
contact with the cytoplasm; this marks the end of prophase.
Advanced Biology with Vernier 3-1

Computer 3
Metaphase: The chromosomes, still doubled, become arranged so that each

centromere is on the equatorial region of the spindle. Each chromosome is
attracted to the spindle fibers by its centromere; often the arms of the
chromosome point toward one of the two poles. Some of the spindle fibers
pass from one pole to the other and have no chromosome attached.
Anaphase: The chromatids separate from one another and become

individual chromosomes. First, the centromere divides and the two daughter
chromosomes move away from the equator toward opposite poles. Their
centromeres, which are still attached to the spindle fiber, move first, and the
arms drag behind. The two daughter chromosomes pull apart; the tips of the
longer arms separate last. The spindle fibers attached to the chromosomes
shorten as the chromatids divide and the chromosomes separate. The fibers
appear to move, but in fact the microtubules are continuously formed at one
end of the spindle fiber and disassembled at the other. In the process, it
appears as if the spindle fibers were tugging the chromosomes toward the
poles by their centromeres. By the end of anaphase, the two identical sets of
chromosomes have separated and moved to opposite poles.
Telophase: The separation is made final; the nuclear envelopes are

organized around the two identical sets of chromosomes. The spindle
apparatus disappears. Nucleoli also reform at this time. The
chromosomes become increasingly indistinct, uncoiling to become
slender threads again.
Cytokinesis: As mitosis ends, cytokinesis begins, resulting in the

formation of two daughter cells. The cleaved membrane slowly draws
together, forming a narrow bridge, then separates the cell into two
daughter cells. The cells now enter interphase.
Mitosis ends when the processes are complete and the chromosomes
have once more disappeared from view. The two daughter cells enter
interphase. The two daughter nuclei produced are identical to one
another and to the nucleus that divided to produce them.
In order to investigate the process of mitosis, plant and animal tissues where cells are dividing
rapidly must be examined. In animals, the most rapidly growing and dividing tissues are found in
the embryonic stages of development. Although most animal tissues continue to undergo mitosis
throughout the life cycle of the organism, they do so very slowly when compared to their
embryos. Some animal cells, like most plant tissues, rarely replicate after the organism reaches
maturity.
In plants, these tissues are primarily found in the tips of stems and roots. The root tip plants are
exceptionally good places to look for cells undergoing mitosis. Plant root tips consist of several
different zones where various developmental and functional processes of the root are performed.
3-2 Advanced Biology with Vernier

Mitosis and Meiosis
The primary region for the formation of new cells is the apical meristem. The root cap offers
protection for the rest of the root, the region of elongation is the area where the bulk of cell
growth occurs, and the region of maturation is where tissue differentiation occurs.
Part II Meiosis
Sexual reproduction provides a mechanism to produce genetic variation, as the genes of two
different individuals are arranged in various ways. This requires a reduction in the chromosome
number of the parent cell, normally diploid, to half that, or haploid, in somatic cells. The type of
cell division resulting in half the chromosome number of the parent cell is called meiosis.
In meiosis, a germ cell divides into four

haploid gametes. When two gametes—
egg and sperm—combine during
fertilization, forming a zygote, the diploid
chromosome number is restored. Meiosis
consists of one DNA replication and two
nuclear divisions, meiosis I and II. This
results in the formation of four daughter
cells, each with only half the number of
chromosomes as the parent.
Genetic variability is further increased by

a process called crossing over. In the early
stages of meiosis, the homologous pairs
of chromosomes move close together in
such a way that all four chromatids are
entwined, forming a tetrad. This process,
known as synapsis, allows for the
exchange of chromosome sections
between the homologous pairs.
The example that will be used in the

investigation is Sordaria fimicola, an
ascomycete fungus that is haploid for the
bulk of its life cycle, including the
individual fungal filaments, called
hyphae, which normally exist in a mass
called a mycelium representing the
“body” of the fungus; and the ascospores,
from which mycelia develop. The only
diploid portion of the life cycle of S. fimicola occurs when the nuclei of specialized hyphae come
together.
These hyphae, which belong to different strains of the species, fuse to form a zygote. This zygote
then undergoes meiosis to produce the haploid ascospores, yielding four haploid nuclei contained
in a sac called an ascus. After meiosis, the four nuclei undergo mitosis, resulting in an ascus
containing eight haploid ascospores. Many asci form inside a fruiting body called a perithecium.

Computer 3
One type of genetic variability in S. fimicola is the color of the ascospores. Most strains are the
dark brown, wild-type ascospores, although there are variants. Certain strains have tan or gray
ascospores. A tan ascospore strain mated with the wild-type variety produces a series of
perithecia containing asci with four tan and four wild-type ascospores each.
How these ascospores are arranged within the ascus is a direct representation of whether or not
crossing over has occurred between the centromere and the site for the gene for ascospore color.
If no crossing over has occurred, the ascospores will be arranged in a 4:4 manner. If crossing
over has occurred, they will occur in a 2:4:2 or 2:2:2:2 manner.
By observing the ascospore arrangement, the percentage of asci exhibiting crossover can be
determined. This frequency appears to be affected largely by the distance from the gene to the
centromere. From the crossover frequency, the distance in map units from the gene for ascospore
color, and the chromosome centromere, can be calculated.
OBJECTIVES
• Examine and compare the phases of mitosis in animal and plants cells.
• Determine the relative time cells spend in each phase of mitosis.
• Prepare microscope slides of mitotic cells using onion Allium root tips.
• Follow the processes of mitosis and meiosis in the life cycle of Sordaria.
• Examine the arrangement of Sordaria ascospore microscopically to determine the
frequency of crossing over.
• Calculate the distance, in map units, between a specific gene and the chromosome
centromere.

Mitosis and Meiosis
Part I Mitosis
MATERIALS
Materials for Parts A and B
whitefish mitosis slide
onion mitosis slide
compound microscope
Materials for Part C
onion root tip Bunsen burner
hydrochloric acid 1M clothespin or forceps
toluidine blue 0.5% paper towel
compound microscope pipet
coverslip scalpel
microscope slide
PROCEDURE
Part A: Observing Mitosis in Plant and Animal Cells
1. Observe the prepared microscope slide of onion root tip mitosis, first at 100X, then 400X.
Using the Plant Mitosis Chart as a guide, identify cells that represent each mitotic phase.
2. In the Analysis section, draw each phase of plant cell mitosis that you see. Write a brief
description of each phase below each drawing.
3. Observe the prepared microscope slide of whitefish blastula. Using the Animal Mitosis Chart
as a guide, identify each phase of animal cell mitosis.
4. In the Analysis section, draw each phase of plant or animal cell mitosis that you see. Write a
brief description of each phase below each drawing.
Part B: Relative Lengths of Phases of Mitosis

5. Examine at least three fields of view of the apical meristem of the onion root tip at 400X. In
each view, count the number of cells in the various stages of mitosis. Record this data in
Table 1.
6. Calculate the total number of cells counted and the percentage of total cells counted for each
stage of mitosis. Record this data in Table 1. Record the percentages in Table 2, as well.
7. Assuming that it takes an average of 24 hours (1,440 minutes) for onion root tip cells to
complete the cell cycle, calculate the amount of time cells spent in each phase of the cycle.
Use the formula provided below. Enter your results in Table 1.
Percent of Cells in Phase × 1,440 minutes = _________ minutes cell spent in phase

Computer 3
Part C: Preparation of an Onion Root Tip Squash

8. Wrap a fresh onion set with paper toweling and place it in a plastic sandwich bag. Add 10 mL
of water before sealing the bag.
Note: The set used must have root primordia present or it will not produce root tips. The root
tips will grow within 36 to 72 hours. Common onions have a mitotic cycle of approximately
20 hours.
9. Place the plastic bag in a box or dark place until the root tips have grown to a length of about
4 to 5 mm. It is important that the onion grows in the dark to ensure that it produces roots
rather than shoots.
10. Remove the onion from the box approximately one half-hour before performing the
experiment to expose the root tips to light.
11. Blot as much excess water from the root tips as possible. Any excess water on the slide will
affect your results. Do not allow the root tips to dry out.
12. Using a scalpel or razor blade, cut off the end of one of the emergent root tips; the section
should be approximately 1 to 2 mm long. Place the root tip on a clean microscope slide and
apply one to two drops of HCl on the root tip.
13. Holding the slide with a clothespin or forceps, pass it through the flame of a Bunsen burner
for several seconds. Note: Do not hold the slide directly over the flame.
14. Without harming the root tip, blot the specimen with a paper towel to remove the excess HCl.
Note: You may wish to touch a corner of the paper towel to the edge of the root tip and allow
the paper towel to wick up the solution.
15. Add one to two drops of 0.5% aqueous toluidine blue stain to the root tip. Note: Toluidine
blue is a mild irritant, avoid direct physical contact with this stain.
16. Pass the slide through the flame of a Bunsen burner for one to two minutes. Let the slide
stand and cool for one minute. Note: Do not hold the slide directly over the flame.
17. Remove excess stain with a paper towel in a motion similar to the one used in Step 14.
18. With a scalpel or razor blade, slice the root tip longitudinally down the middle into two
mirror segments.
19. Add a drop of toluidine blue and cover the root tip with a coverslip. Using the scalpel handle
or other blunt instrument, gently press down on the coverslip to squash and spread out the
root tip. Blot off excess stain, if any, that may come out from under the coverslip.
20. View the slide under a microscope using the 10X objective. Locate the apical meristem.
Examine the slide using the 40X objective. Locate cells in the various stages of mitosis.
Sketch the nuclear regions of individual cells in the five different stages and describe key
features of each in the Analysis section. Keep in mind that since the root tip has been
squashed, the meristem may not be readily recognizable.

Mitosis and Meiosis
Part II Meiosis
MATERIALS
microscope slide
coverslip
inoculating loop
sordaria demonstration cross plate (shared)
PROCEDURE
1. Place a drop of water on a clean slide with an inoculating loop.
2. With an inoculating loop, scrape several perithecia from the

demonstration cross plate. Scrape the perithecia from the interface of
two crossing strains close to the edge of the plate and place in the
drop of water on the slide. Avoid picking up agar along with
perithecia; it will interfere with results.
3. Cover the slide with a coverslip. Using a pencil eraser or other blunt
instrument, gently press down on the coverslip to squash and spread
out the perithecia. The pressure should be sufficient to squeeze the
asci from the perithecia, but not enough to crush the asci themselves. Note: It may be helpful
to slide the coverslip around on top of the sample, with slight pressure, to spread out the asci
and make them easier to observe. Keep in mind, however, that applying too much pressure
may rupture the asci, releasing the individual ascospores.
4. View the slide under a microscope at 100X. Locate the asci. You may wish to view the slide
at 400X to determine the color of some ascospores. The slide preparation should show
collapsed perithecia and asci clusters (rosettes), with mature ascospores in various
arrangements. Immature ascospores will all be light colored. Since S. fimicola is homothallic,
the preparation will show both hybrid and self-fertilized perithecia of both parental types.
Hybrid perithecia, however, will not occur very far from the line of contact between the two
varieties. Prepare three slides to get an adequate sampling of hybrids, if possible.
5. Count approximately 50 hybrid asci from at least three fields of view, preferably from
different slides. Record the data in Table 2.

Computer 3
ANALYSIS
Stage: ___________ Stage: ___________ Stage: ___________

Description: ______ Description: ______ Description: ______
_________________ _________________ _________________
_________________ _________________ _________________
_________________ _________________ _________________
_________________ _________________ _________________
Stage: ___________ Stage: ___________

Description: ______ Description: ______
_________________ _________________
_________________ _________________
_________________ _________________
_________________ _________________

Mitosis and Meiosis
Table 1
Cells in each stage of mitosis
Stage # of cells in # of cells in # of cells in Total # of % of total Time of each

Field 1 Field 2 Field 3 cells # of cells stage (min)
Interphase
Prophase
Metaphase
Anaphase
Telophase
Total number of cells counted __________
Table 2
Percentage of cells in each stage of mitosis
Stage % of total # of cells
Interphase
Prophase
Metaphase
Anaphase
Telophase
Total
Table 3
Observed Sordaria Asci
# of 4:4 # of crossover Total % asci showing Gene distance from

crossover centromere
Using your data, determine the distance in map units from the gene for ascospore color to the
chromosome centromere. Calculate the percentage of asci that showed crossover. This
percentage crossover must be divided by two, since only half the ascospores in each hybrid ascus
are the result of crossing over. Dropping the % symbol gives you the map distance from the gene
to the centromere. Record the data in Table 3.
% Crossover = # showing crossover × 100%
total counted
Gene Distance from Centromere = % Crossover
2

Computer 3
QUESTIONS
1. Referring to the percentage of total cells counted in each phase of mitosis, determine which
phase takes the longest for the cell to complete, and explain why. Sketch a pie graph of the
percentage of cells in each phase to illustrate. Be sure to title your graph and include a key.
2. What is the relationship between the processes of mitosis and cytokinesis?
3. Which of the following is significantly different between plant and animal cell mitosis?
a. metaphase
b. anaphase
c. cytokinesis
d. prophase
4. How does meiosis lead to genetic variability within a population? Use S. fimicola as an
example.
5. Define the following terms.

somatic cell
germ cell
chromatin
centromere
diploid
haploid
zygote
6. Create a Venn diagram showing at least two similarities and two differences between mitosis
and meiosis.
3 - 10 Advanced Biology with Vernier

Mitosis and Meiosis
ANIMAL CELL MITOSIS
Interphase Prophase
Metaphase Anaphase
Telophase Cytokinesis
Advanced Biology with Vernier 3 - 11

Computer 3
PLANT CELL MITOSIS
Interphase Prophase
Metaphase Anaphase
Telophase Cytokinesis

Experiment
Mitosis and Meiosis
This experiment correlates with Lab 3 in the 2001 College Board’s AP Biology Lab Manual.
TIME REQUIREMENTS
Part I: A – 20 min.
B – 20 min.
C – 45 min.
Part II: 45 min.
TIPS
1. As a general laboratory practice, it is recommended that students wear proper protective
equipment such as gloves, safety goggles, and a lab apron to avoid staining any clothing or
skin.
2. It is recommended that the magnification of the fields of view be standardized for the entire
class to ensure accurate and comparable data for all lab groups. A random or straight-line
method may be used; always make sure that none overlap.
3. For the Part I activity involving preparing onion root tips, it is necessary to begin growing the
onions two to three days prior to performing the experiment. The student version contains
directions for preparing the onion. If time is a concern, you may grow the onion root tips in
advance following the directions in the student procedure. Starting growth on a Thursday will
assure root tips for Monday.
4. Special handling for the sordaria cross plate: If the lines of growth between the agar cubes
appear dark and well developed, store the plate in a refrigerator, agar side up, until ready to
use. If the lines of growth appear light, incubate at room temperature until it resembles the
illustration below. Then store the plate in a refrigerator, agar side up, until ready to use.
5. To perform the first part of the meiosis lab—creating models of chromosomes with pop
beads or other items—in accordance with AP Biology requirements, we recommend
WARD’S Chromosome Simulation Kit, available separately (36 W 1602).
Advanced Biology with Vernier 3-1T

Experiment 3
SAMPLE DATA
Table 1
Cells in each stage of mitosis
# of cells in # of cells in # of cells in Total # of % of total # Time of each

Stage
Field 1 Field 2 Field 3 cells of cells stage (min)
Interphase 61 64 63 188 88 1,267
Prophase 4 5 4 13 6 86
Metaphase 1 3 2 6 3 43
Anaphase 2 1 1 4 2 29
Telophase 0 1 1 2 1 14
Total number of cells counted 213
Table 2
Percentage of cells in each stage of mitosis
Stage % of total # of cells
Interphase 88
Prophase 6
Metaphase 3
Anaphase 2
Telophase 1
Total 100
Table 3
Observed sordaria asci
% Asci showing Gene distance from

# of 4:4 # of crossover Total
crossover centromere
47 29 76 38 19
3-2T Advanced Biology with Vernier

Teacher Information Mitosis and Meiosis
1. Prophase is normally the longest phase of mitosis, due to the complexity of the events
occurring during prophase. These complex events take a relatively long time for the cell to
perform: chromatin condensing and thickening, nuclear membrane dissolving, and the early
stages of spindle development.
% of cells in each phase
Interphase
Prophase
Metaphase
Anaphase
Telophase
2. Mitosis is the division of the cell nucleus and its genetic material; cytokinesis refers to the
division of the cytoplasm and its constituent organelles, and structures between the two
daughter cells.
3. C. In plants, a cell plate is formed that develops into the cell wall between the two new
daughter cells. In animals, the formation of a microtubule-constructed cell furrow, a
narrowing between the two daughter cells, occurs and eventually pinches the parent cell in
two, bringing about the division of the cytoplasm of the parent cell.
4. Meiosis leads to genetic variability through the segregation of gene alleles, the independent
assortment of genes, and crossing over, as well as the variability that results from the
combination of the genetic material from the gametes of two genetically different individuals.
5. With increased variability among individuals in a population comes an increased probability

that the population would be able to survive under changing environmental and evolutionary
pressures. This ability, in turn, gives the species a better chance of surviving and thriving in
the future.
6. somatic cell – any cell in a multicellular organism that does not produce gametes.
germ cell – a gamete or any cell capable of producing a gamete in a multicellular organism.
chromatin – a combination of DNA and specialized proteins, called histones, within the
nucleus of a cell.
centromere – the site at which two chromatids are held together during chromosome
duplication.
diploid – containing the full complement of an organism’s chromosomes, the diploid number
of chromosomes is referred to as 2N.
haploid – containing half of number of chromosomes normally found in an organism,
gametes are haploid and the haploid chromosome number is referred to a N.
zygote – a cell resulting from the union of a male and female gamete.

Experiment 3
7. Create a Venn diagram showing at least two similarities and two differences between mitosis
and meiosis.
Mitosis Meiosis
Results in diploid Results in haploid

daughter cells cells
Starts with a
diploid cell
Yields two cells Yields four cells

Involves chromosome
replication
Does not involve
Can lead to
crossing-over
crossing-over
8. The fact that S. fimicola displays both haploid and diploid stages of reproduction allows
scientists to easily manipulate different strains of the organism. The colored ascospores are
easily identified and the ascospore patterns readily indicate when crossing over has occurred.
9. Unlike eukaryotic cells, prokaryotic cells utilize neither mitosis nor meiosis. Prokaryotic cells
replicate through a process known as binary fission. The prokaryotic DNA, which is found
free in the cell and not confined in a membrane-bound nucleus, replicates and each copy
attaches to a different part of the cell’s plasma membrane. The cell grows and, when it is
approximately double its original size, the membrane grows inward, dividing the cell into
two genetically identical daughter cells.
10. Student answers will vary. Students may choose materials such as pipe cleaners, yarn, etc.

Computer
Plant Pigment Chromatography

4A
Paper chromatography is a technique used to separate substances in a mixture based on the
movement of the different substances up a piece of paper by capillary action. Pigments extracted
from plant cells contain a variety of molecules, such as chlorophylls, beta carotene, and
xanthophyll, that can be separated using paper chromatography. A small sample of plant pigment
placed on chromatography paper travels up the paper due to capillary action. Beta carotene is
carried the furthest because it is highly soluble in the solvent and because it forms no hydrogen
bonds with the chromatography paper fibers. Xanthophyll contains oxygen and does not travel
quite as far with the solvent because it is less soluble than beta carotene and forms some
hydrogen bonds with the paper. Chlorophylls are bound more tightly to the paper than the other
two, so they travel the shortest distance.
The ratio of the distance moved by a pigment to the distance moved by the solvent is a constant,
Rf. Each type of molecule has its own Rf value.
distance traveled by pigment
Rf =
distance traveled by solvent
OBJECTIVES
• Separate plant pigments.
• Calculate the Rf values of the pigments.
MATERIALS
50 mL graduated cylinder cork stopper
chromatography paper pencil
spinach leaves scissors
coin solvent
goggles ruler
PROCEDURE
Obtain and wear goggles! Caution: The solvent in this experiment is flammable and poisonous.
Be sure there are no open flames in the lab during this experiment. Avoid inhaling fumes. Wear
goggles at all times. Notify your teacher immediately if an accident occurs.
1. Obtain a 50 mL graduated cylinder with 5 mL of solvent in the bottom.
2. Cut the chromatography paper so that it is long enough to reach the solvent. Cut one end of
the paper into a point.
3. Draw a pencil line 2.0 cm above the pointed end of the paper.
4. Use the coin to extract the pigments from the spinach leaf. Place a small section of the leaf on
top of the pencil line. Use the ribbed edge of the coin to push the plant cells into the
chromatography paper. Repeat the procedure 10 times making sure to use a different part of
the leaf each time.

Computer 4A
5. Place the chromatography paper in the cylinder so the pointed end just touches the solvent.
Make sure the pigment is not in the solvent.
6. Stopper the cylinder and wait until the solvent is approximately 1 cm from the top of the
paper. Remove the chromatography paper and mark the solvent front before it evaporates.
7. Allow the paper to dry. Mark the bottom of each pigment band. Measure the distance each
pigment moved from the starting line to the bottom of the pigment band. Record the distance
that each of the pigments and the solvent moved, in millimeters.
8. Identify each of the bands and label them on the chromatography paper.
• beta carotene: yellow to yellow orange
• xanthophyll: yellow
• chlorophyll a: bright green to blue green
• chlorophyll b: yellow green to olive green
9. Staple the chromatogram to the front of your lab sheet.
10. Discard the solvent as directed by your teacher.
DATA
Table 1
Band number Distance traveled Band color Identity

(mm)
5*
Distance solvent front moved = mm
* The fifth band may not appear.

PROCESSING THE DATA

Calculate the Rf values and record in Table 2.
Table 2
Molecule Rf
beta carotene
xanthophyll
chlorophyll a
chlorophyll b
QUESTIONS
1. What factors are involved in the separation of the pigments?
2. Would you expect the Rf value to be different with a different solvent?
3. Why do the pigments become separated during the development of the chromatogram?

Experiment
1. This experiment correlates with Exercise A of Lab 4 in the 2001 College Board’s AP Biology
Lab Manual.
2. The student pages can be found in Word® format on the CD that accompanies this book. See
Appendix A for more information.
3. Prepare the solvent for chromatography by mixing 9 parts (by volume) petroleum ether with
1 part acetone. Distribute 5 mL of solvent to each of the cylinders Warning: Be very careful
with the petroleum ether as it is highly flammable. Extinguish all flames in the room.
HAZARD ALERT: Petroleum Ether: Flammable liquid; flammable; dangerous fire risk.
Hazard code: C—Somewhat hazardous.
HAZARD ALERT: Acetone: Dangerous fire risk; flammable; slightly toxic by ingestion
and inhalation. Hazard code: C—Somewhat hazardous
The hazard information reference is: Flinn Scientific, Inc., Chemical & Biological Catalog
Reference Manual, 2000, (800) 452-1261, www.flinnsci.com. See Appendix D of this book,
Advanced Biology with Vernier, for more information.
4. Remember to handle paper strips by the edges only.
5. Remind students to wear goggles and to keep the tubes stoppered.
6. Refer to the MSDS for proper disposal techniques.
Ideas for Inquiry

• Repeat the paper chromatography with various species of plants. What similarities do you
see? What differences are there?
• Repeat the paper chromatography with different colored plant leaves such as coleus and
zebrina.
• Repeat the paper chromatography with different colored petals from plant flowers.

Experiment 4A
SAMPLE RESULTS
Table 1
Band number Distance traveled Band color Identity

(mm)
1 141 faint yellow carotene
2 60 yellow xanthrophyll
3 30 bright green chlorophyll a
4 15 olive green chlorophyll b
5*
Distance solvent front moved = 150 mm
* band 5 may not appear

Table 2
Molecule Rf
beta carotene 0.94
xanthophyll 0.40
chlorophyll a 0.20
chlorophyll b 0.10
1. The distance that each pigment migrates up the paper is determined by its solubility and its
attraction to the fibers in the paper. Pigments with the greatest solubility and least attraction
to the fibers in the paper travel the greatest distance up the paper.
2. Since the Rf factor depends on a molecule’s ability to dissolve in the solvent, the pigment
may not travel as far up the paper.
3. The pigments become separated because they are not equally soluble in the solvent. They are
attracted, to different degrees, to the fibers in the paper through the formation of
intermolecular bonds, such as hydrogen bonds.

Computer
Photosynthesis
4B
The process of photosynthesis involves the use of light energy to convert carbon dioxide and
water into sugar, oxygen, and other organic compounds. This process is often summarized by the
following reaction:
6 H2O + 6 CO2 + light energy → C6H12O6 + 6 O2
This process is an extremely complex one, occurring in two stages. The first stage, called the
light reactions of photosynthesis, requires light energy. The products of the light reactions are
then used to produce glucose from carbon dioxide and water. Because the reactions in the second
stage do not require the direct use of light energy, they are called the dark reactions of
photosynthesis.
In the light reactions, electrons derived from water are “excited” (raised to higher energy levels)
in several steps, called photosystems I and II. In both steps, chlorophyll absorbs light energy that
is used to excite the electrons. Normally, these electrons are passed to a cytochrome-containing
electron transport chain. In the first photosystem, these electrons are used to generate ATP. In
the second photosystem, excited electrons are used to produce the reduced coenzyme
nicotinamide adenine dinucleotide phosphate (NADPH). Both ATP and NADPH are then used in
the dark reactions to produce glucose.
In this experiment, a blue dye (2,6-dichlorophenol-indophenol, or DPIP) will be used to replace

NADPH in the light reactions. When the dye is oxidized, it is blue. When reduced, however, it
turns colorless. Since DPIP replaces NADPH in the light reactions, it will turn from blue to
colorless when reduced during photosynthesis.
OBJECTIVES
• Use a Colorimeter or Spectrometer to measure color changes due to photosynthesis.
• Study the effect of light on photosynthesis.
• Study the effect that the boiling of plant cells has on photosynthesis.
• Compare the rates of photosynthesis for plants in different light conditions.
Figure 1

Computer 4B
MATERIALS
computer two small test tubes
Vernier computer interface 5 mL pipet
Logger Pro pipet pump or bulb
Colorimeter or Spectrometer two Beral pipets
four cuvettes with lids 10 mL DPIP
aluminum foil phosphate buffer solution
100 watt floodlight unboiled chloroplast suspension
stopwatch boiled chloroplast suspension
600 mL beaker ice
250 mL beaker distilled water
PROCEDURE
2. Mark one Beral pipet and one test tube with a U (unboiled). Mark the other Beral pipet and
test tube with a B (boiled).
3. Fill the 250 mL beaker with ice and place the test tubes in the beaker.
4. Mark the four cuvette lids with with a BL (blank), a U (unboiled), a D (dark), and a
B (boiled).
5. Cover all four sides and the bottom of one of the cuvettes with aluminum foil. This will be
the Dark cuvette.
6. Use the information in Table 1 to add the phosphate buffer, distilled H2O, and DPIP to each
cuvette. Important: Do not add chloroplasts at this time.
Table 1
BL U D B
Blank Unboiled Unboiled Boiled
(no DPIP) light dark light
Phosphate Buffer 1 mL 1 mL 1 mL 1 mL
Distilled H2O 2 mL 1 mL 1 mL 1 mL
DPIP — 1 mL 1 mL 1 mL
Unboiled chloroplasts 3 drops 3 drops 3 drops —
Boiled chlorophlasts — — — 3 drops
7. Locate the unboiled and boiled chloroplast suspension prepared by your instructor. Before
removing any of the chloroplast suspension, gently swirl to resuspend any chloroplast that
may have settled out. Using the pipet marked U, draw up ~1 mL of unboiled chloroplast
suspension and dispense into the U test tube. Using the pipet maked B, draw up ~1 mL of
boiled chloroplast suspension and dispense into the B test tube. Both test tubes should remain
in the ice.

Photosynthesis
8. Finish preparing the Blank cuvette by adding three drops of unboiled chloroplasts. Place the
lid marked with BL on the cuvette and gently invert three times to mix. To correctly use a
cuvette, remember:
• Wipe the outside of each cuvette with a lint-free tissue.
• Handle cuvettes only by the top edge of the ribbed sides.
• Dislodge any bubbles by gently tapping the cuvette on a hard surface.
• Always position the cuvette so the light passes through the clear sides.
Spectrometer Users Only (Colorimeter users proceed to the Colorimeter section)
9. Calibrate the Spectrometer.
a. Use a USB cable to connect the Spectrometer to your computer. Choose New from the
File menu.
b. To calibrate the Spectrometer, place the blank cuvette into the cuvette slot of the
Spectrometer, and choose Calibrate ►Spectrometer from the Experiment menu. The
calibration dialog box will display the message: “Waiting 90 seconds for lamp to warm
up.” After 90 seconds, the message will change to “Warmup complete.” Click .
10. Determine the optimum wavelength for examining the DPIP solution.
a. Empty the Blank cuvette. Fill it with 1 mL phosphate buffer, 1 mL distilled water, 1 mL
DPIP, and 3 drops of unboiled chloroplast. Place it in the spectrometer.
b. Click . A full spectrum graph of the solution will be displayed. Note that one area
of the graph contains a peak absorbance. Click to complete the analysis.
c. To select a wavelength for analysis, click on the Configure Spectrometer Data Collection
icon, , on the toolbar.
d. Click Abs vs. Concentration (under the Set Collection Mode). The wavelength of
maximum absorbance (λ max) will be selected. (It should be close to 605 nm.) Click
. Remove the cuvette from the spectrometer and dispose of the solution as directed.
e. Proceed to Step 11.
Colorimeter Users Only

9. Connect the Colorimeter to the computer interface. Prepare the computer for data collection
by opening the file “04B Photosynthesis” from the Advanced Biology with Vernier folder of
Logger Pro.
10. Calibrate the Colorimeter.

a. Open the Colorimeter lid.
b. Holding the Blank cuvette by the upper edges, place it in the cuvette slot of the
Colorimeter. Close the lid.
c. Press the < or > button on the Colorimeter to select a wavelength of 635 nm (Red) for this
experiment. Note: If your Colorimeter has a knob to select the wavelength instead of
arrow buttons, ask your instructor for calibration information.
d. Press the CAL button until the red LED begins to flash, then release. When the LED stops
flashing, the calibration is complete. Proceed to Step 11.

Computer 4B
Both Colorimeter and Spectrometer Users

11. Obtain a 600 mL beaker filled with water and a flood lamp. Arrange the lamp and beaker as
shown in Figure 2. The beaker will act as a heat shield, protecting the chloroplasts from
warming by the flood lamp. Do not turn on the lamp until Step 14.
Figure 2
12. Finish preparing the cuvettes. Important: Perform the following steps as quickly as possible
and proceed directly to Step 13.
a. Cuvette U: Add 3 drops of unboiled chloroplasts. Place the lid on the cuvette and gently
mix; try not to introduce bubbles in the solution. Place the cuvette in front of the lamp as
shown in Figure 2. Mark the cuvette's position so that it can always be placed back in the
same spot.
b. Cuvette D: Add 3 drops of unboiled chloroplasts. Place the lid on the cuvette and gently
mix; try not to introduce bubbles in the solution. Place the foil-covered cuvette in front of
the lamp as shown in Figure 2 and mark its position. Make sure that no light can penetrate
the cuvette.
c. Cuvette B: Add 3 drops of boiled chloroplasts. Place the lid on the cuvette and gently mix;
try not to introduce bubbles in the solution. Place the cuvette in front of the lamp as
shown in Figure 2. Mark the cuvette's position so that it can be placed back in the same
spot.
13. Take absorbance readings for each cuvette. Invert each cuvette two times to resuspend the
chloroplast before taking a reading. If any air bubbles form, gently tap on the cuvette lid to
knock them loose.
a. Cuvette U: Place the cuvette in the device (close the lid if using a Colorimeter). Allow
10 seconds for the readings displayed in the meter to stabilize. Record the absorbance
value in Table 2. Remove the cuvette and place it in its original position in front of the
lamp.
b. Cuvette D: Remove the cuvette from the foil sleeve and place it in the device (close the lid
if using a Colorimeter). Wait 10 seconds and record the absorbance value in Table 2.
Remove the cuvette and place it back into the foil sleeve. Place the cuvette in its original
position in front of the lamp.
c. Cuvette B: Place the cuvette in the device (close the lid if using a Colorimeter). Wait
10 seconds and record the absorbance value in Table 2. Remove the cuvette and place it in
its original position in front of the lamp.
14. Turn on the lamp and note the time.
15. Repeat Step 13 after 5, 10, 15, and 20 minutes have elapsed.

Photosynthesis
PROCESSING THE DATA

1. Go to Page 2 of the experiment file. Colorimeter users will already have this file open.
Spectrometer users need to open the file “04B Photosynthesis” from the Advanced Biology
with Vernier folder of Logger Pro. Manually enter the data recorded in Table 1 into the
appropriate column in the table. Click on the table cell to enter values.
2. Calculate the rate of photosynthesis for each of the three cuvettes tested.
a. Click the Linear Fit button, , to perform a linear regression. A dialog box will appear.
Select the three data sets on which you wish to perform a linear regression and click
. A floating box will appear with the formula for a best-fit line for each data set
selected.
b. In Table 3, record the slope of the line, m, as the rate of photosynthesis for each data set.
c. Close the linear regression floating boxes.
DATA
Table 2 Table 3
Absorbance Absorbance Absorbance

Time Rate of
unboiled unboiled boiled Chloroplast
(min) photosynthesis
light dark light
0 Unboiled
5 Dark
10 Boiled
15
20
QUESTIONS
1. Is there evidence that chloroplasts were able to reduce DPIP in this experiment? Explain.
2. Were chloroplasts able to reduce DPIP when kept in the dark? Explain.
3. Were boiled chloroplasts able to reduce DPIP ? Explain.
4. What conclusions can you make about the photosynthetic activity of spinach?

Experiment
Photosynthesis
1. This experiment correlates with Exercise B of Lab 4 in the 2001 College Board’s AP Biology
Lab Manual.
2. The student pages with complete instructions for using a Colorimeter or Spectrometer with
LabQuest App and Logger Pro (computers) can be found on the CD that accompanies this
book. Instructions for using a Colorimeter with EasyData (calculators) can also be found on
the CD (EasyData does not support the use of Spectrometers). See Appendix A for more
information.
3. It is necessary to suspend the spinach chloroplasts in a 0.5 M sucrose solution. To prepare the
0.5 M sucrose solution, add 171 g of sucrose to distilled water to make a total volume of 1 L.
Store this solution in the refrigerator overnight before preparing the chloroplast solution.
Place the blender and beaker in the freezer overnight also.
4. To prepare the phosphate buffer solution:
a. Add 174 g of K2HPO4 (dibasic) to distilled water to make a total volume of 1 liter.
b. Add 136 g of KH2PO4 (monobasic) to distilled water to make a total volume of 1 liter.
c. Combine the two solutions until the pH is 6.5. About 685 mL of monobasic should be
added to 315 mL of dibasic to obtain a solution with a pH of 6.5.
5. To prepare the DPIP (2,6-dichlorophenol-indophenol ) solution, add 0.072 g of DPIP to
distilled water to make a total volume of 1 L. Store this solution in a dark bottle and
refrigerate.
6. To prepare a chloroplast suspension,
a. Place fresh spinach leaves in the light for a few hours. Be sure they remain cool and
hydrated.
b. Cover the blades of a blender with cold 0.5 M sucrose.
c. Pack the spinach leaves into the blender until they are about three centimeters above the
blades.
d. Blend the spinach with three 10 second bursts. Wait 30 seconds between bursts.
e. Filter the mixture through cheesecloth into a cold beaker. Keep the beaker on ice. You will
need to squeeze the cheesecloth to release as much liquid as possible.
f. Split the chloroplast suspension into two equal parts. Set one part aside for Item 7 below.
g. Distribute 2 mL portions into covered, cooled vials. The vials should be light-tight. Black
electrical tape works well to cover the vials.
h. Keep the chloroplast suspensions on ice.
7. Obtain the spinach solution that was set aside in Item 7 and boil it for 5 minutes. Distribute
2 mL portions of boiled chloroplasts into darkened, taped vials. Keep these suspensions on
ice.
8. Test the chloroplast suspension prior to use. If the DPIP is reduced too rapidly, further dilute
the suspension before distributing it into the vials.

Experiment 4B
9. The success of this lab is greatly dependent on the ability of the students to synchronize taking
absorbance readings. Encourage the students to read over the procedure carefully before
beginning.
10. There are two models of Vernier Colorimeters. The first model (rectangular shape) has three
wavelength settings, and the newest model (a rounded shape) has four wavelength settings.
The 635 nm wavelength of either model is used in this experiment. The newer model is an
auto-ID sensor and supports automatic calibration (pressing the CAL button on the
Colorimeter with a blank cuvette in the slot). If you have an older model Colorimeter, see
www.vernier.com/til/1665.html for calibration information.
11. If incandescent bulbs are unavailable, the experiment can be modified to use halogen, compact
fluorescent, or LED bulbs.
Ideas for Inquiry

• How do different plant types affect photosynthesis? (i.e., C3, C4, CAM plants)
• What effect does the use of red, blue, and green light have on the photosynthetic activity of
spinach?
• What effect do different types of lights have on photosynthetic activity?
• What is the effect of a change in concentration of the unboiled chloroplasts?
• What is the effect of changing the concentration of the buffer?
SAMPLE RESULTS
Table 1
Time Unboiled Dark Boiled
(min) absorbance absorbance absorbance
0 0.480 0.456 0.480
5 0.394 0.466 0.470
10 0.326 0.466 0.490
15 0.250 0.451 0.466
20 0.139 0.456 0.466

Teacher Information Photosynthesis
Table 2
Rate of
Chloroplast
photosynthesis
Unboiled 0.0165
Dark 0.0003
Boiled 0.0007
1. Yes, there is evidence that spinach chloroplasts were able to reduce DPIP in this experiment,
as the color of the solution changed when the light intensity changed.
2. No, chloroplasts were not able to reduce DPIP while kept in the dark, as evidenced by the
lack of color change of DPIP and the low rate of photosynthesis. The reduced form of DPIP is
colorless, and since the solution remained blue, the DPIP did not participate in the light
reactions of photosynthesis.
3. Boiled chloroplasts were not able to reduce DPIP, as the heat destroyed the photosynthetic
machinery of chloroplasts. The chloroplasts were inactive, even in the presence of light.
4. Based upon student data, chloroplasts should reduce DPIP when exposed to light.
Chloroplasts should not be able to reduce DPIP when placed in the dark, nor when destroyed.
The amount of reduced DPIP is proportional to the amount of light the chloroplasts are
exposed to. The implication is that the amount of sugar produced in photosynthesis depends
upon the duration of exposure to light.

Computer
Cell Respiration
5
(Method 1–CO2 and O2)
Cell respiration refers to the process of converting the chemical energy of organic molecules into
a form immediately usable by organisms. Glucose may be oxidized completely if sufficient
oxygen is available according to the following equation:
C6H12O6 + 6O2(g) → 6 H2O + 6 CO2(g) + energy
All organisms, including plants and animals, oxidize glucose for energy. Often, this energy is
used to convert ADP and phosphate into ATP. Peas undergo cell respiration during germination.
Do peas undergo cell respiration before germination? Using your collected data, you will be able
to answer the question regarding respiration and non-germinating peas.
OBJECTIVES
• Measure gas production.
• Study the effect of temperature on cell respiration.
• Determine whether germinating peas and non-germinating peas respire.
• Compare the rates of cell respiration in germinating and non-germinating peas.
MATERIALS
computer 25 germinating peas
Vernier computer interface 25 non-germinating peas
Logger Pro 250 mL respiration chamber
Vernier CO2 Gas Sensor ice cubes
Vernier O2 Gas Sensor thermometer
BioChamber 250 two 100 mL beakers
PROCEDURE
Using the CO2 Gas Sensor and O2 Gas Sensor, you will monitor the carbon dioxide produced and
the oxygen consumed by peas during cell respiration. Both germinating and non-germinating
peas will be tested. Additionally, cell respiration of germinating peas at two different
temperatures will be investigated.
Advanced Biology with Vernier 5 - 1 (CO2 and O2)

Computer 5
Figure 1
1. If your CO2 Gas Sensor has a switch, set it to the Low (0–10,000 ppm) setting. Connect the
CO2 Gas Sensor to Channel 1 and the O2 Gas Sensor to Channel 2 of the Vernier computer
interface.
2. Prepare the computer for data collection by opening the file “05 Cell Resp M1 CO2 O2”
from the Advanced Biology with Vernier folder of Logger Pro.
3. Obtain 25 germinating peas and blot them dry between two pieces of paper towel. Use the
thermometer to measure the room temperature. Record the temperature in Table 1.
4. Place the germinating peas into the respiration chamber.
5. Place the O2 Gas Sensor into the BioChamber 250 as shown in Figure 1. Insert the sensor
snugly into the grommet. The O2 Gas Sensor should remain vertical throughout the
experiment. Place the CO2 Gas Sensor into the neck of the respiration chamber as shown in
Figure 1.
6. Wait four minutes for readings to stabilize, then begin collecting data by clicking .
Collect data for ten minutes and click .
7. When data collection has finished, remove the sensors from the respiration chamber. Place
the peas in a 100 mL beaker filled with cold water and ice.
8. Fill the respiration chamber with water and then empty it. Thoroughly dry the inside of the
respiration chamber with a paper towel.
9. Determine the rate of respiration:
a. Click anywhere on the CO2 graph to select it. Click the Linear Fit button, , to perform a
linear regression. A floating box will appear with the formula for a best fit line.
b. Record the slope of the line, m, as the rate of respiration for germinating peas at room
temperature in Table 2.
c. Close the linear regression floating box.
d. Repeat Steps 9a–c for the O2 graph.
menu.
11. Obtain 25 non-germinating peas and place them in the respiration chamber
12. Repeat Steps 5–10 for the non-germinating peas.
5 - 2 (CO2 and O2) Advanced Biology with Vernier

Cell Respiration (CO2 and O2)
Part II Germinating peas, cool temperatures

13. Remove the peas from the cold water and blot them dry between two paper towels.
14. Repeat Steps 5–9 to collect data with the germinating peas at a cold temperature.
15. To print a graph of concentration vs. volume showing all three data runs:
a. Click anywhere on the CO2 graph. Label all three curves by choosing Text Annotation
from the Insert menu, and typing “Room Temp Germinated” (or “Room Temp Non-
germinated”, or “Cold Germinated”) in the edit box. Then drag each box to a position near
its respective curve. Adjust the position of the arrow head.
c. Click on the O2 graph and repeat the process to print a copy of the O2 graph.
DATA
Table 1
Condition Temperature (°C)
Room
Table 2
Peas CO2 O2
Rate of respiration Rate of consumption
(ppt/min) (ppt/min)
Germinating, room temperature
Non-germinating, room temperature
Germinating, cool temperature
QUESTIONS
1. Do you have evidence that cell respiration occurred in peas? Explain.
2. What is the effect of germination on the rate of cell respiration in peas?
3. What is the effect of temperature on the rate of cell respiration in peas?
4. Why do germinating peas undergo cell respiration?
Advanced Biology with Vernier 5 - 3 (CO2 and O2)

Computer 5
EXTENSIONS
1. Compare the respiration rate among various types of seeds.
2. Compare the respiration rate among seeds that have germinated for different time periods,
such as 1, 3, and 5 days.
3. Compare the respiration rate among various types of small animals, such as insects or
earthworms.
5 - 4 (CO2 and O2) Advanced Biology with Vernier

Experiment
Cell Respiration
(Method 1–CO2 and O2)
3. If you are using calculators for data collection, this activity can be performed with calculators
from the TI-83 Plus or TI-84 Plus families and a LabPro or CBL 2. It can not be performed
with Easy products because the CO2 Gas Sensor is not supported by EasyLink.
4. Allow the seeds to germinate for three days prior to the experiment. Prior to the first day,
soak them in water overnight. On subsequent days, roll them in a moist paper towel and place
the towel in a paper bag. Place the bag in a warm, dark place. Check each day to be sure the
towels remain very moist. If time is short, the peas can be used after they have soaked
overnight. For best results, allow them to germinate for the full three days.
5. The O2 Gas Sensor should always be stored upright in the box in which it was shipped.
6. The morning of the experiment, fill a 1 L beaker with ice and water so that students will have
cold water. Students will also need access to ice.
7. The calibrations stored in this experiment file for both sensors work well for this experiment.
Initial readings that seem slightly high or low will still reflect an accurate change in gas
levels.
8. The stopper included with the older-style CO2 Gas Sensor is slit to allow easy application and
removal from the probe. When students are placing the probe in the respiration chamber, they
should gently twist the stopper into the chamber opening. Warn the students not to twist the
probe shaft or they may damage the sensing unit.
9. To conserve battery power, we suggest that AC Adapters be used to power the interfaces
rather than batteries when working with the older-style CO2 Gas Sensor.
Ideas for Inquiry

• How does temperature affect pea respiration rate?
• What is the optimal temperature for pea respiration?
• How does germination duration affect the rate of CO2 production?
• How does the presence of gibberellin (kinetin, zeatin) in the germination medium affect
cellular respiration in peas?
• How does the presence of mannitol (sucrose) in the germination medium affect cellular
respiration in peas?
Advanced Biology with Vernier 5 - 1 T (CO2 and O2)

Experiment 5
SAMPLE RESULTS
O2 consumed by germinated peas
CO2 levels of germinating and non-germinating peas
Table 1
room 22.4
cold water 13.7
5 - 2 T (CO2 and O2) Advanced Biology with Vernier

Teacher Information Cell Respiration (CO2 and O2)
Table 2
CO2 rate of respiration O2 rate of consumption

Peas
(ppt/min) (ppt/min)
Germinating, room
0.249 –0.152
temperature
Non-germinating,
0.003 –0.002
room temperature
Germinating, cool
0.066 –0.087
temperature
1. Yes, the oxygen concentration vs. time graph clearly indicates that oxygen is being consumed
at a steady rate when germinating peas are in the respiration chamber. The carbon dioxide
concentration vs. time graph indicates that carbon dioxide is being produced at a steady rate.
2. Germination greatly accelerates the rate of cellular respiration. This reflects a higher rate of
metabolic activity in germinating seeds. In most experiments, non-germinating seeds do not
seem to be respiring.
3. Warm temperatures increase the rate of respiration. This reflects a higher rate of metabolic
activity in warm germinating seeds than in cooler seeds.
4. It is necessary for germinating seeds to undergo cellular respiration in order to acquire the
energy they need for growth and development. Unlike their mature relatives, seeds do not yet
have the necessary photosynthetic abilities needed to produce their own energy sources.
Advanced Biology with Vernier 5 - 3 T (CO2 and O2)

Computer
Cell Respiration
5
(Method 2–CO2 Gas Sensor)
OBJECTIVES
MATERIALS
Vernier CO2 Gas Sensor ice cubes
two 100 mL beakers thermometer
PROCEDURE
1. If your sensor has a switch, set it to the Low (0–10,000 ppm) setting. Connect
the CO2 Gas Sensor to Channel 1 of the Vernier computer interface.
2. Prepare the computer for data collection by opening the “05 Cell Resp M2
CO2” file in the Advanced Biology with Vernier folder of Logger Pro.
3. Obtain 25 germinating peas and blot them dry between two pieces of paper
towel. Use the thermometer to measure the room temperature. Record the
5. Place the shaft of the CO2 Gas Sensor in the opening of the respiration
chamber. Figure 1
Advanced Biology with Vernier 5 - 1 (CO2)

Computer 5
6. Wait one minute, then begin measuring carbon dioxide concentration by clicking .
Data will be collected for 10 minutes.
7. Remove the CO2 Gas Sensor from the respiration chamber. Place the peas in a 100 mL beaker
filled with cold water and an ice cube. The cold water will prepare the peas for part II of the
experiment.
8. Use a notebook or notepad to fan air across the openings in the probe shaft of the CO2 Gas
Sensor for 1 minute.

a. Move the mouse pointer to the point where the data values begin to increase. Hold down
the left mouse button. Drag the mouse pointer to the end of the data and release the mouse
button.
c. Record the slope of the line, m, as the rate of respiration for germinating peas at room
menu.

15. Repeat Steps 5–11 to collect data with the cold germinating peas.
16. To print a graph showing all three data runs:
“Room Temp Germinated” (or “Room Temp Non-germinated”, or “Cold Germinated”) in
the edit box. Then drag each box to a position near its respective curve. Adjust the
position of the arrowhead.
5 - 2 (CO2) Advanced Biology with Vernier

Cell Respiration (CO2)
DATA
Table 1
Temperature
Condition
(°C)
Room
Table 2
Rate of Respiration
Peas
(ppm/min)
QUESTIONS
EXTENSIONS
earthworms.
Advanced Biology with Vernier 5 - 3 (CO2)

Experiment
Cell Respiration
(Method 2–CO2 Gas Sensor)
3. If you are using calculators for data collection, this activity can be performed with calculators
from the TI-83 Plus or TI-84 Plus families and a LabPro or CBL 2. It can not be performed
with Easy products because the CO2 Gas Sensor is not supported by EasyLink.
5. Heavy condensation buildup in the respiration chamber can interfere with readings from the
CO2 Gas Sensor. This can be a source of error if the peas are very wet when placed in the
respiration chamber. Before placing the peas in the respiration chamber, blot them dry with a
paper towel.
6. The stopper included with the older-style CO2 Gas Sensor is slit to allow easy application and
removal from the probe. When students are placing the probe in the respiration chamber, they
should gently twist the stopper into the chamber opening. Warn the students not to twist the
probe shaft or they may damage the sensing unit.
7. The CO2 Gas Sensor relies on the diffusion of gases into the probe shaft. Students should
allow a couple of minutes between trials so that gases from the previous trial will have exited
the probe shaft. Alternatively, the students can use a firm object such as a book or notepad to
fan air through the probe shaft. This method is used in Step 8 of the student procedure.
8. The morning of the experiment fill a 1 L beaker with ice and water so that students will have
cold water. Students will also need access to ice.
9. The calibration stored in this experiment file works well for this experiment. Initial readings
that seem slightly high or low will still reflect an accurate change in gas levels.
10. To conserve battery power, we suggest that AC Adapters be used to power the interfaces
rather than batteries when working with the older-style CO2 Gas Sensor.
Ideas for Inquiry

Advanced Biology with Vernier 5 - 1 T (CO2)

Experiment 5

SAMPLE RESULTS
CO2 respired by germinating and non-germinating peas
Table 1
room 22
cold water 10
Table 2
Peas Rate Respiration (ppm/min)
Germinating, room temperature 194.5
Non-germinating, room temperature 6.6
Germinating, cool temperature 122.1
1. Yes, the carbon dioxide concentration vs. time graph clearly indicates that carbon dioxide is
being produced at a steady rate when germinating peas are in the respiration chamber.
seem to be respiring. Occasionally, however, some respiration is detectable.
5 - 2 T (CO2) Advanced Biology with Vernier

Teacher Information Cell Respiration (CO2)
activity in warm germinating seeds than in cool seeds.
have the necessary photosynthetic abilities needed to product their own energy sources.
Advanced Biology with Vernier 5 - 3 T (CO2)

Computer
Cell Respiration
5
OBJECTIVES
MATERIALS
Vernier O2 Gas Sensor ice cubes
two 100 mL beakers thermometer
PROCEDURE
1. Connect the O2 Gas Sensor to the computer interface.
2. Prepare the computer for data collection by opening the file “05 Cell
Resp M3 O2” from the Advanced Biology with Vernier folder of
Logger Pro.
3. Obtain 25 germinating peas and blot them dry between two pieces of
paper towel. Use the thermometer to measure the room temperature.
Record the temperature in Table 1.
5. Place the O2 Gas Sensor into the bottle as shown in Figure 1. Gently push
the sensor down into the bottle until it stops. The sensor is designed to
seal the bottle without the need for unnecessary force. Figure 1

Computer 5
6. Wait two minutes, then begin collecting data by clicking . Data will be collected for
10 minutes.
7. When data collection has finished, remove the O2 Gas Sensor from the respiration chamber.
Place the peas in a 100 mL beaker filled with cold water and an ice cube.

a. Click the Linear Fit button, , to perform a linear regression. A floating box will appear
b. Record the slope of the line, m, as the rate of respiration for germinating peas at room
c. Close the linear regression floating box.
menu.

14. Repeat Steps 5–9 to collect data with the germinating peas at a cold temperature.
15. To print a graph showing all three data runs:

“Room Temp Germinated” (or “Room Temp Non-germinated”, or “Cold Germinated”) in
the edit box. Then drag each box to a position near its respective curve. Adjust the
position of the arrow head.

Cell Respiration (O2)
DATA
Table 1
Temperature
Condition
(°C)
room
Table 2
Rate of Respiration
Peas
(%/min)
QUESTIONS
EXTENSIONS
earthworms.

Experiment
Cell Respiration
4. To extend the life of the O2 Gas Sensor, always store the sensor upright in the box it was
shipped in.
5. The morning of the experiment fill a 1 L beaker with ice and water so that students will have
cold water for Step 7. Students will also need access to ice.
6. The calibration stored in the data-collection software works well for this experiment. The
calibration is for the O2 Gas Sensor (%).
Ideas for Inquiry


Experiment 5
SAMPLE RESULTS
O2 respired by germinated peas
Table 1
Temperature
Condition (°C)
room 24
cold water 9
Table 2
Peas Rate of Respiration

(%/min)
Germinating, room temperature –0.057
Non-germinating, room temperature –0.002
Germinating, cool temperature –0.039
1. Yes, the oxygen concentration vs. time graph clearly indicates that oxygen is being consumed
at a steady rate when germinating peas are in the respiration chamber.

Teacher Information Cell Respiration (O2)

Computer
Cell Respiration
5
Cellular respiration refers to the process of converting the chemical energy of organic molecules
into a form immediately usable by organisms. Glucose may be oxidized completely if sufficient
oxygen is available and is summarized by the following reaction:
C6H12O6 + 6 O2(g) ←
⎯→ 6 H2O + 6 CO2(g) + energy
used to convert ADP and phosphate into ATP.
To measure the rate of cellular respiration, the pressure change due to the consumption of oxygen
by peas will be measured with a Gas Pressure Sensor. It is not possible to directly measure
pressure changes due to oxygen, since the Gas Pressure Sensor measures the total pressure
change. Carbon dioxide is produced as oxygen is consumed. The pressure due to CO2 might
cancel out any change due to the consumption of oxygen. To eliminate this problem, a chemical
will be added that will selectively remove CO2. Potassium hydroxide, KOH, will chemically
react with CO2 by the following equation:
2 KOH + CO2 ⎯
⎯→ K2CO3 + H2O
This will allow you to monitor pressure changes exclusively due to the consumption of oxygen.
A respirometer is the system used to measure cellular respiration. Pressure changes in the
respirometer are directly proportional to a change in the amount of gas in the respirometer,
providing the volume and the temperature of the respirometer do not change. If you wish to
compare the consumption of oxygen in two different respirometers, as we will in this experiment,
you must keep the volume and temperature of the air equal in each respirometer.
Both germinating and non-germinating peas will be tested. Additionally, cellular respiration of
germinating peas at two different temperatures will be tested.
OBJECTIVES

Computer 5
MATERIALS
computer glass beads
Vernier computer interface ice
Logger Pro non-absorbent cotton
2 Vernier Gas Pressure Sensors thermometer
15% KOH in a dropper bottle test tube rack
25 germinating peas timer with a second hand
25 non-germinating peas three 18 ×150 mm test tubes
100 mL graduated cylinder two 1-hole rubber stopper assemblies
absorbent cotton two 1 L beakers
forceps ring stand
2 utility clamps
PROCEDURE
Figure 1
1. Connect the plastic tubing to the valves on the Gas Pressure Sensors
2. Connect the Gas Pressure Sensors to the computer interface. Prepare the computer for data
collection by opening the file “05 Cell Resp M4 Pressure” from the Advanced Biology with
Vernier folder of Logger Pro.
To test whether germinating peas undergo cell respiration, you will
need to
• set up two water baths.
• prepare a respirometer for the germinating peas.
• prepare a second, control respirometer containing glass beads.
3. Set up two water baths, one at about 25°C and one at about 10°C.
Obtain two 1 liter beakers and place about 800 mL of water in each.
Add ice to attain the 10°C water bath.
4. To be sure the volumes of air in all respirometers are equal, you will
Peas
need to measure the volume of the twenty-five peas that will be in the Cotton non-absorbent
experimental respirometer. The control respirometer must have an equal
volume of glass beads (or other non-oxygen consuming material) to
make the air volume equal to the respirometer with germinating peas.
Cotton with KOH
Similarly, glass beads will be used to account for any volume difference
between the germinating and non-germinating peas.
Figure 2

Cell Respiration (Gas Pressure)
5. Obtain three test tubes and label them “T1”, T2”, and “T3”.
6. Place a 3 cm wad of absorbent cotton in the bottom of each test tube. Using a dropper pipette,
carefully add a sufficient amount of KOH to the cotton to completely saturate it. Do not put
so much that liquid can easily run out of the tube. Note: Do not allow any of the KOH to
touch the sides of the test tube. The sides should be completely dry, or the KOH may damage
the peas. CAUTION: Potassium hydroxide solution is caustic. Avoid spilling it on your
clothes or skin.
7. Prepare the test tube containing germinating peas (T1):

a. Add 50 mL of water to a 100 mL graduated cylinder.
b. Place 25 germinating peas into the water.
c. Measure the volume of the peas by water displacement. Record that volume in Table 1.
d. Gently remove the peas from the graduated cylinder and blot them dry with a paper towel.
e. Add a small wad of non-absorbent dry cotton to the bottom of the test tube to prevent the
peas from touching the KOH saturated cotton.
f. Add these germinating peas to the respirometer labeled “T1”.
8. Prepare the test tube containing non-germinating peas (T2):
a. Refill the graduated cylinder with 50 mL of water.
b. Place 25 non-germinating peas into the water.
c. Measure the volume of the peas by water displacement. Record the volume in Table 1.
d. Add a sufficient number of glass beads to the non-germinating peas and water until they
displace exactly the same volume of water as the germinating peas.
e. Gently remove the peas and glass beads from the graduated cylinder and dry them with a
paper towel.
f. Add a small wad of dry non-absorbent cotton to the bottom of the test tube to prevent the
g. Add the non-germinating peas and glass beads to the respirometer labeled “T2”.
9. Prepare the test tube containing glass beads (T3):
a. Refill the graduated cylinder with 50 mL of water.
b. Add a sufficient number of glass beads to the water until they displace exactly the same
volume of water as the germinating peas.
c. Remove the glass beads from the graduated cylinder and dry them.
d. Add a small wad of dry non-absorbent cotton to the bottom of the test tube to prevent the
e. Add the glass beads to the respirometer labeled “T3”.

Computer 5
Part I Germinating peas, room temperature

10. Insert a single-holed rubber-stopper into test tube T1 and T3. Note: Firmly twist the stopper
for an airtight fit. Secure each test tube with a utility clamp and ring-stand as shown in
Figure 1.
11. Arrange test tubes T1 and T3 in the warm water bath using the apparatus shown
in Figure 1. Incubate the test tube for 10 minutes in the water bath. Be sure to
keep the temperature of the water bath constant. If you need to add more hot or
cold water, first remove about as much water as you will be adding, or the beaker
may overflow. Use a basting bulb to remove excess water. Record the resulting
temperature of the water bath once incubation has finished in Table 2.
Note: Be sure the tubes are submerged to an equal depth, just up to the rubber
stoppers. The temperature of the air in the tube must be constant for this
experiment to work well.
12. When incubation has finished, connect the free-end of the Pressure 1 plastic
tubing to the connector in the rubber stopper in test tube T1 (the experimental
test tube) as shown in Figure 3. Connect the free end of the Pressure 2 plastic
tubing to the connector in the rubber stopper in T3 (the control test tube).
13. Click to begin data collection. Maintain the temperature of the water bath
during the course of the experiment.
Figure 3
14. Data collection will end after 20 minutes. Monitor the pressure readings
displayed in the live readouts on the toolbar. If the pressure exceeds 130 kPa, the pressure
inside the tube will be too great and the rubber stopper is likely to pop off. Disconnect the
plastic tubing from the Gas Pressure Sensor if the pressure exceeds 130 kPa.
15. The rate of respiration can be measured by examining the slope of the pressure change vs.
time plot at the right of the screen. Calculate a linear regression for the pressure change vs.
time graph:
a. Click the Pressure Change vs. Time graph to select it.
c. Record the slope of the line, m, in Table 3 as the rate of oxygen consumption by
germinating peas.
16. Move your data to a stored data run. To do this, choose Store Latest Run from the
Experiment menu.
Part II Non-germinating peas, room temperature

17. Disconnect the plastic tubing connectors from the rubber stoppers. Remove the rubber
stopper from each test tube.
18. Repeat Steps 10–16, using test tubes T2 and T3.
Part III Germinating peas, cool temperatures

19. Disconnect the plastic tubing connectors from the rubber stoppers. Remove the rubber
stopper from each test tube.

Cell Respiration (Gas Pressure)
20. Repeat Steps 10–16, using test tubes T1 and T3 in a cold water bath.
21. (optional) If instructed by your teacher, print out a copy of the graph with each of the three
trials.
DATA
Table 1
Volume
Peas
(mL)
Germinating
Non-germinating
Table 2
Temperature
Water bath
(°C)
warm
cool
Table 3
Rate of respiration
Peas
(kPa/min)
Germinated, room temperature
Non-germinated, room temperature
Germinated, cool temperature
QUESTIONS
1. Do you have evidence that cellular respiration occurred in peas? Explain.
2. What is the effect of germination on the rate of cellular respiration in peas?
3. What is the effect of temperature on the rate of cellular respiration in peas?
4. What was the role of the control respirometer in each series of experiments?
5. Why do germinating peas undergo cellular respiration?

Computer 5
EXTENSIONS
earthworms.

Experiment
Cell Respiration
3. This experiment may take several 50 minute lab periods to complete. A good stopping place
might be at the end of Step 16. If Part III is completed during a second or third lab period,
you may want to cool the respirometers in the refrigerator prior to the class. This will save a
substantial amount of equilibration time for students.
5. To prepare the 15% KOH solution, add 75 grams of solid KOH to distilled water to make a
total volume of 500 mL. If the solution will be stored for an extended time, it will be best to
store it in a plastic container. Strong bases will damage glass containers. HAZARD ALERT:
Corrosive solid; skin burns are possible; much heat evolves when added to water; very
dangerous to eyes; wear face and eye protection when using this substance. Wear gloves.
Hazard Code: B—Hazardous.
The hazard information reference is: Flinn Scientific, Inc., Chemical & Biological Catalog
Reference Manual, 2000, (800) 452-1261, www.flinnsci.com. See Appendix D of this book,
Advanced Biology with Computers, for more information.
connections and when closing valves or twisting the stopper into a test tube.
7. The accessory items used in this experiment are the #1 single hole stopper fitted with a
tapered valve connector and the section of plastic tubing fitted with Luer-lock connectors.
8. The length of plastic tubing connecting the rubber stopper assemblies to each gas pressure
sensor must be the same for all groups. It is best to keep the length of tubing reasonably small
to keep the volume of gas in the test tube low. Note: If pressure changes during data
collection are too small, you may need to decrease the total gas volume in the system.
Shortening the length of tubing used will help to decrease the volume.

Experiment 5
Ideas for Inquiry

SAMPLE RESULTS
Figure 1
The graphs in Figure 1 illustrate cellular respiration of germinating and non-germinating peas at
warm temperatures. The pressure change vs. time graph is calculated from the difference between
the two left graphs.
Table 2
Temperature
Water bath
(°C)
warm 26
cool 10

Teacher Information Cell Respiration (Gas Pressure)
Table 3
Rate of O2 consumption
Peas
(kPa/min)
Germinating, warm 0.1124
Non-germinating, warm 0.0193
Germinating, cool 0.0699
1. Yes. The pressure change vs. time graph indicates that some gas is being removed at a
constant rate from the respirometer when germinating seeds are present.
4. Gas in the control respirometer responded to temperature changes exactly as the experimental
respirometer. By subtracting the control respirometer’s pressure readings from the
experimental respirometer’s pressure readings, only the pressure change due to the removal
of oxygen gas by seeds is detected. Any pressure change due to temperature fluctuations is
eliminated.
If one only looks at the graph of the experimental respirometer, it appears that both
germinating and non-germinating seeds respire. This would be an erroneous conclusion, as
the control respirometer’s graph clearly indicates a change as well. The control would not be
necessary only if the graph indicated no change in pressure throughout the experiment.

Computer
6A
Easy Transformation of
E. coli using the pGLO™ Bacterial
Transformation Kit
Introduction to Transformation
In this lab, you will perform a procedure known as genetic transformation. Genetic transformation
literally means “change caused by genes”, and occurs when the cell incorporates and expresses a
new piece of genetic material – DNA derived from another organism. Transformation involves the
insertion of a gene into an organism in order to alter the recipient organism’s expression. Genetic
transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such
as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation,
bacteria can be genetically transformed with genes enabling them to digest and breakdown
pollutants such as oil spills or heavy metals contamination In medicine, disorders caused by
defective genes are being treated by gene therapy; that is, by genetically transforming a sick
person’s cells with healthy copies of the defective gene.
Your Protein of Interest - The Green Fluorescent Protein, GFP

You will use a procedure to transform bacteria with a gene that codes for Green Fluorescent
Protein, GFP. The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria.
Following the transformation procedure, the bacteria will express their newly acquired gene and
produce GFP, which causes them to glow a brilliant green color under ultraviolet or blue light.
The Host Cell - Escherichia coli (E. coli)

The bacterium, E. coli, is the ideal host for transformation because it is a small, single-celled
organism that reproduces quickly, so its transformation will be seen rather quickly. Also, the
strain of E. coli being used is nonpathogenic, does not make people or animals sick, and it does
not survive outside the laboratory environment. Although it is safe, it requires the use of Standard
Microbiological Practices, as directed by your instructor.
The Plasmid Vector - A Means of Gene Delivery

A plasmid will be used to transfer the GFP gene into the bacteria. A plasmid is a small circular
piece of DNA that is capable of self-replicating. In addition to one large chromosome, many
bacteria naturally contain one or more plasmids. Plasmid DNA usually contains genes for one or
more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids
back and forth allowing them to share these beneficial genes. This natural mechanism allows
bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics
is due to bacterial transmission of plasmids.
In this activity, the pGLO plasmid will be used. The pGLO plasmid is unique because not only
does it encode the gene for GFP, but it also encodes a gene for resistance to the antibiotic
ampicillin, and a special gene regulation system, which is used to control expression of GFP in the
transformed bacteria.

Computer 6A
Antibiotic Selection
The gene, which codes for antibiotic resistance, produces the protein beta-lactamase. Normally
bacteria cannot survive in the presence of antibiotics, such as ampicillin. However, the beta-
lactamase protein inactivates the ampicillin present in the agar environment of the bacteria
allowing it to survive. Only transformed bacteria that contain the pGLO plasmid and express beta-
lactamase can survive on agar plates containing ampicillin. You will observe that a small
percentage of bacterial cells take up the plasmid DNA and are transformed. As untransformed
cells cannot grow in the presence of ampicillin, we will use this as a selection method to calculate
transformation efficiencies and determine the extent to which the E. coli cells were transformed.
Gene Regulation
As previously mentioned, the pGLO plasmid codes for a special gene regulation system which
controls the expression of GFP in transformed bacteria. The gene regulation system is called the
arabinose operon. In nature, this operon contains the machinery and three genes that code for
three digestive enzymes involved in the breakdown of the plant sugar, arabinose, which is a food
source for the bacteria. When arabinose is present, the genes are expressed to digest the sugar;
when arabinose is not present, the bacteria do not express these digestive enzymes because they
are not necessary. This design allows the bacteria to quickly adapt to its environment and to use
its resources wisely.
In this activity, the pGLO plasmid has been designed with a modified arabinose operon. The three
genes for the digestive enzymes have been replaced with the gfp gene which produces GFP.
Therefore, in the presence of arabinose, the bacterial cells which have been transformed by the
pGLO plasmid will fluoresce (when exposed to UV or blue light) because of the production of
GFP. When GFP is not made, the bacterial colonies will appear whitish.
OBJECTIVES
• Use a plasmid vector to transform bacteria with genes for Green Fluorescent Protein (GFP)
and antibiotic resistance in a controlled experiment.
• Use the heat shock method of transforming E. coli.
• Regulate the expression of the GFP gene using arabinose.
• Describe the biological process involved in transforming bacterial cells.
• Calculate your transformation efficiency.
• Learn basic molecular biology techniques.
MATERIALS
E. coli starter plate 5 sterile DPTP pipets
4 agar plates ( 1LB, 2 LB/amp, 1 LB/amp/ara) 7 inoculation loops
transformation solution foam microtube holder/float
LB nutrient broth 37ºC incubator
rehydrated pGLO plasmid DNA 42ºC water bath and thermometer
UV lamp, handheld or BlueView™ Transilluminator cup of crushed ice
2 microcentrifuge tubes marking pen

Easy Transformation of E. coli
PRE-LAB QUESTIONS
1. Define bacterial transformation.
2. In this experiment, what particular type of DNA vector is used to transfer the GFP gene into
E. coli?
3. How is GFP expression induced or “switched on” during the transformation?
4. To genetically transform an entire organism, you must insert the new gene(s) into every cell in
the organism. Which organism is better suited for total genetic transformation: a single-cell or
multi-cell organism? Explain your answer.
5. To learn if a genetically transformed organism can pass its new traits to its offspring and
future generations, which would be a better candidate for your investigation: an organism in
which each generation quickly develops and reproduces or one that does so slowly?
PROCEDURE
The pGLO Bacterial Transformation Kit—Quick Guide

1. Label one closed micro test tube
+pGLO and another -pGLO. Label both
+pGLO
-pGLO
tubes with your group’s name. Place
them in the foam tube rack.
2. Open the tubes and using a sterile

transfer pipet, transfer 250 µL of 250 µL
transformation solution (CaCl2) into
each tube.
Transformation
solution
-pGLO
+pGLO
3. Place the tubes on crushed ice.
+pGLO -pGLO Ice

Computer 6A
4. Use a sterile loop to pick up a single

colony of bacteria from your starter
plate. Pick up the +pGLO tube and
immerse the loop into the
transformation solution at the bottom of
the tube. Spin the loop between your
index finger and thumb until the entire
colony is dispersed in the
transformation solution (with no
floating chunks). Place the tube back in
+pGLO
the tube rack in the ice. Using a new -pGLO
sterile loop, repeat for the -pGLO tube.
5. Examine the pGLO plasmid DNA

solution with the UV lamp. Note your
observations. Immerse a new sterile
loop into the plasmid DNA stock tube.
Withdraw a loopful. There should be a
film of plasmid solution across the ring.
This is similar to seeing a soapy film
across a ring for blowing soap bubbles.
Mix the loopful into the cell suspension
of the +pGLO tube. Optionally, pipet
10 µL of pGLO plasmid DNA. Close Plasmid DNA +pGLO -pGLO
the tube and return it to the face on ice.
Also close the -pGLO tube. Do not add
plasmid DNA to the -pGLO tube. Why
not?
6. Incubate the tubes on ice for

10 minutes. Make sure to push the
tubes all the way down in the rack so
the bottom of the tubes stick out and Rack Ice
make contact with the ice.
7. While the tubes are sitting on ice, label

your four agar plates on the bottom
(not the lid) as indicated on the
diagram.

8. Heat shock. Using the foam rack as a

holder, transfer both the (+) pGLO and
(-) pGLO tubes into the water bath, set
at 42°C, for exactly 50 seconds. Make
sure to push the tubes all the way down
in the rack so the bottom of the tubes
stick out and make contact with the Water bath
warm water. When the 50 seconds are
up, place both tubes back on ice. For Ice Ice
the best transformation results, the 42ºC for 50 seconds
change from the ice (0°C) to 42°C and
then back to the ice must be rapid.
Incubate tubes on ice for two minutes.
9. Remove the rack containing the tubes 250 µL

from the ice and place on the bench top.
Open a tube and, using a new sterile
pipet, add 250 µL of LB nutrient broth
to the tube and close it. Repeat with a
new sterile pipet for the other tube.
Incubate the tubes for 10 min at room LB-Broth +pGLO
-pGLO
temperature.
10. Tap the closed tubes with your finger to

mix. Use a new sterile pipet for each
tube and pipet 100 µL of the
transformation and control suspensions
onto the appropriate plates.
11. Use a new sterile loop for each plate.

Spread the suspensions evenly around
the surface of the agar by quickly
skating the flat surface of a new sterile
loop back and forth across the plate
surface.
12. Stack up your plates and tape them

together. Put your group name and
class period on the bottom of the stack
and place the stack upside down in the
37°C incubator until the next day.

Computer 6A
ANALYSIS
Capturing an Image of the Transformation
1. View the LB/amp and LB/amp/ara plates either with a handheld UV lamp or place them on
the BlueView™ Transilluminator to view the fluorescent green colonies. Photograph them
either with a camera or a Vernier ProScope.
Calculation of Transformation Efficiency

2. Based on your observations above, note the number of colonies and their color on each plate:
a. LB (-pGLO):
b. LB/amp (-pGLO):
c. LB/amp (+pGLO):
d. LB/amp/ara (+pGLO):
3. Count the total number of green fluorescent colonies and enter here: ________________
4. Did you transform bacteria? State your evidence.
5. Calculate the transformation efficiency in the table below:
Calculating Transformation Efficiency
Description Formula Sample calculation Your calculations
Number of
Transformed Count directly from
Colonies LB/amp/ara plate 100
(transformants)
Volume of plasmid (µl)

Mass (µg) of plasmid
used x Concentration 10 µl x 0.08 µg/µl = 0.8 µg
(µg/µl)
Volume spread on
Fraction of plasmid
that was plated plate (µl)/Total volume 100 µl/510 µl = 0.196
in microtube (µl)
Mass of plasmid Mass of plasmid used

plated (µg) (µg) x Fraction of 0.8 µg x 0.196 = 0.156 µg
plasmid plated
Transformation 100/0.156 µg =
Efficiency Number of
(transformants/µg transformants/µg of 641 transformants/µg
plasmid) plasmid plasmid

QUESTIONS
1. In this experiment, which plates are control plates and what purpose does each control plate
serve?
2. If the genetically transformed bacteria have acquired the ability to live in the presence of
ampicillin, then what can be inferred about the other genes on the plasmid that were involved
in the transformation?
3. Very often an organism’s traits are determined by a combination of its genes and its
environment. Think about the green color you saw in the genetically transformed bacteria.
a. What two factors must be present in order for you to see the green color? Hint: one factor
is in the plate, the other is in how you look at the bacteria.
b. What is the advantage to having this type of gene regulation?

Experiment
Easy Transformation of
E. coli using the pGLO™ Bacterial
Transformation Kit
1. This experiment correlates with Lab 6: Exercise 6A in the 2001 College Board’s AP Biology
Lab Manual. The suggested complete kit (pGLO Bacterial Transformation Kit; catalog
number 166-0003EDU) is available from Bio-Rad Laboratories; see Appendix G for contact
information. This kit is sufficient for 8 workstations accommodating four students per
workstation.
2. The student pages are also available on the CD that is located on the inside back cover of this
manual. See Appendix A for more information.
Ideas for Inquiry

• How does varying the concentration of arabinose in the LB/Amp/Ara affect the production of
GFP in cells?
• Can you make the bacteria in the LB/amp plate produce GFP by later adding arabinose?
• Can you influence transformation efficiency by altering the amount of plasmid added to the
transformation (+) tube?
• Design an experiment to transform cells from a liquid culture.
TIME REQUIREMENTS
Pre-lab preparation 90 minutes
Procedure 45 minutes
Analysis and assessment 40 minutes
PRE-LAB PREPARATION
Step Objective Time Required When
1 Prepare agar plates 1 hour 3-7 days prior
Rehydrate E. coli
2 Streak starter plates 15 minutes 24-36 hours prior
Rehydrate pGLO plasmid DNA
3 Aliquot solutions and set up workstations 15 minutes Just prior

Experiment 6A
PRE-LAB PROCEDURE
1. Approximately three to seven days prior to the lab, prepare five agar plates for each student
workstation. Each workstation should have one LB plate, two LB/amp plates, and one
LB/amp/ara plate. The fifth plate will also be labeled LB and will server as the “starter plate”
on which the host bacteria, E. coli, will grow and serve as the source of cells for the
transformation. Agar is prepared according to standard procedures, and a complete
description can be found in the pGLO instruction manual. Briefly, agar is added to 500 mL of
distilled water, then dissolved and heat-sterilized in a microwave. Upon cooling, ampicillin and
arabinose are added, and then plates are poured.
Figure 1: Preparing agar just prior to heat sterilization
Figure 2: Adding ampicillin and/or arabinose to cooled agar and then pouring plates
2. Approximately 24-36 hours prior to the lab, rehydrate the E. coli bacteria and pGLO plasmid
DNA and streak the bacteria onto the starter plates. Using a sterile pipet, rehydrate the
lyophilized bacteria with 250 µL of transformation solution. Mix well and streak 10 µL onto
an LB agar plate prepared in Step 1. The goal of streaking is to generate single colonies from
a concentrated suspension of bacteria. A minute amount of suspension goes a long way! Each
workstation will need one starter plate.
Figure 3: Streaking plates in a quadrant-like fashion using rehydrated E. coli bacteria

Similarly, prepare the pGLO plasmid DNA; use a new sterile pipet to add 250 µL of
transformation solution to the vial of lyophilized pGLO plasmid DNA and store in a
refrigerator until ready to use.

Teacher Information Easy Transformation of E. coli
3. Just prior to performing the experiment, aliquot 1 mL of CaCl2 transformation solution and
1 mL of LB nutrient broth into separate 2 mL microtubes and distribute them to each
workstation. Set up the workstations with the materials as described in the student’s section.
ANSWERS TO PRE-LAB QUESTIONS

1. Insertion of a gene into an organism in order to change that organism’s trait.
2. A plasmid is used to transfer GFP.
3. By adding the sugar arabinose, which is the inducing agent of expression.
4. A single-celled organism would be the best choice because it only contains one cell that needs
to take up the new gene(s).
5. An organism which reproduces quickly will allow you to quickly assess if the new trait has
been passed on.
SAMPLE RESULTS
1. Answers will vary.
2a. A lawn of whitish bacteria

b. No growth, therefore no colonies
c. Multiple whitish colonies. Answers will vary.
d. Multiple colonies that are whitish under normal lighting and fluoresce bright green when
exposed to either long-wave UV light or viewed with the BlueView Transilluminator.
Numbers of colonies will vary.
4. The bacteria have been successfully transformed if they can grow on both the LB/amp
(+pGLO) and the LB/amp/ara (+pGLO) plates and not on the LB/amp (-pGLO) plates.
Additionally, the bacterial colonies on the LB/amp/ara plates should fluoresce when exposed
to UV light.
Transformation is not successful if colonies are not present on the +pGLO plates (which are
LB/amp & LB/amp/ara). This could either be due to not adding the plasmid or not adding a
colony of bacteria to the tube just prior to transformation.
ANSWERS TO QUESTIONS:
1. A control plate is a guide to help interpret experimental results. In this experiment, both –
pGLO plates are control plates
The LB (-pGLO) control plate confirms that the bacteria is viable but cannot grow on
ampicillin, as evidenced by the lack of colonies on the LB/amp (-pGLO) plate. Without this
control, one would not know if the absence of bacteria were due to its inability to grow on
ampicillin or some other cause.

Experiment 6A
By comparing the LB/amp (-pGLO) control plate to the LB/amp (+pGLO) plate, it shows that
only genetically transformed bacteria can grow on ampicillin due to the uptake of the pGLO
plasmid and the expression of the ampicillin-resistance gene.
Finally, the LB (-pGLO) plate can be compared to the other two LB/amp plates to further
demonstrate that the bacteria from the starter culture must uptake the plasmid in order to
grow on ampicillin plates.
2. It can be inferred that those genes can now be expressed by the host’s protein machinery
3a. One factor is the sugar (arabinose) in the LB/amp/arabinose plate, and the other is the UV or
blue light that is necessary to cause the GFP protein within the bacteria to fluoresce.
b. Gene regulation allows an organism to adapt to differing conditions and prevents wasteful
overproduction of unneeded proteins.

Computer
6B
Analysis of
Precut Lambda DNA
Restriction enzymes are a special class of proteins that cut DNA at specific sites and have
become an indispensable tool in molecular biology. Restriction enzymes, also known as
endonucleases, recognize specific sequences of DNA base pairs and cut or chemically separate
the DNA at that site. The specific sequence of DNA recognized by a restriction enzyme is called
a restriction site.
These unique enzymes occur naturally in some bacteria and act to protect them from invading
viruses. Viruses called bacteriophages, phages for short, attack bacteria by inserting their genetic
material into the bacterial cell. The phage commandeers the bacterial cell, replicating rapidly
until the bacterial cell lyses and releases more phages to carry out the same infection process in
neighboring cells. However, if the bacterial strain has restriction enzymes that recognize
restriction sites on the invading phage nucleic acid, then enzymes can destroy the invading
genetic material by digesting and inactivating the phage genes. Bacterial cells protect their own
DNA from being self-digested by modifying certain nitrogen bases along their genome, this
prevents their restriction enzymes from recognizing and digesting their own sequences.
Restriction Enzymes as Molecular Scissors

Scientists use restriction enzymes as tools to assist with DNA splicing. Restriction enzymes are
used to cut both the DNA sequence of interest and the host DNA; they act like molecular
scissors. Their discovery led to the development of recombinant DNA technology that allowed,
for example, the large scale production of human insulin for diabetics using E. coli bacteria.
Many restriction enzymes have been studied in detail, and more than 600 are available
commercially and are used routinely for DNA modification and manipulation in laboratories.
Gel Electrophoresis
Once the DNA has been digested, the “soup” of fragments must be separated in order to learn
more about each fragment, such as its size. Gel electrophoresis is a method used to separate DNA
fragments based on their sizes by applying an electrical field to an agarose gel containing these
DNA fragments. DNA contains many negative electrical charges, and scientists used this
property to separate pieces of DNA. Once the fragments are loaded onto the gel, an electrical
current is applied causing the negatively-charged DNA molecules to move towards the positive
electrode (red). The agarose gel acts as a matrix of tiny pores that allow small particles to move
through it relatively quickly. The larger fragments migrate much more slowly through the gel.
After a set period of exposure to the electrical current, the DNA fragments are separated by size
with the smaller ones located further away from the wells than the larger fragments. Fragments
that are either the same or very similar in size will tend to migrate together through the gel.
Fragment bands form as a result of the various distances the DNA segments migrate.
Stains to visualize DNA

DNA is colorless and must be visualized with a “stain”. In this laboratory activity, DNA bands
may be stained with either Fast Blast™ DNA Stain or SYBR® Safe DNA gel stain then analyzed
with Vernier’s bioimaging systems. Fast Blast stained gels can be viewed with the White Light
Transilluminator and SYBR Safe stained gels can be viewed with the BlueView
Advanced Biology with Vernier 6B - 1 (Lambda)

Computer 6B (Lambda)
Transilluminator. Either transilluminator can be used with the ProScope HR and Logger Pro
software to capture digital images of the stained gel and perform analysis to determine the sizes
of the DNA bands.
Lambda (λ) DNA

In this activity, the DNA you will be working with is the genome of a virus that has already been
digested into segments by restriction enzymes. Lambda DNA comes from a bacteriophage and is
harmless to man and other eukaryotic organisms, and therefore, makes an excellent source of
DNA for experimental study. The individual digests of this bacteriophage, a 48,502 base pair
linear DNA segment, use three common restriction enzymes: EcoRI, HindIII and PstI.
The restriction sites for each of the restriction enzymes are as follows:
Table 1: Restriction sites* recognized by EcoRI, HindIII, and PstI
5’….G AATTC…….3’
EcoRI
3’….CTTAA G……5’
5’….A AGCTT…….3’
HindIII
3’….TTCGA A……5’
5’….CTGCA G…….3’
PstI
3’…G ACGTC……5’
*The four DNA bases are Adenine (A); Cytosine (C); Guanine (G); and Thymine (T)
Your task is to separate the DNA fragments based on size using a procedure known as gel
electrophoresis and then compare the DNA fragments with those of a standard ladder whose base
pair sizes are already known.
Standard Ladder and DNA Fragment Size Determination:

To determine the sizes of the experimental DNA bands, they must be compared to a standard
ladder that has DNA bands with known molecular weights. The standard used during this activity
is the HindIII digest of lambda DNA, a commonly used DNA molecular weight marker. The
standard ladder is run in a lane next to the experimental lanes under the same conditions. Linear
DNA fragment migration distance through a gel is inversely proportional to the log10 of its
molecular weight (base pair number). This process is carried out using the Gel Analysis in
Logger Pro.
6B - 2 (Lambda) Advanced Biology with Vernier

Analysis of Precut Lambda DNA
OBJECTIVES
• Perform agarose gel electrophoresis using three different predigested samples of lambda
DNA and uncut lambda DNA.
• Stain the gel.
• Document and examine gel results with an imaging system.
• Construct a standard curve and determine the size of the DNA fragments from the gel using
Logger Pro.
MATERIALS
computer lambda DNA samples:*
Vernier interface lambda DNA – uncut
Logger Pro lambda DNA – EcoRI digest
Vernier Blue Digital Bioimaging System lambda DNA – HindIII digest
or White Digital Bioimaging System lambda DNA – PstI digest
electrophoresis chamber & power supply electrophoresis buffer, 50x, TAE*
adjustable micropipette, 2–20 µL and tips sample loading dye*
adjustable micropipette, 20–200 µL and tips fast Blast DNA stain*
microwave oven or hot plate multicolor micro test tubes*
microcentrifuge foam micro test tube holders*
gel support film agarose, 5 g*
rocking platform staining trays*
water bath or heat block permanent markers
ruler, millimeter laboratory tape (not regular sticky tape)
* Included in Bio-Rad kit
PRE-LAB ACTIVITY
1. Make a gel. Note: You do not need to do this step if you are using a pre-cast gel.
a. Clean the lab table surface, wash your hands, glove, set the lab mat, and review lab safety
procedures.
b. Measure out 0.5 g of agarose and pour the powder into the flask.
c. Measure out 50 mL of 1X TAE (Tris-Acetate- EDTA) buffer and transfer volume to the
flask containing the agarose. Swirl gently and place a funnel, stem first, into the top of the
flask.
d. Place the flask with its cover in a microwave and heat on high for 40 seconds or until the
solution starts to boil. The agarose solution must be crystal clear and void of suspended
granules, use additional ten-second blasts of the microwave until this condition is attained.
e. With hot-gloves, remove the hot flask and transfer the container to your lab space.
f. While the flask is cooling, prepare the gel tray by taping the two open ends of the gel tray
with lab tape (masking tape and Scotch tape will not work) then place the eight-toothed
comb in position at one end of the tray. The tray needs to be placed level on the surface of
the lab mat.
g. Allow the solution to cool until the flask can comfortably be placed on the back of your
palm (60° to 55°C).

h. Perform this step if you are using SYBR Safe pre-electrophoresis stain. If you are not, skip
to Step i. Pipette 5 μL of concentrated SYBR Safe 10,000x DNA staining solution (1 μL
per 10 mL of agarose solution) to the flask, swirl, and then pour solution into your
prepared gel tray. Pour the gel to a thickness of 0.8 to 1.0 cm, which is approximately half
way up the teeth of the comb.
i. It will take between 15 and 25 minutes for the gel to set and appear cloudy or opaque
when ready to use. Carefully remove the comb from the solidified gel and untape the ends
of the tray.
PROCEDURE
Quick Guide for Analysis of Precut Lambda DNA Kit
Part A Sample Preparation
1. Obtain a set of colored micro test tubes

and label as follows:
yellow L = lambda DNA
violet P = PstI lambda digest
green E = EcoRI lambda digest L P E H
orange H = HindIII lambda digest
2. Using a fresh tip for each sample, pipet

10 µL of DNA sample from each stock
tube and transfer to the corresponding
colored micro test tube.
3. Add 2 µL of sample loading dye to each

tube. Mix the contents by flicking the tube
with your finger.
4. Optional: Heat the DNA samples at 65°C

for 5 minutes.
5. Pulse-spin the tubes in the centrifuge to

bring all of the liquid to the bottom or tap Centrifuge Tap
them gently on the benchtop.
6. At this point you can put the DNA

samples into the refrigerator and run the
agarose gel during the next class or you
can continue with Step 7 and run the
agarose gel today. Ask your instructor if
you are unsure what to do.

Part B Agarose Gel Electrophoresis
7. Remove the agarose gel from the

refrigerator (if necessary), remove the
plastic wrap, and place the gel in the
electrophoresis chamber. Fill the
electrophoresis chamber and cover the gel (-)
with approximately 275 mL of 1x buffer.
8. Check that the wells of the agarose gels (+)

are near the black (–) electrode and that
the bottom edge of the gel is near the red
(+) electrode.
9. Load 10 µL of each sample into separate

wells in the gel chamber in the following
order:
Lane 1: L (yellow tube)
Lane 2: P (violet tube)
Lane 3: E (green tube)
Lane 4: H (orange tube)
10. Place the lid on the electrophoresis
chamber carefully. Connect the electrical
leads into the power supply, red to red and
black to black. (-)
11. Turn on the power and run the gel at (+)

100 V for 30 minutes.
12. When the electrophoresis run is complete,

turn off the power and remove the top of
the chamber.

Part C Staining the Gel
13. Your teacher will let you know which of the four staining options you will use.
Option 1: Overnight staining using 1x Fast Blast stain
Option 2: Quick staining using Bio-Rad’s 100x Fast Blast stain (requires 12-15 minutes)
Option 3: Vernier SYBR Safe Stain post-electrophoresis
Option 4: Vernier SYBR Safe Stain pre-electrophoresis

14. Stain the gel using 1x Fast Blast Stain.
a. Remove the gel and tray from the gel box. Be careful
because the gel is very slippery. Slide the gel into the
staining tray.
b. Add 120 mL of 1x Fast Blast stain into a staining tray
(2 gels per tray).
c. Let the gels stain overnight, with gentle shaking for best
results. No destaining is required.
d. Pour off the water into a waste beaker.
e. Proceed to the Analysis section.
Option 2: Quick staining using Bio-Rad’s 100x Fast Blast

stain
14. Stain the gel using Bio-Rad’s 100x Fast Blast stain.
because the gel is very slippery. Slide the gel into the
staining tray.
b. Add 120 mL of 100x Fast Blast stain into a staining tray
(2 gels per tray).
c. Stain the gels for 2 minutes with gentle agitation. Save
the used stain for future use.
d. Transfer the gels into a large washing container and rinse
with warm (40–55°C) tap water for
approximately10 seconds.
e. Destain by washing twice in warm tap water for
5 minutes each with gentle shaking for best results.
f. Proceed to the Analysis section.

Option 3: SYBR Safe Stain post-electrophoresis stain

14. Post-electrophoresis staining procedure.
because the gel is very slippery.
b. Place the gel in a gel staining tray and measure out
50 mL or enough of the SYBR Safe DNA Gel
Staining solution (0.5x) to cover the gel in the tray.
c. Obtain a piece of Aluminum foil to cover and keep
light from directly shining on the gel in the staining
tray. The gel will be left in this staining solution for
30 minutes.
d. Place gel on rocker or periodically swirl the solution
and gel in the staining tray to obtain a thorough and
uniform stain of the DNA fragments.
e. No destaining is necessary once the thirty-minute
period has elapsed; rinse the gel with water and
proceed to the Analysis section.
Option 4: SYBR Safe Stain pre-electrophoresis stain

14. Your gel has already been stained. Remove the gel and
tray from the gel box. Be careful because the gel is very
slippery. Proceed directly to the Analysis section.
ANALYSIS
Photodocumentation of Gel
1. Connect the transilluminator to AC power and turn it on.
2. Prepare the ProScope for use.

a. Connect the 1–10x lens to the ProScope.
b. Connect the ProScope to the USB port of your computer.
c. Mount the ProScope to the stand and position the stand next to the transilluminator,
opposite the side with the hinge.
d. Level the ProScope so that its lens is parallel to the surface of the transilluminator.
3. Prepare Logger Pro for use.
a. Start Logger Pro.
b. Choose Gel Analysis ► Take Photo from the Insert menu.
c. Check Close Window and Auto Arrange as the Photo Actions.

4. Take a photo of the gel.

a. Transfer the gel to the central portion
of the transilluminator platform.
b. Orient the gel and platform so that the
wells are at the top of the picture in
Logger Pro.
c. Orient and focus the ProScope so both
the bands and lane numbers are clear
and sharp. Note: Lowering the
brightness by clicking Camera Settings
may improve visibility of the gel. Figure 1
d. Place the Imaging Hood over the
ProScope and the transilluminator. Reach through the flap of the hood to make final
adjustments for best position, focus, and resolution.
e. Once satisfied with the image, click . The screen should now resemble Figure 1.
Note: You may want to re-size the photo and graph to increase the size of the photo for
ease of analysis.
5. Indicate the position of the wells on the photograph.
a. Click Set Origin, .
b. Click the photograph just to the left of the first well. A yellow coordinate system will
appear.
c. Position the x-axis directly along the bottom edge of the wells. You can move the origin
by clicking either axis and dragging it to the desired location. The axis can be rotated by
clicking the round handle on the x-axis.
6. Convert the units of distance from pixels to millimeters.
a. Click Set Scale, .
b. Click and drag to draw a line that is one centimeter long using the ruler on the gel tray as
your guide. For example, click and drag between the 1 cm mark and the 2 cm mark to
create a line that is one centimeter long.
c. Enter the distance value and units as millimeters and click .
7. Identify the bands and base pair values of the standard ladder using the HindIII digest lane
base pair values in Table 2.
a. Click Set Standard Ladder, .
b. Click the center of the second band in the HindIII digest lane (the fourth lane).
c. Enter the number of base pairs for this band using the values in Table 2. Click .
d. Click the center of the next band in this lane and enter the base pair value. Click .
e. Repeat this process for each visible band of the standard ladder. Logger Pro will
automatically create a standard curve on the graph.
8. Identity the bands in the remaining lanes.
a. Click Add Lane, , and select Add Lane.
b. Click the center of the second band in the EcoRI digest lane (the third lane). Notice that
when you click, three things happen: a marker with a distinct shape and color appears on
the photograph, a matching marker appears on the standard curve of the graph, and the
distance and number of base pairs are added to the data table.

c. Click the center of the next band in this lane.

d. Continue this process for each visible band in the EcoRI digest lane.
9. Repeat Step 8 for the PstI digest lane (the second lane). You will start with the first band in
this lane. Note: Do not add a lane for the uncut lambda DNA. It contains only one band,
which contains approximately 48,000 base pairs.
10. Record the base pair values for the experimental lanes in Table 2. Not all cells will be filled.
11. (optional) Save and print the results of the gel analysis.
DATA
Record your results in the table below to reflect the migrations patterns on your gel and
determine the size of the fragments. Some lanes will have fewer than six bands.
Table 2 Results
L = uncut lambda P = PstI restriction E = EcoRI restriction H = HindIII

DNA digest of lambda DNA digest of lambda DNA restriction digest of
lambda DNA
Distance Estimated Distance Estimated Distance Estimated Distance Actual

(mm) base (mm) base (mm) base (mm) base
pairs pairs pairs pairs
Band 1 ~48,000 ~23,000 23,130
Band 2 9,416
Band 3 6,557
Band 4 4,361
Band 5 2,322
Band 6 2,027
QUESTIONS
1. What is a restriction enzyme?
2. How are restriction enzymes useful to bacteria in nature?
3. Describe the palindromic property of restriction sites.
4. How are the DNA fragments separated from one another after a restriction digest has been
performed?
5. What is a molecular weight standard (also known as marker or ladder) and what is its role?
6. How is the DNA visualized?

Experiment
Lab Manual.
2. The student pages with complete instructions for analysis using Logger Pro (computers), can
be found on the CD that accompanies this book. See Appendix A for more information.
Note: This activity can only be completed using computers.
The complete teacher’s guide, protocol, and student manual are detailed in the Bio-Rad
instruction manual. The protocol has been summarized in the Quick Guide section in the
student pages.
3. The suggested complete kit, Analysis of Precut Lambda DNA Kit, is available from Bio-Rad
Laboratories. Refer to Appendix G for ordering information. This kit is sufficient for
32 students (eight workstations; four students per workstation).
4. The techniques introduced in this exercise form the basis of recombinant DNA technology
techniques, DNA fingerprinting, and forensic DNA analysis. Analysis is done using either a
Vernier Blue Digital Bioimaging System or White Digital Bioimaging System connected to a
computer running Logger Pro software. From the digital image of the gel, Logger Pro will
create a standard curve and automatically calculate the number of base pairs (molecular
weight) for each experimental DNA band.
5. Tips for using the Vernier Blue Digital Bioimaging System and White Digital Bioimaging
System:
• Use the power supply that shipped with the device. Using the wrong power supply could
damage it.
• If using the BlueView Transilluminator, ensure that the BlueView’s lid is closed, otherwise
it will not function. This is a safety feature.
6. Tips for using Logger Pro to take a photo of the stained gel:
• For the best photo of the gel banding patterns, you may want to adjust the camera settings
before clicking . Click Camera Settings in the Take Gel Photo dialog box, then
click Adjustments. From here, position the brightness slider between 10% and 25%,
making sure that the dimmer switch of the BlueView Transilluminator is turned to full
brightness and the Imaging Hood is placed over the system. Continue to adjust until the
best results are obtained.
• When performing a gel analysis, the sequence of events described in the procedure must be
followed in order. It is not possible to go back and change parameters, with these
exceptions:
• Points placed on the photograph identifying experimental bands can be moved or
deleted. Simply click Select Point, , then click on the point to be edited. Drag it with
the cursor to move it or use the Delete key on your keyboard to delete.
• Points for experimental bands can be added by clicking Add Lane, , selecting the
appropriate lane, then clicking on the band in the photograph.
Advanced Biology with Vernier 6B - 1 T (Lambda)

Experiment 6B
Ideas for Inquiry

• How does changing the concentration of the agarose in your gel effect DNA band
migration?
• How does changing the concentration of running buffer in your gel tank affect DNA band
migration?
PRE-LAB PREPARATION
Table 1 Pre-lab objectives and time requirements
Objective Time required When
Gels may be cast 1 to

30 minutes to
Pour agarose gels (or use precast gels) 7 days in advance.
1 hour; depending
Part A Aliquot loading dye Alternatively, student
on how gels are
Set up student and teacher workstations teams may prepare
prepared
their own gels.
Prepare electrophoresis buffer & stain

Part B Set up electrophoresis chambers, 20 minutes Just prior to lab
Set up teacher and student workstations
Analysis Set up imaging system 15 minutes Just prior to lab
STAINING OPTIONS
After electrophoresis, there are four staining options to view the DNA bands using either
Fast Blast™ DNA Stain (included in the Bio-Rad kit) or SYBR Safe™ DNA gel stain (available
from Vernier). Both stains are convenient, safe, and nontoxic alternatives to ethidium bromide.
• Gels stained with Fast Blast should be analyzed by capturing digital images using a Vernier
White Digital Bioimaging System connected to a computer and Logger Pro software.
• Gels stained with SYBR Safe should be analyzed by capturing digital images using a Vernier
Blue Digital Bioimaging System connected to a computer and Logger Pro software.
• Any stains with an excitation wavelength in the blue range of 450 to 520 nm range will also
work with the Blue Digital Bioimaging System. For use with nucleic acids, these include, but
are not limited to, SYBR Safe, SYBR® Gold, SYBR® Green I, GelGreen™, and GelStar®.
Though each staining method is described in detail in their original documentation, they are
summarized here for your convenience.
Option 1 Using 1x Fast Blast DNA Stain for Overnight Staining
This method requires the gel to sit overnight while it stains. Direct students to complete all Parts
of the student procedure on Day 1 and then perform the analysis on Day 2.
a. Dilute the 500X concentrate to 1X. We recommend using 120 mL of 1X Fast Blast to stain
two 7 X 7 cm or 7 X 10 cm gels in individual staining trays (included in the kit); each tray
can accommodate two gels.
6B - 2 T (Lambda) Advanced Biology with Vernier

b. Carefully remove the gels from their gel trays and slide them into the staining tray; add the
X stain until the gels are completely submerged.
c. Place the staining tray on a rocking platform and gently shake overnight. If a rocking
platform is not available, then periodically swirl the solution and gel a few times during
the staining period. This is crucial since smaller fragments tend to diffuse without shaking.
The bands will begin to develop after two hours, but at least eight hours of staining is
recommended for complete visibility.
d. The gel is ready to be analyzed using a White Digital Bioimaging System.
Option 2 Using 100x Fast Blast DNA Stain for Quick Staining (12–15 minutes)
If you use this option, you will be able to complete the entire lab in a 90-minute lab period. If you
have a 50-minute lab period, direct students to complete all Parts of the student procedure on
Day 1 and then perform the Analysis section on Day 2.
a. Dilute the 500X concentrate to 100X. We recommend using 120 mL of 100X Fast Blast to
stain two 7 X 7 cm or 7 X 10 cm gels in individual staining trays (included in the kit); each
tray can accommodate two gels.
100x stain until the gels are completely submerged.
c. Stain for 2–3 minutes, maximum. Pour the 100X stain into a storage bottle and save for
future use. The 100X stain may be reused at least seven times.
d. Rinse the gels for 10 seconds in 500–700 mL of clean, warm (40–55ºC) tap water
e. Wash the gels for 5 minutes in 500–700 mL of clean, warm (40–55ºC) tap water. Repeat
this step. The bands may appear fuzzy immediately after the second wash, but will begin
to develop into sharper bands within 5–15 minutes after the second wash.
f. To obtain maximum contrast, additional washes in warm water may be necessary. Destain
to the desired level, but do not leave the gel in water overnight. If complete destaining
cannot be done in the allocated time, then transfer the gel to 1X Fast Blast stain overnight.
g. The gel is ready to be analyzed using a White Digital Bioimaging System.
Option 3 Using SYBR Safe DNA Gel Stain (0.5x) Post-Electrophoresis
If you use this option, you will be able to complete the entire lab in a 90-minute lab period. If you
have 50-minute lab periods, direct students to complete all Parts of the student procedure on
Day 1 and then perform the analysis on Day 2. This stain can either be diluted from the 10,000X
concentrate or purchased as a convenient, pre-diluted 0.5X stain.
a. After electrophoresis, carefully remove the gel from its gel tray and slide it into the
staining tray; add sufficient stain until the gels are completely submerged.
b. Cover the staining tray with aluminum foil to protect the gel from light. Place the staining
tray on a rocking platform with gentle shaking and stain for 30 minutes at room
temperature. If a rocking platform is not available, then periodically swirl the solution and
gel to obtain thorough and uniform staining patterns.
c. Rinse the gel with water and place on the Blue Digital Bioimaging System for analysis.
Option 4 Using SYBR Safe DNA Gel Stain Pre-Electrophoresis
If you use this option, you will be able to complete the entire lab in a 50- or 90-minute lab period.
a. Follow the directions for preparing agarose gels and add the appropriate volume of SYBR
Safe using this ratio: 1 µL of SYBR Safe 10,000X concentrate to 10 mL of agarose
solution. Thus for 40 mL of agarose, add 4 µL of SYBR Safe. Swirl to mix then pour the
gels.

Experiment 6B
b. Store SYBR Safe-containing gels protected from light by covering them with aluminum
foil at room temperature for 1–2 days or at 4ºC for up to one week.
c. Gels containing SYBR Safe stain are run in the same buffer system and electrophoresis
conditions as those gels that do not.
d. These gels do not need to be stained or destained. They may be analyzed using the Blue
Digital Bioimaging System.
e. Students load the gel with uncut lambda DNA and three separate digests of lambda DNA,
as indicated below. The HindIII digest serves as the standard.
Table 3 Lane assignments

Load volume
Lane lambda DNA Lambda DNA sample
(μL)
1 10 Tube L: Uncut lambda DNA
2 10 Tube P: PstI restriction digest of lambda DNA
3 10 Tube E: EcoRI restriction digest of lambda DNA
4 10 Tube H: HindIII restriction digest of lambda DNA
SAMPLE DATA
P = PstI restriction E = EcoRI restriction H = HindIII

L = uncut λ DNA digest of λ DNA digest of λ DNA restriction digest of
lambda DNA
Distance Estimated Distance Estimated Distance Estimated Distance Actual

(mm) base (mm) base (mm) base (mm) base
pairs pairs pairs pairs
Band 1 (48,000) 17.5 (9,400) (23,000) (23,130)
Band 2 23.5 4,500 19 7,400 17 9,416
Band 3 28 2,750 21 6,000 20 6,557
Band 4 29 2,500 22.5 5,000 23.5 4,361
Band 5 30.5 2,200 25 3,800 29 2,322
Band 6 31 2,000 31 2,027

Notes:
1. Values in parentheses are known values that are too large for this method of base pair determination.
2. Some lanes will have fewer than six bands.
3. The values listed are ideal, student results may vary due to many factors that include technique, sample
concentrations, photograph band results, band positions in gel analysis presence or absence of specific bands,
concentration of agarose, and the like.
6B - 4 T (Lambda) Advanced Biology with Vernier

1. A restriction enzyme (or restriction endonuclease) is an enzyme that cuts DNA at specific
sequences of DNA known as restriction sites.
2. Restriction enzymes provide a defense mechanism against invading viruses. Inside a bacterial
host, the restriction enzymes selectively cut up foreign DNA; the host DNA is modified to
protect it from the restriction enzyme’s activity.
3. The top strand (when read from left to right) is identical to the bottom strand (when read
from right to left).
4. DNA fragments are separated on an agarose gel which is subjected to an electric current. This
is known as gel electrophoresis. The fragments migrate towards the positive electrode, and
the smallest fragments migrate the fastest.
5. A DNA molecular weight standard is a mixture of DNA fragments of known molecular

weights. Its role is to establish the distance migrated by these known DNA fragments under
experimental conditions (i.e. buffer composition, length of the run, voltage, thickness and
percentage of the gel) such that the sizes of experimental samples can be determined under
those same experimental conditions. The standard used in this activity was the HindIII digest
of lambda DNA.
6. DNA must be stained with a DNA stain, such as SYBR Safe or Fast Blast. SYBR Safe-
stained DNA is then visualized immediately using an imaging system. Fast Blast-stained
DNA can be visualized with the naked eye, without the use of an additional light source.

Computer
6B
Forensic DNA Fingerprinting
Scientists working in forensic labs are often asked to perform DNA profiling or “fingerprinting”
to analyze evidence in law enforcement, mass disasters, and paternity cases. In this laboratory
activity, you will enter into the role of a forensic scientist who has been called upon to help solve
a crime. You will use forensic techniques, and the first steps will be to gather DNA found at the
“crime scene” and obtain DNA samples from five “suspects”. The DNA will be digested with a
fixed set of restriction enzymes, separated on a gel by gel electrophoresis, and then analyzed for
patterns of similarity with the crime scene sample. From these results, you will make
recommendation to identify the perpetrator.
Restriction enzymes are a special class of proteins that cut DNA at specific sites and have
become an indispensable tool in molecular biology. Restriction enzymes, also known as
endonucleases, recognize specific sequences of DNA base pairs and cut, or chemically separate,
DNA at that specific arrangement of base pairs. The specific sequence of DNA is called a
restriction site.
These unique enzymes occur naturally in some bacteria and act to protect them from invading
viruses. Viruses called bacteriophages, phages for short, attack bacteria by inserting their genetic
material into the bacterial cell. The phage commandeers the bacterial cell, replicating rapidly
until the bacterial cell lyses and releases more phages to carry out the same infection process in
neighboring cells. However, if the bacterial strain has restriction enzymes that recognize
restriction sites on the invading phage nucleic acid, then enzymes can destroy the invading
genetic material by digesting and inactivating the phage genes. Bacterial cells protect their own
DNA from being self-digested by modifying certain nitrogen bases along their genome, this
prevents their restriction enzymes from recognizing and digesting their own sequences..
Restriction Enzymes as Molecular Scissors

Scientists use restriction enzymes as tools to assist with DNA splicing. Restriction enzymes are
used to cut both the DNA sequence of interest and the host DNA; they act like molecular
scissors. Their discovery led to the development of recombinant DNA technology that allowed,
for example, the large scale production of human insulin for diabetics using E. coli bacteria.
Many restriction enzymes have been studied in detail, and more than 600 are available
commercially and are used routinely for DNA modification and manipulation in laboratories.
Gel Electrophoresis
Once the DNA has been digested, the “soup” of fragments must be separated in order to learn
more about each fragment, such as its size. Gel electrophoresis is a method used to separate DNA
fragments based on their sizes by applying an electrical field to an agarose gel containing these
DNA fragments. DNA contains many negative electrical charges, and scientists used this
property to separate pieces of DNA. Once the fragments are loaded onto the gel, an electrical
current is applied causing the negatively-charged DNA molecules to move towards the positive
electrode (red). The agarose gel acts as a matrix of tiny pores that allow small particles to move
through it relatively quickly. The larger fragments migrate much more slowly through the gel.
After a set period of exposure to the electrical current, the DNA fragments are separated by size
with the smaller ones located further away from the wells than the larger fragments. Fragments
that are either the same or very similar in size will tend to migrate together through the gel.
Fragment bands form as a result of the various distances the DNA segments migrate.
Advanced Biology with Vernier 6B - 1 (Fingerprint)

Computer 6B
Stains to visualize DNA

DNA is colorless and must be visualized with a “stain”. In this laboratory activity, DNA bands
may be stained with either Fast Blast™ DNA Stain or SYBR® Safe DNA gel stain then analyzed
with Vernier’s bioimaging systems. Fast Blast stained gels can be viewed with the White Light
Transilluminator and SYBR Safe stained gels can be viewed with the BlueView
Transilluminator. Both transilluminators can be used with Vernier’s Proscope and LoggerPro
software to capture digital images of the stained gels and perform analysis of the gels to
determine the sizes of the DNA bands.
DNA Fingerprinting
Your mission begins with the collection and restriction digestion of “Crime Scene” and
“Suspect” DNA. When DNA is mixed with restriction enzymes, the enzymes act as “molecular
scissors” and digest the DNA into smaller pieces. However, these cuts are not random. The
enzymes look for specific DNA sequences, restriction sites, and make specific cuts at those
locations. The resulting fragment sizes are dependent on how often the restriction sites occur
within the DNA. This means that identical DNA sequences will produce identical DNA
fragments when the DNA is digested with the same restriction enzymes and different DNA
sequences will produce different sized fragments when digested with the same restriction
enzymes.
The restriction sites for the restriction enzymes used in today’s lab activity are noted below:
Table 1: Restriction sites* recognized by EcoRI and PstI
5’….G AATTC…….3’
3’….CTTAA G……5’ EcoRI
5’….CTGCA G…….3’
3’…G ACGTC……5’
PstI
*The four DNA bases are Adenine (A); Cytosine (C); Guanine (G); and Thymine (T)
Your task will be to separate the DNA fragments based on size using a procedure known as gel
electrophoresis and then to compare the DNA fragments with those of a standard ladder whose
base pair sizes are already known.
Molecular Weight Standards (also known as Markers or Ladders):

Being the professional forensic scientist that you are, you will quantitate the size of the DNA
fragments in both the crime scene and suspects samples. To determine the sizes of the
experimental DNA bands, they must be compared to a standard ladder that has DNA bands with
known molecular weights. The standard used during activity is the HindIII digest of lambda
DNA, a commonly used DNA molecular weight marker. The standard ladder is run in a lane next
to the experimental lanes under the same conditions. Linear DNA fragment migration distance
through a gel is inversely proportional to the log10 of its molecular weight (base pair number).
This process is carried out using the Gel Analysis feature in Logger Pro.
6B - 2 (Fingerprint) Advanced Biology with Vernier

OBJECTIVES
In this activity, you will
• Digest DNA found at the “crime scene” and the DNA of five “suspects” with two
restriction enzymes.
• Perform agarose gel electrophoresis on DNA samples.
• Stain the gel to visualize the DNA bands.
• Document and examine gel results with an imaging system.
• Evaluate who cannot be excluded from the investigation by constructing a standard curve
and determining the size of the DNA fragments from the gel using Logger Pro.
MATERIALS
computer water bath or heat block
Vernier interface ruler, millimeter
Logger Pro permanent markers
Vernier Blue Digital Bioimaging System laboratory tape (not sticky tape)
or White Digital Bioimaging System stain
DNA samples:* SYBR Safe Stain or
Crime Scene DNA Fast Blast DNA Stain*
Suspect DNA, 5 different suspects multicolor micro test tubes*
EcoRI/PstI restriction enzyme mix clear micro test tubes*
Lambda DNA HindIII digest (DNA standard) foam micro test tube holders*
electrophoresis buffer, 50x, TAE* agarose*
sample loading dye* staining trays*
electrophoresis chamber & power supply sterile water*
adjustable micropipette, 2–20 µL and tips microwave oven or hot plate
adjustable micropipette, 20–200 µL and tips microcentrifuge
rocking platform gel support film
* Included in the Bio-Rad kit
PRE-LAB ACTIVITY
1. Make a gel. Note: You do not need to do this step if you are using a pre-cast gel.
a. Clean the lab table surface, wash your hands, glove, set the lab mat, and review lab safety
procedures.
b. Measure out 0.5 grams of agarose and pour the powder into the flask.
c. Measure out 50 mL of 1X TAE (Tris-Acetate- EDTA) buffer and transfer volume to the
flask containing the agarose. Swirl gently and place a funnel, stem first, into the top of the
flask.
d. Place the flask with its cover in a microwave and heat on high for 40 seconds or until the
solution starts to boil. The agarose solution must be crystal clear and void of suspended
granules, use additional ten-second blasts of the microwave until this condition is attained.
e. With hot-gloves, remove the hot flask and transfer the container to your lab space.
f. While the flask is cooling, prepare the gel tray by taping the two open ends of the gel tray
with lab tape (masking tape and Scotch® tape will not work) then place the eight-toothed
comb in position at one end of the tray. The tray needs to be placed level on the surface of
the lab mat.

Computer 6B
g. Allow the solution to cool until the flask can comfortably be placed on the back of your
palm (60° to 55°C).
h. Perform this step if you are using SYBR Safe pre-electrophoresis stain. If you are not, skip
to Step i. Pipette 5 μL of concentrated SYBR Safe 10,000x DNA staining solution (1 μL
per 10 mL of agarose solution) to the flask, swirl, and then pour solution into your
prepared gel tray. Pour the gel to a thickness of 0.8 to 1.0 cm, which is approximately half
way up the teeth of the comb.
i. It will take between 15 and 25 minutes for the gel to set and appear cloudy or opaque
when ready to use. Carefully remove the comb from the solidified gel and untape the ends
of the tray.
PROCEDURE
Quick Guide for Forensic DNA Fingerprinting Kit

Part A Restriction Digestion
1. Place the tube containing the restriction

enzyme mix, labeled ENZ, on ice.
ENZ Ice
2. Label the colored microtubes as follows:

green tube CS = crime scene
blue tube S1 = suspect 1
orange tube S2 = suspect 2
violet tube S3 = suspect 3 CS S1 S2 S3 S4 S5 Foam
red tube S4 = suspect 4 Rack
yellow tube S5 = suspect 5
Label the tubes with your name, date, and

lab period. Place the tubes in the foam
rack.
3. Using a fresh tip for each sample, pipet

10 µL of each DNA sample from the stock DNA Samples
tubes and transfer to the corresponding +
colored micro test tubes. Make sure the Enzyme Mix
sample is transferred to the bottom of the
tubes.
4. Pipet 10 µL of the enzyme mix (ENZ) into

the very bottom of each tube. Use a fresh
tip to transfer enzyme to each tube.
Stock CS S1 S2 S3 S4 S5

5. Tightly cap the tubes and mix the

components by gently flicking the tubes
with your finger. If a microcentrifuge is
available, pulse-spin in the centrifuge to
collect all the liquid in the bottom of the
tube. Otherwise, gently tap the tube on the Flick Tap
table.
6. Place the tubes in the foam micro tube

holder and incubate for 45 minutes at 37ºC
or overnight at room temperature in a large
volume of water heated to 37ºC.
7. At this point you can put the DNA samples

into the refrigerator and run the agarose gel Water bath
during the next class or you can continue
with Step 9 and run the agarose gel today.
Ask your instructor if you are unsure.
Part B Agarose Gel Electrophoresis
8. Remove the digested DNA samples from

the refrigerator (if necessary).
9. If a centrifuge is available, pulse spin the __________

tubes in the centrifuge to bring all of the
liquid into the bottom of the tube or gently Centrifuge Tap
tap on the table top.
10. Using a separate tip for each sample, add

5 µL of loading dye “LD” into each tube. DNA Loading Dye
Cap the tubes and mix by gently flicking
the tube with your finger. Collect the
sample at the bottom of the tube by tapping
it gently on the table or by pulse-spinning
in a centrifuge. Flick
11. Remove the agarose gel from the

refrigerator (if applicable) and remove the
plastic wrap.
12. Place an agarose gel in the electrophoresis
apparatus. Fill the electrophoresis chamber (-)
with 1x TAE buffer to cover the gel, using (+)
approximately 275 mL of buffer.
13. Check that the wells of the agarose gels are
near the black (–) electrode and the bottom
edge of the gel is near the red (+) electrode.

Computer 6B
14. Using a separate tip for each sample, load

the indicated volume of each sample into
7 wells of the gel in the following order:
Lane 1: M, DNA size marker, 10 µL
Lane 2: CS, green, 20 µL
Lane 3: S1, blue, 20 µL
Lane 4: S2, orange, 20 µL (-)
Lane 5: S3, violet, 20 µL (+)
Lane 6: S4, red, 20 µL
Lane 7: S5, yellow, 20 µL
15. Place the lid on the electrophoresis
chamber carefully. The lid will attach to
the base in only one orientation. The red
and black jacks on the lid will match with
the red and black jacks on the base. Plug
the electrodes into the power supply, red to
red and black to black.
16. Turn on the power and run the gel at 100 V
for 30 minutes.
17. When the electrophoresis run is complete,
turn off the power and remove the top of
the chamber.
Part C Staining the Gel
17. Your teacher will let you know which of the four staining options you will use.
Option 2: Quick staining using Bio-Rad’s 100x Fast Blast stain (requires 12–15 minutes)
Option 3: Vernier SYBR Safe Stain post-electrophoresis
Option 4: Vernier SYBR Safe Stain pre-electrophoresis

18. Stain the gel using 1x Fast Blast Stain.
a. Carefully remove the gel and tray from the gel box. Be careful
because the gel is very slippery. Slide the gel into the staining
tray.
b. Add 120 mL of 1x Fast Blast stain into a staining tray (2 gels per
tray).
c. Let the gels stain overnight, with gentle shaking for best results.
No destaining is required.
d. Pour off the water into a waste beaker.
e. Proceed to the Analysis section.

Option 2: Quick staining using Bio-Rad’s 100x Fast Blast stain

18. Stain the gel using Bio-Rad’s 100x Fast Blast stain.
because the gel is very slippery. Slide the gel into the staining
tray.
b. Add 120 mL of 100 x Fast Blast stain into a staining tray (2 gels
per tray).
c. Stain the gels for 2 minutes with gentle agitation. Save the used
stain for future use.
d. Transfer the gels into a large washing container and rinse with
warm (40–55°C) tap water for approximately 10 seconds.
e. Destain by washing twice in warm tap water for 5 minutes each
with gentle shaking for best results.
f. Proceed to the Analysis section.
Option 3: SYBR Safe Stain post-electrophoresis gel

18. Post-electrophoresis staining procedure.
because the gel is very slippery.
b. Place the gel in a gel staining tray and measure out 50 mL or
enough of the SYBR Safe DNA Gel Staining solution (0.5 x) to
cover the gel in the tray.
c. Obtain a piece of Aluminum foil to cover and keep light from
directly shining on the gel in the staining tray. The gel will be
left in this staining solution for 30 minutes.
d. Place gel on rocker or periodically swirl the solution and gel in
the staining tray to obtain a thorough and uniform stain of the
DNA fragments.
e. No destaining is necessary once the 30-minute period has
elapsed. Rinse the gel with water and proceed to the Analysis
section.
Option 4: SYBR Safe Stain pre-electrophoresis stain

18. Your gel has already been stained. Carefully remove the gel and
tray from the gel box. Be careful because the gel is very slippery.
Proceed directly to the Analysis section.
ANALYSIS
1. Connect the transilluminator to AC power and turn it on.
2. Prepare the ProScope for use.

a. Connect the 1–10x lens to the ProScope.
b. Connect the ProScope to the USB port of your computer.

Computer 6B
c. Mount the ProScope to the stand and position the stand next to the transilluminator,
opposite the side with the hinge.
d. Level the ProScope so that its lens is parallel to the surface of the transilluminator.
3. Prepare Logger Pro for use.
a. Start Logger Pro.
b. Choose Gel Analysis ► Take Photo from the Insert menu.
c. Check Close Window and Auto Arrange as the Photo Actions.
4. Take a photo of the gel.
a. Transfer the gel to the central portion of
the transilluminator platform.
b. Orient the gel and platform so that the
wells are at the top of the picture in
Logger Pro.
c. Orient and focus the ProScope so both
the bands and lane numbers are clear and
sharp. Note: Lowering the brightness by
clicking Camera Settings may improve
visibility of the gel.
d. Place the Imaging Hood over the Figure 1
ProScope and the transilluminator.
Reach through the flap of the hood to make final adjustments for best position, focus, and
resolution.
e. Once satisfied with the image, click . The screen should now resemble Figure 1.
Note: You may want to re-size the photo and graph to increase the size of the photo for
ease of analysis.
5. Indicate the position of the wells on the photograph.
a. Click Set Origin, .
b. Click the photograph just to the left of the first well. A yellow coordinate system will
appear on the photograph.
c. Position the x-axis directly along the bottom edge of the wells. You can move the origin
by clicking either axis and dragging it to the desired location. The axis can be rotated by
clicking the round handle on the x-axis.
6. Convert the units of distance from pixels to millimeters.
a. Click Set Scale, .
b. Click and drag to draw a line that is one centimeter long using the ruler on the gel tray as
your guide. For example, click and drag between the 1 cm mark and the 2 cm mark to
create a line that is one centimeter long.
c. Enter the distance value and units as millimeters and click .
7. Identify the bands and base pair values of the standard ladder using the HindIII digest lane
base pair values in Table 2.
a. Click Set Standard Ladder, .
b. Click the center of the first band in the HindIII digest lane.
c. Enter the number of base pairs for this band using the values in Table 2. Click .

d. Click the center of the next band in this lane and enter the base pair value. Click .
e. Repeat this process for each visible band of the standard ladder. Logger Pro will
automatically create a standard curve on the graph.
8. Identity the bands in the remaining lanes. Logger Pro will plot bands, record distance
migrated and calculate the respective number of base pairs.
a. Click Add Lane, , and choose Add Lane.
b. Click the center of the first band in the first experimental lane. Notice that when you click,
three things happen: a marker with a distinct shape and color is placed on the photograph,
a matching marker is placed on the standard curve of the graph, and the distance and
number of base pairs are added to the data table.
c. Click the center of the next band in this lane.
d. Continue this process for each visible band in the experimental lane.
9. Repeat Step 8 for each remaining lane.
10. Record the base pair values for the experimental lanes in Table 3. Not all cells will be filled.
11. (optional) Save and print the results of the gel analysis.

Standard HindIII
restriction digest of Crime Scene Suspect 1 Suspect 2 Suspect 3 Suspect 4** Suspect 5**
lambda DNA
Computer 6B
Actual Approx Approx Approx Approx Approx Approx

Distance Distance Distance Distance Distance Distance Distance
Band Size Size Size Size Size Size Size
(mm) (mm) (mm) (mm) (mm) (mm) (mm)
(bp) (bp) (bp) (bp) (bp) (bp) (bp)
6B - 10 (Fingerprint)
1 23,130
2 9,416
3 6,557
4 4,361*
5 2,322
6 2,027
*May appear faint if the markers were not headed to 65•C. Lambda HindIII digestion also generates bands of 564 and 125 bp that are usually too faint to see on a gel.
** S4 and S5 DNA lanes may also contain a very faint band of 500 bp.

QUESTIONS
1. What is a restriction enzyme?
2. How are restriction enzymes useful to bacteria in nature?
3. Describe the palindromic property of restriction sites.
4. How are the DNA fragments separated from one another after a restriction digest has been
performed?
5. What is a molecular weight standard (also known as marker or ladder) and what is its role?
6. How is the DNA visualized?
7. Do any of the suspect samples appear to have the same banding pattern as that found at the
crime scene?
8. What can be said of the restriction sites of the crime scene and suspect DNA samples?
9. Based on your analysis, what can you conclude about the DNA found at the crime scene and
the DNA of the five suspects?

Experiment
Lab Manual.
2. The student pages with complete instructions for analysis using Logger Pro (computers), can
be found on the CD that accompanies this book. See Appendix A for more information.
Note: This activity can only be completed using computers.
3. In this activity, students play the role of a forensic scientist and use restriction enzymes to
digest real DNA and identify the culprit among five “suspects”. It models the more elaborate
technique that is performed on complex human DNA samples. Principles of restriction
analysis, plasmid mapping, and DNA fragment size determination can also be documented.
The techniques introduced in this actvity form the basis of recombinant DNA technology
techniques, DNA fingerprinting, and forensic DNA analysis.
4. The suggested complete kit, Forensic DNA Fingerprinting Kit, is available from Bio-Rad
Laboratories. Refer to Appendix G for ordering information. This kit is sufficient for
32 students (eight workstations, four students per workstation).
5. Analysis of the gel is completed using either Vernier’s Blue Digital Bioimaging System or
White Digital Bioimaging System connected to a computer running Logger Pro data-
collection software. From the digital image of the gel, Logger Pro will create a standard
curve and automatically calculate the number of base pairs (molecular weight) for each
experimental DNA band.
6. Tips for using the Vernier Blue Digital Bioimaging System and White Digital Bioimaging
System:
• Use the power supply that shipped with the device. Using the wrong power supply could
damage it.
• If using the BlueView Transilluminator, ensure that the BlueView’s lid is closed, otherwise
it will not function. This is a safety feature.
7. Tips for using Logger Pro to take a photo of the stained gel:
• For the best photo of the gel banding patterns, you may want to adjust the camera settings
before clicking . Click Camera Settings in the Take Gel Photo dialog box, then
click Adjustments. From here, position the brightness slider between 10 and 25%, making
sure that the dimmer switch of the BlueView Transilluminator is turned to full brightness
and the Imaging Hood is placed over the system. Continue to adjust until the best results
are obtained.
• When performing a gel analysis, the sequence of events described in the procedure must be
followed in order. It is not possible to go back and change parameters, with these
exceptions:
• Points placed on the photograph identifying experimental bands can be moved or
deleted. Simply click Select Point, , then click on the point to be edited. Drag it with
the cursor to move it or use the Delete key on your keyboard to delete.
• Points for experimental bands can be added by clicking Add Lane, , selecting the
appropriate lane, then clicking on the band in the photograph.
Advanced Biology with Vernier 6B - 1 T (Fingerprint)

Experiment 6B (Fingerprint)
Ideas for Inquiry

• How does changing the amount of enzyme used affect band patterns and intensity?
• How does changing the amount of DNA used affect band intensity?
• How does changing the reaction time (DNA + enzyme) affect band pattern?
• How does changing temperature of incubation affect band pattern?
TIME REQUIREMENTS
This experiment is divided into three parts. Each part is designed to be completed in a 50-minute
lab period. Parts A and B will each take one day. You will be able to complete Part C and the
analysis on the third day unless you are staining using 1x Fast Blast DNA for Overnight Staining.
If you are staining overnight, you will perform the analysis on a fourth day. If you have 90-
minute lab periods, you should be able to complete Parts A and B in the first day.
PRE-LAB PREPARATION
Table 1 Pre-Lab Objectives and Time Requirements
Objective Time Required When
Gels may be cast 1 to

Prepare electrophoresis TAE buffer
30 minutes to 1 hour; 7 days in advance.
Using the procedure in the Pre-lab
depending on how Alternatively, student
Activity, pour agarose gels (or use precast
gels are prepared teams may prepare
gels)
Part B their own gels.
Rehydrate lyophilized DNA and buffer

samples
30 minutes Just prior to Part B
Rehydrate enzyme mix; aliquot
Set up student and teacher workstations
Prepare DNA molecular weight marker
Aliquot sample loading dye
Part C Dilute DNA Stain 45 minutes Just prior to Part C
Prepare electrophoresis chambers
Set up student and teacher workstations
Analysis Set up imaging system 15 minutes Prior to Analysis
STAINING PROCEDURE
After electrophoresis, there are four staining options to view the DNA bands using either Fast
Blast™ DNA Stain (included in the Bio-Rad kit) or SYBR Safe™ DNA gel stain (available from
Vernier). Both stains are convenient, safe, and nontoxic alternatives to ethidium bromide.
• Gels stained with Fast Blast should be analyzed by capturing digital images using a Vernier
White Digital Bioimaging System connected to a computer and Logger Pro software.
6B - 2 T (Fingerprint) Advanced Biology with Vernier

• Gels stained with SYBR Safe should be analyzed by capturing digital images using a Vernier
Blue Digital Bioimaging System connected to a computer and Logger Pro software.
• Any stains with an excitation wavelength in the blue range of 450 to 520 nm range will also
work with the Blue Digital Bioimaging System. For use with nucleic acids, these include, but
are not limited to, SYBR Safe, SYBR® Gold, SYBR® Green I, GelGreen™, and GelStar®.
Though each staining method is described in detail in their original documentation, they are
summarized here and in Table 2 for your convenience:
Option 1 Using 1x Fast Blast DNA Stain for Overnight Staining
This method requires the gel to sit overnight while it stains.
a. Dilute the 500X concentrate to 1X. We recommend using 120 mL of 1X Fast Blast to stain
two 7 X 7 cm or 7 X 10 cm gels in individual staining trays (included in the kit); each tray
can accommodate two gels.
c. Place the staining tray on a rocking platform and gently shake overnight. If a rocking
platform is not available, then periodically swirl the solution and gel a few times during
the staining period. This is crucial since smaller fragments tend to diffuse without shaking.
The bands will begin to develop after two hours, but at least eight hours of staining is
recommended for complete visibility.
d. The gel is ready to be analyzed using a White Digital Bioimaging System.
Option 2 Using 100x Fast Blast DNA Stain for Quick Staining (12–15 minutes)
If you use this option, you will be able to complete Part C and the Analysis section in a 50 or 90-
minute lab period.
a. Dilute the 500X concentrate to 100X. We recommend using 120 mL of 100X Fast Blast to
stain two 7 X 7 cm or 7 X 10 cm gels in individual staining trays (included in the kit); each
tray can accommodate two gels.
c. Stain for 2–3 minutes, maximum. Pour the 100X stain into a storage bottle and save for
future use. The 100X stain may be reused at least seven times.
d. Rinse the gels for 10 seconds in 500–700 mL of clean, warm (40–55ºC) tap water
e. Wash the gels for 5 minutes in 500–700 mL of clean, warm (40–55ºC) tap water. Repeat
this step. The bands may appear fuzzy immediately after the second wash, but will begin
to develop into sharper bands within 5–15 minutes after the second wash.
f. To obtain maximum contrast, additional washes in warm water may be necessary. Destain
to the desired level, but do not leave the gel in water overnight. If complete destaining
cannot be done in the allocated time, then transfer the gel to 1x Fast Blast stain overnight.
g. The gel is ready to be analyzed using a White Digital Bioimaging System.
Option 3 Using SYBR Safe DNA Gel Stain (0.5x) Post-Electrophoresis
minute lab period. This stain can either be diluted from the 10,000X concentrate or purchased as a
convenient, pre-diluted 0.5X stain.
a. After electrophoresis, carefully remove the gel from its gel tray and slide it into the
staining tray; add sufficient stain until the gels are completely submerged.
Advanced Biology with Vernier 6B - 3 T (Fingerprint)

b. Cover the staining tray with aluminum foil to protect the gel from light. Place the staining
tray on a rocking platform with gentle shaking and stain for 30 minutes at room
temperature. If a rocking platform is not available, then periodically swirl the solution and
gel to obtain thorough and uniform staining patterns.
c. Rinse the gel with water and place on the Blue Digital Bioimaging System for analysis.
Option 4 Using SYBR Safe DNA Gel Stain Pre-Electrophoresis
minute lab period.
a. Follow the directions for preparing agarose gels and add the appropriate volume of SYBR
Safe using this ratio: 1 µL of SYBR Safe 10,000X concentrate to 10 mL of agarose
solution. Thus for 40 mL of agarose, add 4 µL of SYBR Safe. Swirl to mix then pour the
gels.
b. Store SYBR Safe-containing gels protected from light by covering them with aluminum
foil at room temperature for 1–2 days or at 4ºC for up to one week.
c. Gels containing SYBR Safe stain are run in the same buffer system and electrophoresis
conditions as those gels that do not.
d. These gels do not need to be stained or destained. They may be analyzed using the Blue
Digital Bioimaging System.
SAMPLE DATA
Students load the gel with digests of DNA found at a “crime scene” and DNA of five “suspects,”
as indicated below. The HindIII lambda DNA digest serves as the standard.
Table 3 Lane assignments
Lane Load volume DNA samples & marker

λ DNA (μL)
HindIII lambda DNA molecular
1 10
weight marker/standard
2 20 CS (Crime Scene) DNA
3 20 S1 (Suspect 1) DNA

Standard HindIII
restriction digest of Crime Scene Suspect 1 Suspect 2 Suspect 3 Suspect 4*** Suspect 5***
lambda DNA
Actual Approx Approx Approx Approx Approx Approx

Distance Distance Distance Distance Distance Distance Distance
Band Size Size Size Size Size Size Size

(mm) (mm) (mm) (mm) (mm) (mm) (mm)
(bp) (bp) (bp) (bp) (bp) (bp) (bp)
1 11.0 23,130 19.0 3679 21.0 2860** 21.0 2860** 19.0 3679 21.0 2860** 21.0 2860**
2 13.0 9,416 20.5 2860** 23.5 1199 25.0 1700 20.5 2860** 29.5 1093 24.0 1986
3 15.0 6,557 32.0 828 30.5 941 28.5 1159 32.0 828 29.5 1093
4 18.0 4,361*
5 23.0 2,322
6 24.0 2,027
*May appear faint if the markers were not headed to 65•C. Lambda HindIII digestion also generates bands of 564 and 125 bp that are usually too faint to see on a gel.
** The measured migration distance for these bands varies depending upon the thickness of the bands. See Appendix D of the Bio-Rad manual to understand why the
bands are so intense in S4 and S5.
*** S4 and S5 DNA lanes may also contain a very faint band of 500 bp.
6B - 5 T (Fingerprint)
1. A restriction enzyme (or restriction endonuclease) is an enzyme that cuts DNA at specific
sequences of DNA known as restriction sites.
2. Restriction enzymes provide a defense mechanism against invading viruses. Inside a bacterial
host, the restriction enzymes selectively cut up foreign DNA; the host DNA is modified to
protect it from the restriction enzyme’s activity.
3. The top strand (when read from left to right) is identical to the bottom strand (when read
from right to left).
4. DNA fragments are separated on an agarose gel which is subjected to an electric current. This
is known as gel electrophoresis. The fragments migrate towards the positive electrode, and
the smallest fragments migrate the fastest.
5. A DNA molecular weight standard is a mixture of DNA fragments of known molecular

weights. Its role is to establish the distance migrated by these known DNA fragments under
experimental conditions (i.e: buffer composition, length of the run, voltage, thickness and
percentage of the gel) such that the sizes of experimental samples can be determined under
those same experimental conditions. The standard used in this activity was the HindIII digest
of lambda DNA.
6. DNA must be stained with a DNA stain, such as SYBR Safe or Fast Blast. SYBR Safe-
stained DNA is then visualized immediately using an imaging system. Fast Blast-stained
DNA can be visualized with the naked eye, without the use of an additional light source.
7. Yes: Samples in Lanes 2 and 5 match (CS and S3).
8. Both the crime scene DNA and the DNA of suspect 3 have EcoRI and PstI recognition sites
in the same location. While the DNA samples of the other four suspects do contain EcoRI
and PstI recognition sites (as evidenced by the fragments), they are not found in the same
locations as that of the crime scene DNA because they result in different banding patterns.
9. We can conclude that the DNA of suspect 3 matches that found at the crime scene.

Computer
Genetics of Drosophila
7
In 1865, Gregor Mendel published a paper on the patterns of genetic inheritance in the common
garden pea. This revolutionary work provided the basis for future study of genetics. Mendel
hypothesized that heredity was passed on by discrete particles, rather than by the blending of
parental traits, as was believed at the time, strongly affecting the argument over Darwin’s theory
of evolution.
Mendel proposed two very basic laws which serve as the cornerstones of modern genetics:
Mendel’s Law of Segregation and Law of Independent Assortment.
Mendel’s Laws of Genetic Inheritance

Mendel’s Law of Segregation states that for each trait (gene), each organism carries two factors
(alleles), and that each of the organism’s gametes contains one and only one of these factors. In
this way, the alleles segregate during meiosis, providing for genetic variability among the
organism’s offspring. This is apparent in monohybrid crosses—matings involving only one trait.
Mendel’s Law of Independent Assortment, found in dihybrid crosses (crosses involving two
traits), states that the alleles for one trait will separate independently of the alleles in another trait.
This means that with two genes (A and B), each with two possible alleles (A, a and B, b), there
are four possible combinations gametes can receive (AB, Ab, aB, or ab). This helps to ensure
genetic variability among offspring.
Although Mendel’s laws have proven to be true, they have their limitations. It must be assumed
that the genes involved are on different chromosomes. If they are on the same chromosome, the
assortment of their alleles will be closely linked instead of being independent. It must also be
assumed that the genes in question are not located on the X chromosome; this could cause the
expression of the trait to be linked to the sex of the offspring. Color blindness in humans is a
good example of this type of heredity. None of the traits Mendel selected in his pea plant
investigations were sex linked, or located on the same chromosome.
Since Mendel’s time, our knowledge of genetics, as well as the tools we use, have advanced
drastically. Instead of crossing varieties of pea plants and observing the results as Mendel did, we
now use restriction enzymes, electrophoresis, and basepair sequencing to learn more about traits
and their expression. Yet even with these advances, the basic concepts of genetic inheritance
described by Mendel so many years ago still stand.
Drosophila Use in Genetic Research

Drosophila melanogaster, the common fruit fly, was first used in genetic experiments in 1907 by
Thomas Hunt Morgan of Columbia University, and has been a staple of genetic research ever
since. Drosophila specimens are well suited to investigations into Mendelian patterns of
inheritance; they are small, produce large numbers of offspring, have many easily discernible
mutations, have only four pairs of chromosomes, and complete their entire life cycle in
approximately 12 days. Additionally, Drosophila are relatively easy to maintain, as they are hardy
and have simple food requirements.
Since the fruit fly was selected for study nearly a hundred years ago, a great deal has been learned
about its genome. In fact, the first chromosome map of any kind was constructed to detail the

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fruit fly. Chromosomes 1 (the X chromosome), 2, and 3 are

very large, and the Y chromosome—number 4—is extremely
small. Thousands of genes reside on these four
chromosomes, many of which are universal in nature,
existing in most eukaryotic forms, including humans. The
similarities between the Drosophila genome and those of
other species is helpful in determining patterns of evolution
and species divergence.
Drosophila embryos develop in the egg membrane. The egg
hatches and produces a larva which feeds by burrowing
through the medium. The larval period consists of three
stages, or instars, the end of each stage marked by a molt.
The first instar is the newly hatched larva; the third instar is
the final larval stage, where the larva may attain a length of
4.5mm. Near the end of the larval period, the third instar
larva will crawl up the sides of the culture vial, attach
themselves to a dry surface (the jar, the filter paper, etc.) and
form pupae. After a period of time the adults emerge.
It takes one or two days for Drosophila eggs to hatch into larvae, four to five days for the larvae
to enter pupae, and four days for the pupa stage. The duration of these stages, however, vary with
the temperature; at 20°C the average length of the egg–larval period is eight days, while at 25°C
it is reduced to five days. Thus at 25°C the life cycle may be completed in about 10 days; at
20°C, 15 days are required.
Different body features characterize the
male and female flies. The females are
slightly larger and have a light-colored,
pointed abdomen. The abdomen of the
males will be dark and blunt. The male
flies also have dark bristles on the upper
portion of the forelegs, which are known
as sex combs.
In the following experiment, parental
generations (P1) of wild-type and mutant
strains of Drosophila with various traits to demonstrate basic genetic principles are used.
Monohybrid, dihybrid, and sex-linked crosses are performed; the offspring of the first cross, the
F1 generation, are normally allowed to breed among themselves to produce the F2 generation.
Members from the F1 and F2 generations are collected and their traits observed to draw
conclusions about genetic inheritance.
The second filial generation, or F2, represents the flies that result form self-fertilization or
inbreeding among members of the F1 generation.
Punnett Square
Based on the laws of segregation and independent assortment, a Punnett square is extremely
important in determining the outcome of crosses in Mendelian genetics; it clearly displays the
possible combinations in chart form.

The simplest Punnett square to construct is one for a monohybrid cross. A good example of this
is a cross between female fruit flies with vestigial wings and male wild-type fruit flies.
Determining the genotype of the flies being crossed is vital to the accuracy of the results of a
Punnett square; if it is known that the trait for vestigial wings is a recessive mutation, the flies
with vestigial wings must be homozygous recessive for the first trait, and therefore have a
genotype of vv. Assuming that the wild-type flies are heterozygous dominant, they will have a
genotype of Vv. According to the Law of Segregation, only one of the alleles for the trait can be
passed on to a gamete for each parental fly. Therefore, the male wild-type fly could pass either
the V or the v allele on to its offspring. Likewise, the female, vestigial fly can pass on only one of
her alleles for the trait. In her case, however, they are both v. Therefore, the possible allelic
combinations in the offspring are Vv and vv. This is diagrammed in a Punnett square, below.
Males
V v
Females
v Vv Vv
v Vv vv
The two possible genotypes, Vv an vv, will exist in a 1:1 ratio, and the phenotypic ratio will also
be 1:1 with as many offspring with vestigial wings as with normal wings.
Punnett squares become more complicated when diagramming a dihybrid cross. Due to the
complex nature of dihybrid crosses—or even worse, crosses with three or more traits—it is even
more important to diagram the crosses with a Punnett square.
For an example of a dihybrid cross, females with normal eyes and vestigial wings can be crossed
with male flies that have sepia (dark brown) eyes and normal wings, assuming that the females
have the genotype Ssvv, and the males have the genotype ssVV. Again, each parent can donate
only one allele for each trait to the offspring, but by applying Mendel’s Law of Independent
Assortment, the alleles for the two traits should be distributed without regard to the distribution
of the other. There are, therefore, four possible combinations of alleles each parent can donate to
any one gamete. The females can donate the set of alleles Sv or sv; the males can only donate the
set of alleles sV. The Punnett square for this cross is diagrammed below.
Males
sV sV sV sV
Sv SsVv SsVv SsVv SsVv

Females
sv ssVv ssVv ssVv ssVv
sv ssVv ssVv ssVv ssVv

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Chi-Square Test
The chi-square test is a statistical tool that compares experiment results with an accepted set of
data to determine how much the experimental values deviated from the accepted ones and
whether or not that deviation can be explained solely by chance.
The square of the difference between the observed and expected values (O – E)2 for each data
point (phenotype category in this case) is calculated. Then, by dividing this by the expected
value, the amount of deviation between the experiment data and the accepted value for that data
point can be determined. Adding the statistic for each data point yields a value known as the X2
(chi-square) statistic.
X2 = ∑ (O – E)2/E
Because all the values for each category, or data point, are being added together, the value of X2
will rise as the number of data points used increases. For this reason, “degrees of freedom” must
be included in the parameters of the analysis. The number of degrees of freedom (v) for a chi-
square test is equal to the number of data points minus one. The number of degrees of freedom
does not have an impact on the value of X2 itself, but rather is used in the interpretation of the
importance of the value, as shown in the chi-square table, below. The numbers get larger as you
go down and the value for degrees of freedom increases.
Chi-Square Table
DF a
v P=0.99 0.95 0.80 0.50 0.20 0.05 0.01
1 0.00016 0.00393 0.06420 0.45500 1.64200 3.84100 6.63500
2 0.02010 0.10300 0.44600 1.38600 3.21900 5.99100 9.21000
3 0.11500 0.35200 1.00500 2.36600 4.64200 7.81500 11.34500
4 0.29700 0.71100 1.64900 3.35700 5.98900 9.44800 13.27700
5 0.55400 1.14500 2.34300 4.35100 7.28900 11.07000 15.08600
6 0.87200 1.63500 3.07000 5.34800 8.55800 12.59200 16.81200
7 1.23900 2.16700 3.82200 6.34600 9.80300 14.06700 18.47500
8 1.64600 2.73300 4.59400 7.34400 11.03000 15.50700 20.09000
9 2.08800 3.32500 5.38000 8.34300 12.24200 16.91900 21.66600
10 2.55800 3.94000 6.17900 9.34200 13.44200 18.30700 23.20900
15 5.22900 7.26100 10.30700 14.33900 19.31100 24.99600 30.57800
20 8.26000 10.85100 14.57800 19.33700 25.03800 31.41000 37.56600
25 11.52400 14.61100 18.94000 24.33700 30.67500 37.65200 44.31400
30 14.95300 18.49300 23.36400 29.33600 36.25000 43.77300 50.89200
First two hypotheses, H0 and H1, must be formulated, with H1 stating that the variations cannot
be explained solely by chance, and H0 stating that they can be determined solely by chance. This
will be determined by the value of “a”, which is defined as the probability that H1 can be
accepted as true. In order to justify H0, “a” must be quite low, depending on the specific needs of
the experiment and the confidence level required in the data.

The value of “a” is determined using the chi-square chart: the value of X2 is found in the row for
the correct number of degrees of freedom (v). It is likely that it will be between values; in this
case the value of “a” should be approximated. The probability that H0 can be accepted as true can
then be defined as 1 – a.
For an experiment with a relatively small sample size, a confidence level of between 50% and
80% is acceptable.
Example:
Assuming only two phenotypic categories, vestigial wings and normal wings, use the following
table of results for the F2 generation:
Phenotype No. of Males No. of Females
Vestigial Wings 14 12
Normal Wings 36 38
The calculation needs to be performed only twice—once for each phenotype. Adding the results
together provides the value for X2.
Total number of flies observed: 100

Flies with vestigial wings: 26
Flies with normal wings: 74
Expected ratio: 3:1
Expected number of flies with normal wings: 75
Expected number of flies with vestigial wings: 25
X2 = (74–75)2 + (26–25)2 = 0.013 + 0.04 = 0.053

75 25
Since only two phenotypes are used, there will be only one degree of freedom, as the number of
degrees of freedom is one less than the number of phenotypic categories. Since the X2 value is
less than the X2 value for one degree of freedom, at 0.05% (3.841), the null hypothesis can be
accepted as true. Since the X2 value (0.053) is between 0.00393 and 0.0642, the values for 80%
and 95% confidence respectively, there is an 80 to 95% confidence that the deviations from the
expected experiment values are due solely to chance.
OBJECTIVES
• Learn basic handling and culture techniques for working with Drosophila.
• Apply concepts and principles of Mendelian inheritance patterns.
• Diagram monohybrid, dihybrid, and sex-linked crosses.
• Gain experience sorting, sexing, and crossing Drosophila through two generations.
• Perform a chi-square statistical analysis of experimental results.

Computer 7
MATERIALS
culture vial of wild-type drosophila thermo-anesthetizer
culture vial of monohybrid cross petri dish
or culture vial of dihybrid cross 2 drosophila vials and labels
or culture vial of sex-linked cross drosophila medium
100 mL 10% isopropyl alcohol fly morgue
camel hair brush forceps
PROCEDURE
Part A: Working with Drosophila
You will need to observe wild type Drosophila to familiarize yourself with the wild type
phenotype. You will eventually be assigned a cross without being told what strain, genotype, or
the type of experimental cross that will be performed. You will examine the flies in the parental
generation of your cross, noting any phenotype variations from the wild type, and name the
mutations.
1. Thermally immobilize a vial of wild-type Drosophila. Your instructor will demonstrate the
proper immobilization technique. Note: Your instructor may have immobilized the flies in
advance. If this is the case, begin with step 2.
2. Observe the flies’ traits, particularly body features that distinguish males and females, eye
color, and wing size and shape. Record your observations in Table 1 in the Analysis section.
If, at any time during your observations, the flies begin to become active, re-immobilize them
according to your instructor. Note: Use the camel’s hair brush to move the flies when making
observations.
Part B: Performing a Drosophila Cross
3. Obtain a vial of a prepared Drosophila cross. Note: These flies have been mated and may
exhibit one or more mutations. They are the parental generation for your experiment. The
offspring of this generation, which should already exist as eggs or larvae in the vial, are the
F1 generation.
4. Record the letter written on the vial in Table 2 in the Analysis section of the lab. This will
help you to keep track of which cross you have received. This will aid in determining
expected results as well as allow your instructor to identify any problems you may be having
and to help correct them.
5. Immobilize the parental generation of your cross and observe the flies under a
stereomicroscope. If, at any time during your observations, the flies begin to become active,
re-immobilize them according to your instructor.
6. Separate the males from the females. Note any mutations from the wild-type phenotype, as
well as whether the mutation is apparent in the male or female flies. Record your
observations in Table 2. Note: You may be sharing the parental generation with another
group for observation. If this is the case, do not perform the next step until all groups have
had a chance to make their observations.
7. Place the parental generation in the morgue.
8. Place the vial (with the parental generation removed) in a warm place to incubate to allow the
F1 generation to mature. Observe the vial occasionally and record your observations. Note:
Do not allow the temperature to exceed 30°C.

9. When the adult flies emerge, collect, immobilize, and examine them. Note the sex of each
one, as well as the presence of any mutations. Record your observations in Table 3. Be sure
every group assigned to that cross has a chance to observe and count the F1 progeny.
10. Prepare a fresh culture vial: Place approximately one tablespoon of medium in the bottom of
a vial. Add an equal amount of water and let it absorb. You may want to add a piece of
plastic mesh to give the flies something to crawl on, but it is not essential. Insert a foam plug
in the vial.
11. Place five mating pairs (one virgin female and one male) from the F1 generation into the fresh
culture vial. Label the vial with your name(s), date, and letter of cross. Place the culture vial
in a warm place to incubate to allow the F1 generation to mature.
12. Transfer the remaining nonmating F1 flies to the morgue.
13. Leave the F1 adults in the vial for about one week to mate and lay their eggs. Once they have
laid their eggs, remove the adults, place them in the morgue, and wait for the F2 generation
adults to emerge over the next several days. Note: Do not allow the temperature to exceed
30°C.
14. As the F2 generation flies begin to emerge as adults, immobilize and examine them. Record
the number of males and females, noting any mutations which may be present. Record your
findings in Table 4. Note: Try to collect as many adults as possible.
DATA
Table 1
Phenotypes of Wild Type Drosophila
Eye color Wing size and shape
Male
Female
Table 2
Phenotypes of the Parental Generation
Phenotype No. of males No. of females
Cross Letter:______

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Table 3
Phenotypes of the F1 Generation
Cross Letter:______
Table 4
Phenotypes of the F2 Generation
Cross Letter:______
Polytene Chromosome Investigation

ANALYSIS
1. Describe the parental cross you received; use genetic symbols. Example: A cross between
vestigial and wild-type flies would be expressed as vv x VV. Draw a Punnett square to show
the possible allelic combinations for this gene in the F1 generation
2. Identify the genotype the F1 flies should exhibit. Identify the phenotype. Compare your
experiment results by counting the members of the F1 generation.
3. Describe the F1 cross you performed, and draw a Punnett square to show allelic combinations
possible in the F2 generation.
4. Identify the genotype ratio the F2 flies should exhibit. Identify the phenotype ratio. Compare
your experiment results by counting the members of the F2 generation.
5. Identify the type of cross you received: monohybrid or dihybrid, autosomal or sex linked,
mutations dominant or recessive.
6. Using a chi-square test, determine whether or not the variation between the observed and
expected number of individuals of each phenotype can adequately be explained by chance
alone. Use the following formula, and apply it to the chi-square table in the Introduction to
determine the confidence that the variation is due solely to chance.
X2 = ∑ (O–E)2/E
O = observed number of offspring for the phenotypic category
E = expected number of offspring for the phenotypic category
X2 = _____________________
Confidence that variability is due entirely to chance = ____________ %
QUESTIONS
1. How are the alleles for genes on different chromosomes distributed to gametes? What genetic
principle does this illustrate?
2. Why was it important to have virgin females for the first cross (yielding the F1 generation),
but not the second cross (yielding the F2 generation)?
3. What did the chi-square test tell you about the validity of your experiment data? What is the
importance of such a test?

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OPTIONAL EXERCISE
Polytene Chromosome Investigation

In this exercise, you will investigate chromosomes and their activity by examining the salivary
glands of Drosophila larvae, which contain giant polytene chromosomes as a result of the
repeated replication of the DNA. They make excellent organisms for chromosome observation,
and for investigations into chromosome function and structure. They are most evident in D.
virilis larvae, but can be observed in any species of Drosophila.
MATERIALS
8% HCl coverslip
30 mL aceto-orcein stain drosophila chromosome prepared slide
15 mL piccolyte ii gloves
teasing needle goggles
pipet lab aprons
forceps stereomicroscope
microscope slide distilled water
PROCEDURE
1. Place a microscope slide on a flat surface. Add two or three drops of distilled water to the
center of the slide.
2. With forceps, grasp a fully-grown Drosophila larva from the wall of the wild-type Drosophila
culture vial. Place the larva in the drop of water on the microscope slide.
3. Under a stereomicroscope, locate the head of the larva. It will appear darker than the rest of
the body, and the mouthparts should be evident. The larva will also be moving in that
direction.
4. Grasp the larva in the midsection with the forceps. Squeeze it gently to force the head out.
5. Pierce the head with the teasing needle and pull the head away from the body. The salivary
glands, and likely the digestive tract also, will detach from the body. The salivary glands will
appear clear and cellular, but may be confused with the darker, opaque fat bodies. The
intestine may be present as a tubular, branched structure.
6. Remove all the excess materials from the slide.
7. Touch a corner of a paper towel to the slide to blot off only the excess water. Note: Do not let
the salivary glands dry out.
8. Add two or three drops of 8% HCl to hydrolyze the genetic material for better penetration by
the stain. Let it sit for three minutes. Caution: Avoid skin or eye contact with HCl. Wear
safety goggles, gloves, and a lab apron.
9. Touch a corner of a paper towel to the slide to blot off the excess HCl.

10. Add two or three drops of aceto-orcein stain to the salivary glands and let them to sit for four
to five minutes. Do not let the stain dry out. Add more stain if necessary. Caution: Aceto-
orcein stain is an irritant. Avoid skin or eye contact. Wear safety goggles, gloves, and a lab
apron.
11. Blot excess stain from the slide; leave a small amount of stain on the glands.
12. Place the slide on a smooth, flat surface and cover with a coverslip. Tuck the slide into the
fold of a paper towel and press down firmly on the coverslip with your thumb or a pencil
eraser.
13. Observe the preparation under a compound microscope. Note the bands in the polytene
chromosomes, the “puff” regions, and the chromocenter of the chromosome. Draw what you
see in the space below.
QUESTIONS
1. What do the giant polytene chromosomes look like?
2. What are the various bands believed to be?
3. What chemical compound is the major substance of chromosomes? What is its double
function?

Experiment
TIME REQUIREMENTS
Two to three weeks for culturing. Note: May require extra lab time outside of class for
investigations to coincide with Drosophila life cycle and the hatching of virgin females.
PRE-LAB PREP
Part A: Working with Drosophila
1. Prepare a Petri dish for each lab group: Place a Petri dish on an ice pack and place in the
freezer for at least 24 hours before the lab. Note: Cut an opening in the cover of the ice pack
to allow better contact between the ice pack and the Petri dish.
2. Prepare fly morgue: Add 10 to 15 mL of 10% isopropyl alcohol to the morgue. Secure the lid
tightly to prevent evaporation. Note: You may perform the next several steps in advance or, if
time permits, have the students perform them in class.
3. Thermally anesthetize wild-type Drosophila: Invert the vial of flies and place it in a
refrigerator or tight-sealing cooler with several ice packs for 10 to 20 minutes or until the
flies appear lifeless. Note: Chilling the vial inverted makes it easier to see the flies as they
become immobilized and also prevents them from becoming stuck in the media.
4. Prepare one culture vial per group: Place approximately one tablespoon of medium in the
bottom of a vial. Add an equal amount of water and let it absorb. You may want to add a
piece of plastic mesh to give the flies something to crawl on, but it is not essential. Insert a
foam plug in the vial to prevent any contamination.
5. Subculture wild-type Drosophila: Using one prepared culture vial per lab group, divide the
Drosophila into equal amounts.
6. Thermally anesthetize the subcultured Drosophila just prior to performing the lab. When the
flies are immobile and have collected on the foam plug, remove the ice pack with the Petri
dish from the freezer. Quickly dump the flies into the Petri dish. Note: You may thermally
anesthetize the Drosophila to have them ready for students to examine, or you may have the
students thermally anesthetize the Drosophila themselves as part of the lab. The flies will
remain immobile for 10 to 15 minutes. If more time is required, cover the Petri dish and
return the dish and the ice pack to the freezer for two to three minutes. The flies can be
anesthetized this way repeatedly without being harmed.
Part B: Performing a Drosophila Cross

The Drosophila crosses provided by WARD’S contain the parental (P) generation for each type
of cross. The flies have already mated and the larvae present on the side of the vials represent the
F1 progeny. You must assign one of the three types of crosses to each group. Groups sharing a
cross must share the anesthetized parental flies for the observation portion of the lab. When the
F1 generation emerges, each group may count the offspring separately and compare results. After

Experiment 7
every group has counted the F1 for their respective cross, they may then each obtain several
mating pairs from the F1 specimens for the initiation of the next generation
1. Remove and retain the top label from the culture vials of the crosses before giving them to
the lab groups, so the lab groups receive vials labeled only “A”, “B”, or “C”; they will
identify the crosses. Be sure to note the initiation date on the top label of each cross. These
crosses must be allowed to sit for five days from the initiation date before the students can
proceed with their observations.
2. Prepare culture vial: Place approximately one tablespoon of medium in the bottom of a vial.
Add an equal amount of water and let it absorb. You may want to add a piece of plastic mesh
to give the flies something to crawl on, but it is not essential. Insert a foam plug in the vial to
prevent any contamination. Note: You may prepare the culture vials in advance or you may
wish to have the students prepare their own vials prior to performing their cross.
TIPS
1. Note the initiation date on the Drosophila crosses upon receipt. Allow the Drosophila crosses
to develop five days from the initiation date prior to performing part B of the experiment.
2. In the optional exercise, students investigate chromosomes and their activity by examining
the salivary glands of Drosophila larvae, which contain giant polytene chromosomes as a
result of the repeated replication of the DNA. They make excellent organisms for
chromosome observation, and for investigations into chromosome function and structure.
They are most evident in D. virilis larvae, but can be observed in any species of Drosophila.
SAMPLE DATA
(A) Monohybrid Cross Males Females Conclusions
Sepia eyed 16 15 Answers will vary

Student results should approximate a ratio of:
3: wild eye
Wild eyed 46 51 1: sepia eye
(B) Dihybrid Cross Males Females Conclusions

Wild eyed/normal wing 63 66 Answers will vary
Student results should approximate a ratio of:
Sepia eyed/normal wing 19 21 9: wild eye/normal wing
Wild eyed/vestigial wing 20 20 3: sepia eye/normal wing
3: wild eye/vestigial wing
Sepia eyed/vestigial wing 7 8 1: sepia eye/vestigial wing
(A) Sex Linked Cross Males Females Conclusions

Answers will vary
White eyed 31 29 Student results should approximate a ratio of:
1: wild eye female
1: white eye female
Wild eyed 28 32 1: wild eye male
1: white eye male

Teacher Information Genetics of Drosophila
ANSWERS TO DATA ANALYSIS

1. Example: A cross between vestigial and wild-type flies would be expressed as vv x VV.
a. Monohybrid Cross–Cross between female flies with sepia-colored eyes and wild-type
males: SS (males) x ss (females)
Males
S S
s Ss Ss
Females
s Ss Ss
b. Dihybrid Cross–Cross between males with sepia-colored eyes and normal wings, and
females with normal eyes and vestigial wings: ssVV (males) x SSvv (females)
Males
sV sV sV sV

Females
c. Sex-Linked Cross–Cross between female flies with white eyes and wild-type male flies:
XWY (males) x XwXw (females)
Males
XW Y
Xw XWXw XwY
Females
Xw XWXw XwY
2. a. Monohybrid Cross–All the F1 individuals should be heterozygous dominant (Ss), and

therefore have normal wild-type eye color
b. Dihybrid Cross–All the F1 individuals should be heterozygous dominant for both traits,
and should therefore exhibit the phenotype of wild-type flies.
c. Sex-Linked Cross–All the F1 female flies should be wild type, and all F1 male flies should
have white eyes.

Experiment 7
3. a. Monohybrid Cross– F1 cross: Ss x Ss

Males
S s
S SS Ss
Females
s Ss ss
b. Dihybrid Cross– F1 cross: SsVv (males) x SsVv (females)
Males
SV Sv sV sv
SV SSVV SSVv SsVV SsVv

Sv SSVv SSvv SsVv Ssvv
Females
sV SsVV SsVv ssVV ssVv
sv SsVv Ssvv ssVv ssvv
c. Sex-Linked Cross– F1 cross: XWXw x XwY
Males
Xw Y
XW XWXw XWY
Females
Xw XwXw XwY
4.
a. Monohybrid Cross– The genotype ratio should be 1:2:1. The phenotype ratio should be
3:1, with 75% of the F2 individuals having normal wild-type eyes, and 25% having sepia
eyes.
b. Dihybrid Cross–The genotype ratio can be determined from the Punnett square, and the
phenotype ratio for the F2 generation should be 1:3:3:9, with one individual with both
sepia eyes and vestigial wings, three flies with normal wings and sepia eyes, three flies
with vestigial wings and normal eyes, and nine wild-type flies.
c. Sex-Linked Cross–The female flies should exist in a 1:1 ratio, with half having the
genotype XWXw, and half having the genotype XwXw. The males also exist in a 1:1
ratio, with half being XWy, and half Xwy. The phenotypes of the F2 generation: half of the
males and half of the females will have white eyes, and half of each sex will have wild-
type eyes.

Teacher Information Genetics of Drosophila
5. a. Monohybrid, autosomal, recessive.

b. Dihybrid, autosomal, both traits recessive.
c. Monohybrid, sex-linked, recessive.
6. a. Monohybrid Cross– X2 = 0.111, with v = 1, for a 50 to 80% confidence that the variations
in the data are due entirely to chance
b. Dihybrid Cross– X2 = 0.451, with v = 3, for an 80 to 95% confidence that the variations in
the data are due entirely to chance.
c. Sex-Linked Cross–X2 = 0.06375, with v = 3, for better than 99% confidence that the
variation in the data is due entirely to chance. In this case, there are three degrees of
freedom; because it is a sex-linked trait, expected ratios for white vs. wild-type eyes for
both males and females must be calculated, resulting in four data points.
1. The alleles for several genes on different chromosomes are distributed independent of each
other. This demonstrates Mendel’s Law of Independent Assortment.
2. In the first cross, it was important that the two pure strains of Drosophila were not allowed to
breed among themselves, but breed instead with individuals of the other strain. For this
reason, virgin females were required for this cross. For the second cross, the only male flies
the females could have been exposed to were also of the F1 generation, so all possible
breeding would yield the desired cross.
3. It should have either shown whether the variability between the experiment values and those
expected could be explained solely by chance or by some other factor. The test is important
because it can be used to determine the importance of experiment findings, and can be used
to help prove or disprove scientific hypotheses.
ANSWERS TO OPTIONAL EXERCISE QUESTIONS

1. Large, red-banded, multi-armed structure, with conspicuous light and dark banding.
2. Individual genes.
3. The chemical compound that is the major substance of chromosomes is DNA. It contains the
genetic code and transmits the hereditary pattern.

Computer
Population Genetics and Evolution

8
As early as the 500s B.C., several Greek philosophers theorized about the union of male and
female traits to form offspring. In the 17th century, Leeuwenhoek concluded that semen and eggs
carried hereditary factors conveyed to the offspring. Throughout the next century, scientists
developed theories on the processes of development; LaMarck was one of the first to discuss the
possibility of acquiring changed traits from parents. For example, he thought that if giraffes had
to stretch to eat the tops of trees, their offspring would be born with longer necks.
During the 19th century, Darwin published his theory of evolution, stating that members of a
population vary considerably in their genetic makeup. Those that are the “fittest” for their
environment are better able to survive and reproduce, and therefore pass these suitable traits on
to the next generation. This “natural selection” creates a population that is different from the
previous generations. Since Darwin’s theories were published, several others have expounded on
his work, leading to the ideas of adaptation and mutation. Recent research has determined that
chromosomes, present in each sex’s reproductive material, carry the genes that determine
individual characteristics.
A population—all the individuals of a species that live in the same place at the same time—are
affected by their own characteristics. Population genetics is the effect that heredity has on a
population. What happens to that group of people over the course of a number of generations is
expressed mathematically.
There are three key elements of any population: size, density, and dispersion. Population size is
important to the groups’ ability to reproduce without a lot of inbreeding. Inbreeding can be the
downfall of a population if recessive traits, many of which are harmful, become a common
occurrence. Population density can affect the ability of individuals to reproduce, based on
whether they ever encounter another to mate with. Dispersion, or how populations are arranged,
can also affect populations.
Populations evolve by responding to their surroundings through natural selection. This change
actually occurs in the frequency of gene alleles in the population. William Castle, an American
scientist; Geoffrey Hardy, a British mathematician; and Wilhelm Weinberg, a German physician,
independently determined that the frequencies of genes in a population remain constant unless
certain forces act on the population. Dominant alleles will not replace recessive alleles, and the
ratio of heterozygous and homozygous individuals does not change over the course of several
generations. This theory has come to be known as the Hardy–Weinberg principle; it is the basis
of the study of population genetics.
The Hardy–Weinberg principle is normally stated as a mathematical equation:
p2 + 2pq + q2 = 1
The frequencies of the dominant and recessive alleles are represented by p and q, respectively.
For example, if a diploid individual has two alleles, “A” and “a”, at a particular locus, only three
possible genotypes can be the result: AA, Aa, and aa. The probability of receiving the “A” alleles
from both parents is p x p, or p2; for the “a” alleles, q x q, or q2. Those who received the Aa
combination are described by 2pq, since it is possible for the “A” or the “a” to come from either
parent, thereby doubling the chance.

Computer 8
To apply the principle, at least one of the allele frequencies must be known. For example, if the
frequency of the recessive allele for cystic fibrosis is one in 2,080 Caucasian North Americans, or
0.00048, this is equal to q2. After calculating the square root, q = 0.022. Then the frequency of
the dominant allele is calculated: since p + q = 1, p = 1 – q or p = 1 – 0.022, p = 0.978.
Having identified the values of p and q, the frequency of heterozygotes in the population can be
determined: 2pq = 2(0.978)(0.022) = 0.043. Therefore, 43 out of every 1,000 Caucasian North
Americans are heterozygous for cystic fibrosis.
If the relationship between p and q are constant through randomly mating generations, the
population is said to be in Hardy–Weinberg equilibrium; no evolution occurs. However, five
evolutionary forces act on a population to affect it: mutation, migration, non-random mating,
genetic drift, and natural selection. If any of these conditions are present, the proportions of
heterozygotes and homozygotes can differ. Therefore, the Hardy–Weinberg principle is a useful
tool for measuring the degree of genetic change or evolution occurring in a population.
Mutation is a fairly uncommon occurrence, which will not significantly change allele frequencies
itself, but is the main source of change and therefore evolution. Migration of individuals in or out
of a population is the cause of gene flow, which introduces new alleles to a group and removes
others. Non-random mating can be the result of either inbreeding or an organism’s ability to
choose a mate based on certain characteristics such as size, coloration, or lifestyle. In either case,
the proportion of homozygotes can increase, upsetting the equilibrium. Genetic drift refers to the
possibility that by chance, certain alleles could be eliminated from a population. For example,
two heterozygous parents, Aa, could possibly have two children, both AA, thus eliminating the
aa allele from the next generation. Normally this is balanced by similar events that might
eliminate the A allele from another family, but in small populations it is possible to lose the same
allele at a large number of loci, causing evolution to take place. Natural selection is the
predominant force in creating evolutionary change. An allele can increase or decrease in
frequency based on its effect on reproduction and survival. This can change from one generation
to the next, and from one area to another, since a trait that is harmful to its carrier in one
circumstance could be beneficial in another.
OBJECTIVES
• Investigate a genetically inherited trait and apply the Hardy-Weinberg Principle to a
population.
• Calculate allele frequencies and genotypes for a population using the Hardy-Weinberg
formula.
• Compare allele frequencies within the classroom to North American averages.
• Demonstrate the stability of allele frequencies over five generations in an ideal Hardy-Weinberg
population.
• Examine the effects of natural selection, heterozygous advantage, and genetic drift on allele
frequencies in a simulated mating exercise.
MATERIALS
PTC paper
control paper
4 index cards
coin

PROCEDURE
Part I Calculating Allele Frequencies Using the Hardy–Weinberg Principle

Testing different individuals’ ability to taste PTC (phenylthiocarbamide) is a good way to
demonstrate the Hardy–Weinberg principle. Homozygous-dominant (AA) and heterozygous (Aa)
individuals can taste this bitter chemical, although homozygous-recessive (aa) individuals
cannot. Use your class as a representative population to calculate the frequencies of the two
alleles with the Hardy–Weinberg equation
1. Obtain a piece of PTC test paper. Note: Use each strip of PTC and control test paper only
once. Do not share test papers with other students in your class.
2. Place it on your tongue and note whether you can detect a bitter taste. Note: Used test papers
may be disposed of with general waste.
3. Obtain a piece of control paper and place it on your tongue. Comparing the control paper
with the PTC paper will help determine whether you detected a taste on the PTC paper.
4. Record your results in Table 1 in the Analysis section.
5. Fill in the results for each individual in the class and enter the results in Table 2.
6. Tally the results for the entire class and calculate the frequencies for each allele, using the
Hardy-Weinberg equation, in the Analysis section. Be sure to show your work.
Part II Testing the Hardy–Weinberg Principle

Case A: Testing an Ideal Population
1. Obtain four index cards. Label two “A” and two “a”. These will be your haploid
chromosomes.
2. Randomly pair off with another student for “breeding”. Choose any other student; your
gender, and your partner’s, doesn’t matter in the simulation.
3. Turn your cards upside down and shuffle them. Turn over the top card in your pile. Pair this
card with your partner’s card; this will be the genotype of your first offspring. Record this on
your data sheet.
4. Turn over the next card in your pile. Pair this card with your partner’s card; this will be the
genotype of your second offspring. Record this on your data sheet.
5. Now begin the second generation, taking the genotype of your offspring. For example, if you
produced offspring with the genotype AA, begin the next generation with four A cards.
Record the results in the Analysis section of the lab.
6. Repeat the above procedure for the second generation. Record the genotypes in the Analysis
section.
7. Repeat the procedure for three more generations, for a total of five generations. Record the
genotypes for each.

Computer 8
8. Combine the genotype of your fifth generation results with the rest of the students’ fifth
generation results and enter the totals in the Analysis section.
9. Using the table in the Analysis section, calculate the allele frequencies after five generations
of random mating.
Case B: Selection
The previous exercise was conducted with ideal parameters. For a more realistic situation,
selection must be used. There is 100% selection against homozygous-recessive offspring. If
offspring are recessive (if they receive two mutated alleles), they will never live long enough to
reach a reproductive age; offspring that are either heterozygous or homozygous dominant will
survive long enough to reproduce.
10. Follow the same procedure as the previous exercise, with one difference: If offspring is
produced with the genotype aa, this offspring will not survive; eliminate the alleles from the
population. To maintain population size, you must produce two surviving offspring. If two
alleles are eliminated, draw two new alleles from the extra cards.
11. Repeat the procedure for a total of five generations, selecting against homozygous-recessive
offspring in each generation. Record the genotypes after every generation in the Analysis
section.
12. Combine your fifth generation results with the rest of the students’ fifth generation results
and record in the Analysis section.
13. Using the table in the Analysis section, calculate the allele frequencies after five generations
of random mating.
Case C: Heterozygote Advantage
The previous exercise showed how selection against homozygous-recessive individuals clearly
alters the allelic frequencies in a population. Another form of selection that operates within a
gene pool is diseases, such as a deadly form of malaria, that affect homozygous-dominant
individuals more severely than heterozygous individuals. The heterozygote is therefore favored in
a population.
14. Follow the same procedure, eliminating homozygous-recessive individuals as before. In

addition, if a homozygous-dominant individual is produced, flip a coin. If the result is heads,
the offspring dies; if it is tails, the offspring survives.
15. Repeat the procedure for a total of five generations. Record the genotypes after every
generation in the Analysis section.
16. Combine your fifth generation results with the rest of the students’ fifth generation results
and record in the Analysis section.
17. Continue the procedure for five more generations, for a total of ten generations, this time
starting with the genotypes from the end of the fifth generation. Record the genotypes in the
Analysis section.
18. Using the table in the Analysis section, calculate the allele frequencies after ten generations
of random mating.

Case D: Genetic Drift

Genetic drift is a phenomenon where an allele is lost solely from chance instead of through
selection. The most important factor in genetic drift is population size; smaller populations have
a much greater potential for genetic drift.
19. Your instructor will divide the class into several smaller populations. Within your smaller
population, follow the mating procedure, as in the first exercise, for a total of five
generations. Record the genotypes after every generation in the Analysis section.
20. Combine your group’s fifth generation results with those of the other small populations and
calculate the new allele frequencies.
ANALYSIS
Part I Calculating Allele Frequencies Using the Hardy–Weinberg Principle
Table 1
Taster Nontaster
PTC
Control
Table 2
Phenotypes Allele frequency based on the

Hardy-Weinberg equation
Tasters Nontasters
2 2 p q
(p +2pq) (q )
# % # %
Class
population

Computer 8
Part II Testing the Hardy–Weinberg Principle

Case A
Testing an Ideal Population
Initial Class Frequencies: AA:__________ Aa:__________ aa:__________
My Initial Genotype: ________
F1 Genotype: ________
Final Class Frequencies: AA:__________ Aa:__________ aa:__________
p:________ q:________
Number of A alleles present at the fifth generation
Number of offspring with genotype AA ____________ x 2 = __________ A alleles
Number of offspring with genotype Aa _____________x 1 = __________ A alleles
Total = __________ A alleles

p= TOTAL number of A alleles = _____________
TOTAL number of alleles in the population
Number of a alleles present at the fifth generation
Number of offspring with genotype aa ____________ x 2 = __________ a alleles
Number of offspring with genotype Aa ____________ x 1 = __________ a alleles
Total = __________ a alleles
q= TOTAL number of a alleles = _____________

TOTAL number of alleles in the population

Case B
Selection
Final Class Frequencies: AA: ________ Aa: ________ aa:__________
p:________ q:________
Case C
Heterozygote Advantage
Final Class Frequencies:

AA:__________ Aa:__________ aa:__________
(After five generations)
p:________ q:________
Final Class Frequencies:

AA:__________ Aa:__________ aa:__________
(After ten generations)
p:________ q:________

Computer 8
Case D
Genetic Drift
p:________ q:________
Final Class Frequencies: AA:__________ Aa:__________ aa:__________
QUESTIONS
1. Using the PTC tasting results for the entire class, how close were the frequencies of each
phenotype (taster vs. non-taster) to those found in the North American population? If there
was variation, what could have accounted for this?
2. In the first exercise in Part B, ‘Testing an Ideal Hardy-Weinberg Population’, what would be
the expected values of P and Q after the five generations?
3. How close were your class’s results to an ideal population.
4. What do you think would happen if you carried this simulation out for 10 more generations?
5. How do the frequencies of p and q compare after the factor of selection was added to the
simulation?
6. What do you think would happen if you carried this simulation out for another
10 generations?
7. Do you think that the a allele would ever be totally eliminated from the population? Why or
why not?
8. Suppose there was a medical advance that allowed most individuals with the double recessive
condition to survive and reach reproductive age. How would this affect the allele
frequencies?

9. Explain the difference in the results of the simulation showing selection to the simulation
favoring heterozygotes.
10. Why is the heterozygous condition important in maintaining genetic variation within a
population?
11. In the simulation demonstrating genetic drift, what do the resulting allele frequencies suggest
about population size as an evolutionary force?
12. You are a population geneticist and you have recently visited a remote Pacific island where
you have discovered a race of giant purple-skinned, seven-toed, three-horned dragons. In
examining the new race you have discovered that a small number of the dragons have only
six toes as opposed to seven and it seems to be a genetically inherited trait. After a population
survey, you have found that in a population of 1,378 dragons, 174 of them have only six toes.
Assuming the six-toed organisms are carrying a recessive trait, calculate the gene frequencies
and the percentage of homozygous dominant, heterozygous, and recessive individuals on the
island.
13. Below are four pie charts representing the percentage of homozygous dominant,
heterozygous, and recessive individuals within a population over a period of 400 years.
Examine the charts and explain below what you believe is happening within the population.
14. In this lab you examined the effects of selection, the heterozygous advantage, and genetic
drift on a population. Several other factors can affect population genetics, including mutation,
inbreeding, geographic isolation, and migration. Research one of these factors and explain
how it may affect a population over time.
15. Divide the class in half and discuss the following statement: “All mutations are detrimental to
a population.”

Experiment
TIPS
1. During Part II, the entire class will be used as a representative population. The four cards
each student uses, two “A” and two “a”, represent haploid chromosomes contributed by
parents in a simulated breeding exercise. Each parent begins with the genotype Aa, providing
initial genotype frequencies of 0.25 AA, 0.50 Aa, and 0.25 aa.
2. In Case B and C, since selection can lead to the elimination of certain alleles, it will be
necessary to have extra alleles (index cards) in the event of death of an offspring.
3. In Case D, divide the class into several smaller populations; for example, if you have a class
of 30 students, divide them into three groups of ten.
1. The class results should approximate the frequencies found in the North American
population. However, a limited sample size, for example a class of thirty students, is minute
when compared to the entire North American population and may lead to some statistical
variance.
2. Since you started with equal numbers of each allele and there were no outside influences such
as selection or genetic drift, ideally the frequency of alleles would remain the same after five
generations. If p + q = 1, remembering you started with equal numbers of each allele, then
p=0.5 and q=0.5.
3. The class results in should resemble an ideal population. Chance could lead to a slight
variation due to a limited population size but the should always remain close to the starting
frequencies.
4. Being an ideal population with no outside influences, the frequencies of p and q should
remain constant regardless of how many generations the simulation was carried on for.
5. The frequency of the p allele is much higher and the frequency of the q allele is lower.
Discrimination against the aa genotype resulted in a large decrease in the frequency of the a
allele.
6. Discrimination against the a allele would continue and each successive generation should
reduce the number of a alleles in the population.
7. Heterozygous (Aa) individuals survive to reproductive age so may still pass the a allele on to
successive generations. Though after hundreds of generations, the frequency of the a allele
may greatly diminish, it will never totally disappear.

Experiment 8
8. This would greatly reduce selection against the double recessive condition though some
selection would still occur. The a allele would still decrease in frequency, albeit at a much
slower rate.
9. The p allele frequency rapidly increased and the q allele frequency rapidly decreased when
selection against the recessive was the controlling factor. However, when selection favored
the heterozygote, it encouraged putting both the A and a allele back into the population. Since
some of the homozygous dominant individuals (AA) did have a chance to reproduce while
the recessive (aa) individuals did not, the a allele was still discriminated against. This would
cause the frequency of p to increase and q to decrease although it would occur at a much
slower rate than in the case showing selection only against the recessive condition.
10. Individuals with the heterozygous condition can contribute either allele to the subsequent
generation in a population. If an allele were totally eliminated then all individuals would only
be able to contribute only that allele, ending any opportunity for variation.
11. In a smaller population, there is a greater chance of a sampling error occurring. When
beginning with fewer individuals, any chance discrepancies in the initial gene frequencies
would only be amplified through successive generations. A much larger population has a
better chance of maintaining a stable (assuming no other selective factors are occurring)
balance of alleles within the population.
12. Using the Hardy- Weinberg equation, first determine the value of q2, the frequency of the
recessive condition:
q2 = 174/1,378 (recessive/total population) = 0.13 or 13% recessive
q = square root of 0.13 or 0.36
Since p+q = 1.0 then p = 1.0-q or p = 1.0-0.36 = 0.64
p2 = 0.64 X 0.64 = 0.41 or 41% homozygous dominant
2pq = 2(0.64)(0.36) = 0.46 or 46% heterozygous
13. The percentage of the recessive individuals is rapidly decreasing so there is obviously
selection against the recessive condition. However, the percentage of homozygous dominant
individuals in also decreasing so there is some selection against these individuals as well. The
percentage of heterozygotes is increasing through time indicating that there is an advantage to
having the heterozygous condition.

Computer
Transpiration
9
Water is transported in plants, from the roots to the leaves, following a decreasing water potential
gradient. Transpiration, or loss of water from the leaves, helps to create a lower osmotic
potential in the leaf. The resulting transpirational pull is responsible for the movement of water
from the xylem to the mesophyll cells into the air spaces in the leaves. The rate of evaporation of
water from the air spaces of the leaf to the outside air depends on the water potential gradient
between the leaf and the outside air.
Various environmental factors, including those conditions which directly influence the opening
and closing of the stomata, will affect a plant’s transpiration rate. This experiment will measure
transpiration rates under different conditions of light, humidity, temperature, and air movement.
The data will be collected by measuring pressure changes as the plant takes up water into the
stem.
OBJECTIVES
• Observe how transpiration relates to the overall process of water transport in plants.
• Use a Gas Pressure Sensor to measure the rate of transpiration.
• Determine the effect of light intensity, humidity, wind, and temperature on the rate of
transpiration of a plant cutting.
Pressure Sensor
Biology Gas
Figure 1

Computer 9
MATERIALS
computer metric ruler
Vernier computer interface masking tape
Logger Pro 100 watt light source
Vernier Gas Pressure Sensor plastic gallon size bag with twist tie
utility clamps heater, small electric
ring stand fan with slow speed
plant cuttings aerosol spray container or plant mister
plastic tubing clamps plastic syringe
dropper or Beral pipette ProScope (optional)
razor blade or scalpel
PROCEDURE
1. Position the ring stand, utility clamps, and Gas Pressure Sensor as shown in
Figure 1.
2. Prepare the plastic tubing.

a. Connect the plastic syringe to one end of a 36–42 cm piece of plastic
tubing.
b. Place the other end of the tubing into water and use the syringe to draw
water up into the tubing until it is full. Tap the tubing to expel any air
bubbles that form inside the tube.
c. Slip a plastic tubing clamp onto the tubing as shown in Figure 2. Figure 2
d. Bend the tubing into a U shape with both ends up. Remove the syringe, leaving the tubing
full of water.
3. Select a plant that has a stem roughly the same diameter as the opening of the plastic tubing.
Using a scalpel or razor blade, carefully cut the plant one inch above the soil. Place the plant
under water against a hard surface and make a new cut at a 45° angle near the base of the
stem.
4. Connect the plant to the tubing.

a. The plastic tubing has a white plastic connector at one end that allows you to connect it to
the valve on the Gas Pressure Sensor. Raise the end of
the tubing with the connector until you see water
beginning to drip out of the other end.
b. Carefully push the cut stem of the plant down into the
end of the tubing where the water is dripping out. Be
careful not to allow any air bubbles to form between
the cut portion of the stem and the water in the tube.
c. Push the plant down as far as it will go without
damaging the plant. At least one centimeter of the plant
stem should fit into the tubing. If the stem is too large
for the tubing, cut the stem at a higher point where it is
smaller.
d. Squeeze the tubing clamp shut as tight as possible as
shown in Figure 3. Figure 3

Transpiration
5. When the tubing clamp is shut tight, invert your plant cutting to check for any leaks. If water
does leak out, turn the plant right side up and try tightening the clamp further.
Important: Be sure the tubing is filled completely with water. The water column must be
flush with the stem. There should be no air visible at the base of the stem. If water moves
down the tube away from the stem after it has been inserted, check for a leak in the system.
6. Connect the plastic tubing to the sensor valve. Caution: Do not allow water to enter the valve
of the Gas Pressure Sensor.
7. Secure the plant in an upright position with the utility clamps as shown in Figure 1. It should
be positioned so that the cut stem is about 8 cm below the water level at the other end of the
tubing, as shown in Figure 1.
8. Place a mark on the tube at the starting water level to allow you to refill the tube to the proper
level when you repeat data collection.
9. Place your plant setup in an area where the wind, humidity, and temperature are reasonably
constant. This will be your control setup.
10. Allow the system 5 minutes to adjust to the environment. While the system is adjusting, set
up the computer.
11. Connect the Gas Pressure Sensor to the computer interface. Prepare the computer for data
collection by opening the file “09 Transpiration” from the Advanced Biology with Vernier
folder of Logger Pro.
12. Check the base of the plant stem in the water tube to make sure that no air bubbles or air
pockets have formed that will prevent the plant from taking up water. If an air pocket has
formed, refit the plant in the tubing before initiating data collection in Step 13.
13. After the plant has equilibrated for 5 minutes, click to begin data collection. Data will
be collected for 15 minutes.
14. When data collection has finished, find the rate of transpiration for your plant. To do this,
a. Move the mouse pointer to the point where the pressure values begin to decrease. Click
the mouse button and drag the pointer to the end of the data, then release the mouse
button.
c. Record the slope of the line, m, in Table 1 as the rate of transpiration for the control. Close
the floating box.
15. (Optional) Double click anywhere on the graph and enter “Transpiration: Control” as the
graph title. Print a copy of your graph. Enter your name(s) and the number of copies of the
graph.

Computer 9
16. Design an experiment to simulate one of the following environmental factors, as assigned by
your teacher:
• the effect of light intensity
• the effect of the wind blowing on the plant
• the effect of humidity
• the effect of temperature
• the effect of another self-identified environmental variable
Be sure to address the following questions in your design:

• What is the essential question being addressed?
• What assumptions are made about the system being measured?
• Can those assumptions be easily verified?
• Will the measurements provide the necessary data to answer the question under study?
17. After checking your procedure with your teacher, obtain the materials needed for the
experiment and perform the tests. Refill the water level in the tube to the same level marked
in Step 8. Record your values in Table 1.
PROCESSING THE DATA

1. Determine the surface area of all the leaves on your plant cutting by using one of the
following methods:
Method 1 – Using Logger Pro to Determine Leaf Surface Area
This method requires a ruler, a digital camera such as a ProScope HR with the 1–10X lens,
and Logger Pro software.
a. Carefully remove the leaves from the stem.
b. Select a sample leaf and place it on a flat surface with a ruler along the horizontal axis of
the leaf to provide scale. Place a clear Plexiglas plate over the leaf specimen (optional).
c. Select Video Capture from the Insert menu. Adjust the ProScope HR lens making sure that
both the leaf and the ruler are in focus. The ProScope handle should be parallel to the table
surface to avoid angular distortion.
d. Click to capture the leaf
image. Note: The captured image may
be hidden behind the Video Capture
window. Close the Video Capture
window.
e. Select Auto Arrange from the Page
menu. Note: If there are no analysis
buttons along the right side of the
captured image as seen in Figure 4,
double-click on the image. Select
Standard Analysis from the Picture
Analysis options and select OK.
Figure 4

Transpiration
f. Set the scale of the image by clicking Set

Scale, . Click and drag the cursor
between two distinct points on the ruler
separated by several centimeters. Enter
the distance between the points and the
units. Select OK.
g. Click Add Point, . Move your cursor to
the edge of the leaf and click to add a
point. Circumscribing the leaf with points
in a sequential clockwise manner as
shown in Figure 5. Points need to be
close enough to register all directional
changes along the leaf edge. Place the
final point directly on top of the starting Figure 5
point.
h. Click the Integral button, . The displayed integral value is equal to the surface area of
the leaf. Record this value in the Individual Leaf Surface Areas table below.
i. Repeat Steps 1a–h for each leaf.
j. Add the surface areas of all the leaves and record the total surface area in Table 1.
Individual Leaf Surface Areas

Leaf Surface Area Leaf Surface Area Leaf Surface Area Leaf Surface Area
2 2 2 2
Number (cm ) Number (cm ) Number (cm ) Number (cm )
Method 2 – Using Mass to Determine Leaf Surface Area
This method requires a balance.

a. Cut all the leaves (not stems) off your plant and determine their mass using a balance.
b. Estimate the total leaf surface area in cm2 for your plant by cutting out a section of leaf
5 cm × 5 cm.
c. Determine the mass for this leaf section and divide by 25 cm2 to find the mass of l cm2 of
leaf.
d. Divide the total mass of the leaves by the mass of l cm2 to find the total leaf surface area.
e. Record the calculated surface area in Table 1.
2. Calculate the rate of transpiration/surface area. To do this, divide the rate of transpiration by
the surface area for each plant. These rate values can be expressed as kPa/min/cm2. Record
the rate/area in Table 1.
3. Subtract the control (rate/area) value from the experimental value. Record this adjusted rate
in the last column of Table 1.
4. Record the adjusted rate for your experimental test on the board to share with the class.
Record the class results in Table 2 for each of the environmental conditions tested. If a
condition was tested by more than one group, take the average of the values and record in
Table 2.

Computer 9
5. Make a bar graph that shows the effect of different environmental conditions on the
transpiration of water in plant cuttings. Using the data in Table 2 plot the adjusted rate for
each test on the y-axis and the test label on the x-axis.
DATA
Table 1
Slope Surface area Rate/area Adjusted rate
Test 2 2 2
(kPa/min) (cm ) (kPa/min/cm ) (kPa/min/cm )
Experimental ______________
Control
Table 2
Class Data
Test Adjusted rate

2
(kPa/min/cm )
Light
Humidity
Wind
Temperature
QUESTIONS
1. How was the rate of transpiration affected in each of the experimental situations as compared
to the control?
2. Which variable resulted in the greatest rate of water loss? Explain why this factor might
increase water loss when compared to the others.
3. What adaptations enable plants to increase or decrease water loss? How might each affect
transpiration?

Transpiration
EXTENSIONS
1. Using a compound microscope, identify the vascular tissues of a plant stem. Describe the
function of each tissue type identified.
a. Obtain a section of stem from the plant you used during the transpiration experiment.
b. Using a nut-and-bolt microtome, carefully cut 6 cross sections of the plant stem. The cross
sections should be cut as thin as possible.
c. Place each of the cross sections in a dish or cup of 50% ethanol solution for 5 minutes.
d. Remove the cross sections from the alcohol and place them in a dish containing toludine
blue O stain for 5 minutes.
e. Rinse the cross sections with distilled water and mount them on a microscope slide with a
drop of 50% glycerin. Place a cover slip on the slide and examine the cross sections using
a compound microscope.
f. On a separate sheet of paper, make a drawing of the cross sections. Identify and label the
cell and tissue types described by your teacher.
2. Test cuttings from a variety of different plant species. How does each compare?
3. Count the number of stoma/cm2 for each of the plants in Extension 1. How does this relate to
the plant’s ability to transpire water?
4. Design an experiment to test for the variables in Question 3.

Experiment
Transpiration
3. You should leave water out overnight in a beaker or cup to allow any excess dissolved air to
escape. This will ensure that no air bubbles form in the tube at the cut end of the stem. If air
bubbles form, it may be necessary to restart your experiment. If bubbles do form, remove the
plant and tubing from the two utility clamps and allow the plant to hang towards the ground
with the other end of the tubing pointing up. Carefully tap on the sides of the tubing to loosen
any bubbles—they will float to the water’s surface at the other end. Once all bubbles are
removed, check the plant’s seal at the tube. Secure your plant in the tubing and restart the
data collection.
4. There is not always an immediate change in the transpiration rate. Allow the plant to spend a
few extra minutes under a particular condition before initiating data collection. This will give
the plant the necessary time to adjust. When the transpiration rate changes drastically the
stomata will close, decreasing the transpiration rate. If the data-collection length is extended,
you will be able to see on the graph when the stomata have closed and the rate slows down.
5. Many plants work well for this experiment. Plants that have been used include tomato,
strawberry, bean, geranium, cyclamen, and even honeysuckle. For best results, we recommend
using plants with numerous leaves. Tomato plants work very well and have been used to
collect the sample data for this activity. One possible extension of this experiment would be to
have the students use different plant species under similar conditions and evaluate how
different plants have adapted to prevent water loss.
6. The thick-wall plastic tubing that comes with the Gas Pressure Sensor is well suited for this
lab. The inner diameter of the tubing is 3 mm and may be too small for some plant specimens.
Science supply companies carry thick-wall plastic tubing, with a larger inner diameter, that
will work well on larger plant stems. They also sell tubing connectors that will allow you to
connect the larger tubing to the tubing provided with the Gas Pressure Sensor.
connections.
8. The Vernier Barometer sensor can also be used to perform this experiment. If you already
have a Barometer and wish to do this activity, you will need to order the following parts from
Vernier Software:
Pressure Sensor Accessories Kit (order code PS-ACC)
Plastic 3-Way Pressure Sensor Valve (order code PSV)
9. The plastic tubing clamps used in the student procedure may be purchased from Vernier
Software & Technology:
Plastic Tubing Clamps (order code PTC: package of 100)

Experiment 9
10. There are two methods for determining leaf area. If you have a ruler, a digital camera such as
a ProScope HR with the 1–10X lens, and Logger Pro software, use Method 1. Otherwise,
follow the Method 2 directions, which require the use of a balance.
11. If incandescent bulbs are unavailable, use halogen, compact fluorescent, or LED bulbs if you
want to study light. If you want to study heat, a heat lamp works well.
Ideas for Inquiry

researchable variables can be found in the Procedure.
SAMPLE DATA
Table 1
Adjusted rate
Test
(kPa/min/cm2)
Control –2.39 × 10-3

Light –5.63 × 10-3
–0.52 × 10
-3
Humidity
Wind –0.16 × 10-3
1. It is typically predicted that the light and wind will increase the rate of transpiration. This may
not be apparent until after correction for surface area differences. Sometimes the wind, if too
strong, may cause the leaves to droop or fold up, and in this case they may transpire less.
Stomates may close to counter the dehydration. If this happens, discuss the nature of science
experimentation, e.g., the expected may not always be the result. Usually, after correction for
surface area, the high humidity plant will transpire less than a control. A student may question
whether the light increased the temperature of the leaf. If the light was too close to the plant,
temperature may indeed be a variable without a control.
2. Answers will vary—usually the light will produce the greatest rate of water loss. High light
intensity increases water loss due to increased photosynthesis. Wind removes water vapor
from the surface of the leaf more rapidly. It may increase the evaporation rate by increasing
the gradient between water in the leaf air spaces and water vapor in the air.
3. Plants can increase or decrease water loss by
• closing the stomata during water stress.
• reducing the number of stomata.
• waxy cuticles.
• fleshy, thick leaves.
• hairy surfaces.
• reducing the overall leaf surface area.

Computer
Blood Pressure as a Vital Sign

10A
Blood pressure is a measure of the changing fluid pressure within the circulatory system. It varies
from a peak pressure produced by contraction of the left ventricle, to a low pressure, which is
maintained by closure of the aortic valve and elastic recoil of the arterial system. The peak
pressure is called systole, and the pressure that is maintained even while the left ventricle is
relaxing is called diastole (see Figure 1).
Mean arterial pressure (MAP) is not a simple average of the two pressures, because the duration
of diastole is twice that of systole. MAP is used by emergency room and intensive care unit
personnel as a measure of the adequacy of blood supplied to vital tissues (such as the brain, heart,
and kidneys) when the blood pressure is dangerously low.
Blood pressure is traditionally reported with the systolic pressure stated first and the diastolic
pressure stated second. In adults, 120/80 and below is considered normal blood pressure. High
blood pressure is 140/90 or above. The seriousness of low blood pressure, as well as the health
risks of high blood pressure (also called hypertension), have been elucidated over the past several
decades. High blood pressure is a major risk factor for a number of health problems including
strokes and congestive heart failure. Diet and exercise are beneficial, but many people require
medication for optimal blood pressure control.
In this experiment, you will examine your blood pressure using the Vernier Blood Pressure
Sensor. You will compare blood pressures taken before and after exposure to cold. The cold
stimulus activates the sympathetic nervous system, resulting in hemodynamic changes that
prepare the body for a “fight or flight” response (i.e., when fighting or running from danger).
The sensitivity of blood pressure to harmful external or internal injuries makes it useful as a vital
sign, an indicator of health, disease, excitement, and stress.
Figure 1

Computer 10A
OBJECTIVES
• Obtain graphical representation of blood pressure.
• Compare blood pressure before and after exposure to cold stimulus.
• Observe an example of sympathetic nervous system activation (“fight or flight” response).
MATERIALS
computer Vernier Blood Pressure Sensor
Vernier computer interface ice water bath
Logger Pro towel (paper or cloth)
PROCEDURE
Select one or more persons from your lab group to be the subject(s).
Part I Baseline Blood Pressure
1. Connect the Blood Pressure Sensor to the Vernier computer interface. There are two rubber
tubes connected to the pressure cuff. One tube has a black Luer-lock connector at the end and
the other tube has a bulb pump attached. Connect the Luer-lock connector to the stem on the
Blood Pressure Sensor with a gentle half turn.
2. Open the file “10A Blood Press Vital Sign” from the Advanced Biology with Vernier folder
of Logger Pro.
3. Attach the Blood Pressure cuff firmly around the upper arm, approximately
2 cm above the elbow. The two rubber hoses from the cuff should be
positioned over the biceps muscle (brachial artery) and not under the arm
(see Figure 2).
4. Have the subject sit quietly in a chair with his or her forearm resting on a
table surface. The person having his or her blood pressure measured must
remain still during data collection; there should be no movement of the arm
or hand during measurements.
5. Click to begin data collection. Immediately pump the bulb pump
until the cuff pressure reaches at least 160 mm Hg. Stop pumping. The cuff
will slowly deflate and the pressure will fall. During this time, the systolic, Figure 2
diastolic, mean arterial pressures, and pulse will be calculated by the
software. These values will be displayed on the computer screen. When the cuff pressure
drops below 50 mm Hg, the program will stop calculating blood pressure. At this point, you
can terminate data collection by clicking . Release the pressure from the cuff, but do
not remove it.
6. Enter the pulse and the systolic, diastolic, and mean arterial pressures in Table 1.

Part II Blood Pressure Response to Cold

8. Prepare an ice water bath for use in the next step. The subject will be instructed to place his
or her opposite hand (the one to which the Blood Pressure cuff is not attached) in the ice
water bath for 15 s.
9. Collect data to examine the body’s response to cold.

a. With the cuff still attached, have the subject from Part I put the hand of his or her
non-cuffed arm in the ice water bath.
b. As soon as the subject’s hand enters the ice water bath, click .
c. Pump the bulb until the cuff pressure reaches at least 160 mm Hg, then stop pumping.
d. When data have been collected for 15 s, have the subject remove his or her hand from the
ice water bath.
e. The systolic, diastolic, and mean arterial pressures will be calculated by the software.
These values will be displayed on the computer screen. When the blood pressure readings
have stabilized (after the pressure drops to 50 mm Hg), the program will stop calculating
blood pressure. At this point, you can terminate data collection by clicking .
Release the pressure from the cuff, and remove the cuff from the subject’s arm.
10. Enter the systolic, diastolic, and mean arterial pressures, and the pulse in Table 2.
DATA
Table 1–Baseline Blood Pressure
Systolic pressure Diastolic pressure Mean arterial pressure Pulse

(mm Hg) (mm Hg) (mm Hg) (beats/minute)
Table 2–Blood Pressure Response to Cold

DATA ANALYSIS
1. Describe the trends that occurred in the systolic pressure, diastolic pressure, mean arterial
pressure, and pulse with cold stimulus. How might these be useful in a “fight or flight”
response?
2. Vasovagal syncope is a condition in which severe pain or fright activates the parasympathetic
nervous system instead of the sympathetic nervous system, resulting in fainting. Keeping in
mind that the parasympathetic system causes a response opposite to that of the sympathetic
system, describe the hemodynamic changes that would explain this.

Computer 10A
3. As a vital sign, blood pressure is an indicator of general health. A high blood pressure
(140/90 or higher) increases the risk of cardiovascular disease and strokes. Collect the
systolic and diastolic pressures for the class and calculate the average for each. Rate the class
average blood pressure using the following scale:
Blood Pressure Category
140/90 or higher High
120–139/80–89 Pre-hypertension
119/79 or below Normal
EXTENSION
Blood pressure is traditionally obtained by using a stethoscope to listen to the brachial artery. The
pumping of air into the blood pressure cuff acts to stop the blood flow through this artery. As the
pressure is released, the blood again is allowed to flow. When the blood begins to flow,
pulsations can be heard through the stethoscope. The pressure in the cuff at that time can be
noted, and corresponds closely to the systolic blood pressure. As pressure continues to be
released from the cuff, the pulsations of the artery become less audible. The pressure at which
they disappear has been found to approximate the diastolic pressure. These sounds are known as
Korotkoff Sounds.
With a stethoscope, obtain the blood pressure of a classmate by listening for the appearance and
disappearance of pulsations as the pressure in the cuff is released. Compare this to the blood
pressure you obtained with the Vernier Blood Pressure Sensor.

Experiment

1. This experiment correlates with Lab 10: Exercise 10A in the 2001 College Board’s
AP Biology Lab Manual.
2. This experiment is written for computer and LabQuest only. EasyData does not support data
collection with a Blood Pressure Sensor.
3. Blood pressure may vary from reading to reading for a variety of reasons. It is normal for
blood pressure to vary with the time of day or to be elevated in times of stress. For this
reason, single blood pressure readings are not adequate for a diagnosis of abnormal blood
pressure. It is normal for health care professionals to repeat blood pressure measurements
over a period of several weeks before diagnosing high blood pressure.
4. Alert the students to be sure to secure the cuff around the subjects’ arms very snugly. A
loosely secured cuff will return erratic data.
5. The duration of the experiment is set longer than necessary in order to accommodate
individual differences in the determination of blood pressure. When the blood pressure
readings have stabilized (after the pressure drops to 50 mm Hg), data collection can be
terminated and pressure released from the cuff.
6. If the pressure does not reach 50 mm Hg by the time data collection

ends, the values calculated by the program are not accurate
representations of the subject’s blood pressure. The pressure release
valve is set to release at a rate of 3.0 mm Hg/second on an arm of 32 cm
in circumference at sea level. If you are taking measurements above
6,000 ft, or for arms much larger or smaller, the pressure release valve
will need to be adjusted.
With the bulb in hand and the hose leading away from you, place a screwdriver into the metal
slot on the top of the release valve. To increase the rate of exhaust, turn the screwdriver
clockwise. To decrease the rate of exhaust, turn the screwdriver counter-clockwise.
High Elevation Adjustments
Above 6,000 ft, increase the exhaust rate by turning the valve clockwise about one half turn.
After making adjustments, verify that the pressure reaches 50 mm Hg by the time data
collection ends.
Arm Size Adjustments
If the subject’s arm is greater than 32 cm in circumference, increase the exhaust rate by
turning the valve clockwise. Turn the valve counter-clockwise for smaller arms. After
making adjustments, verify that the pressure reaches 50 mm Hg by the time data collection
ends. Note: If arm circumference is much greater or smaller than 32 cm, adjustments with the
valve may not be sufficient. A smaller cuff (order code CUFF-SM ) is available for subjects
with arm circumferences of 18–27 cm. For subjects with arm circumferences greater than
39 cm order a larger cuff (order code CUFF-LG).
7. In Logger Pro, graphs of oscillatory amplitude are included to aid in assuring that the
pressure sensor is sending enough data to correctly calculate blood pressure.

Experiment 10A
Ideas for Inquiry

• What is the effect of exercise on blood pressure?
• What is the effect of caffeine consumption on blood pressure?
• How does body position affect blood pressure?
• What effect does eating certain foods have on blood pressure?
• How does time of day affect blood pressure?
SAMPLE DATA
Table 1–Baseline Blood Pressure

105 69 82 77
Table 2–Blood Pressure Response to Cold

112 73 88 74
Baseline and response to cold data for cuff pressure and oscillatory amplitude

Teacher Information
ANSWERS TO THE DATA ANALYSIS QUESTIONS

1. Cold exposure caused discomfort or mild pain, which led to activation of the sympathetic
nervous system. Sympathetic nervous system activation increases heart rate and peripheral
vascular resistance (by constricting arterioles), which leads to an elevation of systolic,
diastolic and mean arterial blood pressures.
2. Sudden activation of the parasympathetic nervous system has the opposite effect of
sympathetic activation. The heart rate may slow significantly. Dilatation of peripheral blood
vessels leads to pooling of blood in the legs and lack of blood return to the heart. These two
factors together cause a precipitous fall in blood pressure, lack of adequate blood supply to
the brain, and fainting.
3. A discussion of the general health and well-being of the students in the class (as relates to
blood pressure) would be an appropriate conclusion to this experiment.

Computer
Heart Rate and Physical Fitness

10B
The circulatory system is responsible for the internal transport of many vital substances in
humans, including oxygen, carbon dioxide, and nutrients. The components of the circulatory
system include the heart, blood vessels, and blood. Heartbeats result from electrical stimulation
of the heart cells by the pacemaker, located in the heart’s inner wall of the right atrium. Although
the electrical activity of the pacemaker originates from within the heart, the rhythmic sequence of
impulses produced by the pacemaker is influenced by nerves outside the heart. Many things
might affect heart rate, including the physical fitness of the individual, the presence of drugs such
as caffeine or nicotine in the blood, and the age of the person.
As a rule, the maximum heart rate of all individuals of the same age and sex is about the same.
However, the time it takes individuals to reach that maximum level while exercising varies
greatly. Since physically fit people can deliver a greater volume of blood in a single cardiac cycle
than unfit individuals, they can usually sustain a greater work level before reaching the maximum
heart rate. Physically fit people not only have less of an increase in their heart rate during
exercise, but their heart rate recovers to the resting rate more rapidly than unfit people.
In this experiment, you will evaluate your physical fitness. An arbitrary rating system will be
used to “score” fitness during a variety of situations. Tests will be made while in a resting
position, in a prone position, as well as during and after physical exercise.
Important: Do not attempt this experiment if physical exertion will aggravate a health problem.
Inform your instructor of any possible health problems that might be affected if you participate in
this exercise.
OBJECTIVES
• Determine the effect of body position on heart rates.
• Determine the effect of exercise on heart rates.
• Determine your fitness level.
• Correlate the fitness level of individuals with factors such as smoking, the amount of daily
exercise, and other factors identified by students.

Computer 10B
MATERIALS
computer stepping stool, 45 cm (18 inches) high
Vernier computer interface dropper bottle with saline solution
Logger Pro (only for use with the Exercise HRM)
Vernier Hand-Grip Heart Rate Monitor or
Vernier Exercise Heart Rate Monitor
PROCEDURE
1. Each person in a lab group will take turns being the subject and the tester. When it is your
turn to be the subject, your partner will be responsible for recording the data on your lab sheet
2. Set up the Heart Rate Monitor. Follow the directions for your type of Heart Rate Monitor.
Using a Hand-Grip Heart Rate Monitor
a. The receiver and one of the handles are marked with a
white alignment arrow as shown in Figure 1. Locate
these two arrows.
b. Have the subject grasp the handles of the Hand-Grip
Heart Rate Monitor so that their fingers are in the
reference areas indicated in Figure 2. Hold the handles
vertically.
c. Have someone else hold the receiver near the handles
so that the two alignment arrows are pointing in the
same direction and are at approximately the same Figure 1 Figure 2
height as shown in Figure 1. Note: The receiver must
stay within 60 cm of the handles during data collection.
Using an Exercise Heart Rate Monitor
a. Depending upon your size, select a small- or large-size elastic strap. Secure one of the
plastic ends of the elastic strap to the transmitter belt. It is important that the strap provide
a snug fit of the transmitter belt.
b. Wet each of the electrodes (the two textured oval areas on the
underside of the transmitter belt) with 3 drops of saline solution.
c. Secure the transmitter belt against the skin directly over the base
of the rib cage (see Figure 3). The POLAR logo on the front of
the belt should be centered. Adjust the elastic strap to ensure a
tight fit.
d. Take the receiver module of the Heart Rate Monitor in your
right hand. Note: The receiver must stay within 60 cm of the
handles during data collection. Figure 3
3. Prepare the computer for data collection by opening the file “10B
Heart Rate & Fitness” from the Advanced Biology with Vernier folder of Logger Pro.
4. To determine that everything is set up correctly, click to begin monitoring heart rate.
Note that there may be up to a 30 second delay before data are seen. The readings should be
within the normal range of the individual, usually between 55 and 80 beats per minute. Click
when you have determined that the equipment is operating properly, and proceed to
Step 5.

Standing heart rate

5. Click to begin monitoring heart rate. Stand upright for 2 minutes.
6. Record the resulting heart rate in Table 6.
7. Use the resulting heart rate to assign fitness points based on Table 1 and record the value in
Table 6.
Table 1: Standing Heart Rate

Beats/min Points Beats/min Points
60–70 12 101–110 8
71–80 11 111–120 7
81–90 10 121–130 6
91–100 9 131–140 4
Reclining heart rate

8. Recline on a clean surface or table for 2 minutes. Note: If using the Hand-Grip Heart Rate
Monitor, remember to move the receiver along with the handles to keep the arrows aligned.
9. Record the resulting heart rate in Table 6.
10. Assign fitness points based on Table 2 and record the value in Table 6.
Table 2: Reclining Heart Rate

Beats/min Points Beats/min Points
50–60 12 81–90 8
61–70 11 91–100 6
71–80 10 101–110 4
Heart rate change from reclining to standing

11. Stand up next to the lab table.
12. Immediately record the peak heart rate in Table 6.
13. Subtract the reclining rate value in Step 9 from the peak heart rate after standing to find the
heart rate increase after standing.
14. Locate the row corresponding to the reclining heart rate from Step 9 in Table 3.
15. Use the calculated heart rate increase after standing (from Step 13) to locate the proper
column for fitness points in Table 3. Record the fitness points in Table 6.

Computer 10B
Table 3
Reclining rate Heart rate increase after standing
beats/min 0–10 11–17 18–24 25–33 34+
50–60 12 11 10 8 6
61–70 12 10 8 6 4
71–80 11 9 6 4 2
81–90 10 8 4 2 0
91–100 8 6 2 0 0
101–110 6 4 0 0 0
16. Rest for 2 minutes. Click to end data collection. When the rest period is over, click
to begin data collection.
Step test
17. Before performing the step test, record the subject’s heart rate (Pre-exercise) in Table 6.
18. Perform a step test using the following procedure:
a. Place the right foot on the top step of the stool.
b. Place the left foot completely on the top step of the stool next to the right foot.
c. Place the right foot back on the floor.
d. Place the left foot completely on the floor next to the right foot.
e. This stepping cycle should take 3 seconds to complete.
19. When five steps have been completed, record the heart rate in Table 6. Quickly move to
Step 20.
Recovery rate
20. With a stopwatch or clock, begin timing to determine the subject’s recovery time. During the
recovery period, the subject should remain standing and relatively still. Monitor the heart rate
readings and stop timing when the readings return to the pre-exercise heart rate value
recorded in Step 17. Record the recovery time in Table 6.
21. Click to end data collection.
22. Locate the subject’s recovery time in Table 4 and record the corresponding fitness point value
in Table 6. If the subject’s heart rate did not return to within 10 beats/min from their pre-
exercise heart rate, record a value of 6 points.
Table 4
Time (sec) Points
0–30 14
31–60 12
61–90 10
91–120 8

Step test for endurance

23. Subtract the subject’s pre-exercise heart rate (from Step 17) from his or her heart rate after
5 steps of exercise. Record this heart rate increase in the endurance row of Table 6.
24. Locate the row corresponding to the pre-exercise heart rate in Table 5 and use the heart rate
increase value to determine the proper fitness points. Record the points in the Endurance row
of the Points column in Table 6.
Table 5
Pre-exercise Heart rate increase after exercise
heart rate 0–10 11–20 21–30 31–40 41+
60–70 12 12 10 8 6
71–80 12 10 8 6 4
81–90 12 10 7 4 2
91–100 10 8 6 2 0
101–110 8 6 4 1 0
111–120 8 4 2 1 0
121–130 6 2 1 0 0
131+ 5 1 0 0 0
25. Total all the fitness points recorded in Table 6. Determine the subject’s personal fitness level
using the scale below.
Low Average Exceptional
Fitness Fitness Fitness
20 30 40 50 60
DATA
Table 6
Condition Rate or time Points
Standing heart rate beats/min
Reclining heart rate beats/min
Reclining to standing beats/min
Pre-exercise heart rate beats/min
After 5 steps beats/min
Recovery time seconds
Endurance beats/min
Total points:

Computer 10B
QUESTIONS
1. How did your heart rate change after moving from a standing position to a reclining position?
Is this what you expected? How do you account for this?
2. How did your heart rate change after moving from a reclining position back to a standing
position? Is this what you expected? How do you account for this?
3. Predict what your heart rate might be if you had exercised for twice the length of time that
you actually did. Explain.
4. How does your maximum heart rate compare to other students in your group. Is this what you
expected? How do you account for this?
5. Why would athletes need to work longer and harder before their heart rates were at the
maximum value?
6. In examining the results of your physical fitness test, were you surprised by any of the
findings? If you were, how might you explain them? Based on the results of the test, what
behaviors in your life would you continue to practice? What behaviors might you think about
changing?
7. Current research indicates that most heart attacks occur as people get out of bed after sleep.
Account for this observation.
EXTENSIONS
1. Using a sphygmomanometer, learn how to measure blood pressure. Compare a person’s
blood pressure when reclining, to that of the same person immediately after standing from a
reclined position. Relate the change in blood pressure to the heart rate values measured when
going from reclining to standing.
2. Design an anonymous survey to be taken by each member of your class. In the survey, ask
questions that you think might influence the test results. Examples might include:
• Did you have more than six hours of sleep last night? Gender? Age?
• Do you smoke? If so, how many packs per week do you smoke?
• What was your total number of fitness points?
3. Try to determine whether any of the variables from your survey show a statistical link to
fitness. You may want to use a statistical T-Test to determine whether a relationship between
the variable and physical fitness is due to chance.

Experiment

1. This experiment correlates with Lab 10: Exercise 10B in the 2001 College Board’s AP
Biology Lab Manual.
3. This experiment works equally well with either a Hand-Grip Heart Rate Monitor or an
Exercise Heart Rate Monitor.
4. The receiver module of either type of Heart Rate Monitors will receive signals from the
closest transmitter source. To avoid confusion or erroneous readings, have the test subjects
from different lab teams stay at least 2 m apart.
5. Computer monitors can be a source of electrical interference. Keep the receiver module of the
Heart Rate Monitor as far as possible from any computer monitors in the class.
6. It is possible to alter your heart rate by simply decreasing your respiratory rate and relaxing.
Encourage students to stay alert and to breathe normally.
7. The Exercise Heart Rate Monitor includes a transmitter belt, receiver module, large elastic
strap, and small elastic strap.
8. It is important to have good contact between the transmitter belt and the test subject when
using the Exercise Heart Rate Monitor. It is very important that the belt fit snugly, but not too
tight. Both electrodes should be wet with either saline solution or contact lens solution. A 5%
salt solution works well and can be prepared by adding 5 g of NaCl per 100 mL of solution.
Typical symptoms of inadequate contact with the electrodes are a noisy signal with erroneous
peaks, missing heart beat readings, or a flat-line display. If the students receive a flat reading
with no heart rate detected, have them move the transmitter and the receiver closer together.
The range of the transmitter in the chest belt is 80 cm.
Ideas for Inquiry

• What is the effect of caffeine consumption on heart rate?
• What effect does eating certain foods have on heart rate?
• How does time of day affect heart rate?
• What is the effect of stress (i.e., being frightened) have on heart rate?

Experiment 10B
SAMPLE RESULTS
Sample data from two students are listed below. The first student was a 17 year old male and the
second was a 16 year old female.
Table 6
Sample Student Data
Condition Rate, Student 1 Points Rate, Student 2 Points

(beats/min) (beats/min)
or time or time
Standing heart rate 73 11 93 9
Reclining heart rate 54 12 69 11
Reclining to standing 69 11 84 8
Pre-exercise heart rate 72 93
After 5 steps 87 116
Recovery rate 57 s 12 98 s 10
Endurance 14 10 23 2
Total 56 40
1. The heart rate generally lowers when a student moves from a standing position to a reclining
position. The forces of gravity do not have to be overcome for blood to flow while in a
reclining position.
2. The heart rate generally increases when a student moves from a reclining position to a
standing position. The forces of gravity do have to be overcome while in a standing position.
3. The heart rate generally increases when a student exercises twice as long. It will not increase
to twice the rate, however, because the heart will adjust to the new stress and increase the
blood flow to meet the body’s needs. The blood flow is proportional to the heart rate. When
the blood flow is appropriate, the heart rate will no longer continue increasing.
4. Answers will vary. Factors such as weight, regular exercise, health, etc., may play a part in
determining the maximum heart rate of a student.
5. An athletes heart is more efficient at moving blood through the body. Each contraction of an
athlete’s heart moves a greater volume of blood than an average individual. More blood per
contraction means more oxygen for the body’s cells. Because of this athletes must work
harder to increase their heart rate to its maximum values.
7. The heart rate increases significantly when an individual moves from a reclining position to a
standing position. The force of gravity on the blood makes the heart work harder, as in
Question 2. This increased stress might provoke a heart attack in susceptible people.

Computer
Animal Behavior
11
Perhaps one of the most difficult fields of biology to study is ethology, the study of animal
behavior. Observation of a behavior is simple; interpreting what has been observed requires more
effort; therefore, more than any other division of research in biology, ethology calls for patience,
objectivity, and imagination.
It is important for an ethologist to investigate and interpret the various animal behaviors observed
in terms of the animals’ reaction to their own environment. It is too easy to view animal behavior
in terms of human habits; if a behavior seems to emulate a behavior that humans perform, it is
interpreted as a human interaction. This anthropomorphic view (anthro = human, morph =
change)—bestowing human traits and motivation on animals’ behavior—can lead to inaccurate
interpretations of the animals’ behavior.
For this reason, ethologists assess various behaviors by placing documented behavior in simple
categories. Once the behaviors in these categories are tallied, ethologists can determine the entire
lifestyle of the organism, gaining a complete picture of the animal’s existence. Ethology, then,
can be considered a unique branch of biology, investigating not only what happens in an animal’s
existence, but also why it happens.
In every ecosystem, organisms are influenced by limiting factors—biotic or abiotic factors which
regulate the maximum size of a given population—and a relatively narrow range of
environmental conditions which are favorable to them and their offspring. Since most organisms
cannot change the nature of their environment, they must position themselves in an environment
with favorable conditions. This behavior is called habitat selection.
Many organisms exhibit a tactic response to these environmental factors. Tactic responses may
be positive, toward a favorable environment, or negative, away from an unfavorable
environment. These tactic responses enable an organism to locate prey, avoid predation, seek
shelter, or avoid a toxic environment—all vital to habitat selection.
An animal’s orientation behavior, which allows an animal to be placed in a favorable

environment, occurs in two ways: taxis and kinesis. Taxis is a deliberate movement toward or
away from a stimulus. Kinesis, on the other hand, is a random movement that is not oriented
toward or away from a stimulus. A movement classified as taxis defines the physiological needs
of the organism, its evolutionary history, its nervous system, etc. For example, an insect found
around rotting fruit has most likely responded to a very different environmental stimulus than one
found around a decaying corpse.
Agonistic behavior is characterized as a response, either aggressive or submissive, to another

organism. These behaviors can range from outright confrontation to a simple display of
submission or superiority.
In many ways, mating behaviors are the most complex to observe. Animals have evolved a
complex series of behaviors to find a mate, court, and copulate. It is crucial for a female to select
a male that is well adapted; this ensures that the genes will be passed on to a suitable offspring,
who will in turn pass on his or her genes.

Computer 11
Courtship in Drosophila
Drosophila melanogaster, the common fruit fly, was first used in genetic experiments in 1907 by
T.H. Morgan of Columbia University, and has been a staple of genetic research ever since.
Drosophila specimens are well suited to investigations into Mendelian patterns of inheritance;
they are small, produce large numbers of offspring, have many easily discernible mutations, have
only four pairs of chromosomes, and complete their entire life cycle in approximately 12 days.
Additionally, Drosophila are relatively easy to maintain, as they are hardy and have simple food
requirements.
Since the fruit fly was selected for study nearly a

hundred years ago, a great deal has been learned about its
genome. In fact, the first chromosome map of any kind
was constructed to detail the fruit fly. Chromosomes 1
(the X chromosome), 2, and 3 are very large, and the Y
chromosome—number 4—is extremely small.
Thousands of genes reside on these four chromosomes,
many of which are universal in nature, existing in most
eukaryotic forms, including humans. The similarities
between the Drosophila genome and those of other
species is helpful in determining patterns of evolution
and species divergence.
Drosophila embryos develop in the egg membrane. The

egg hatches and produces a larva which feeds by
burrowing through the medium. The larval period
consists of three stages, or instars, the end of each stage
marked by a molt. The first instar is the newly hatched
larva; the third instar is the final larval stage, where the
larva may attain a length of 4.5mm. Near the end of the
larval period, the third instar larva will crawl up the sides
of the culture vial, attach themselves to a dry surface (the
jar, the filter paper, etc.) and form pupae. After a period
of time the adults emerge.
It takes one or two days for Drosophila eggs to hatch into larvae, four to five days for the larvae
to enter pupae, and four days for the pupa stage. The duration of these stages, however, vary with
the temperature; at 20°C the average length of the egg–larval period is eight days, while at 25°C
it is reduced to five days. Thus at 25°C the life cycle may be completed in about 10 days; at
20°C, 15 days are required.
Drosophila Life Cycle

Different body features characterize the male and female flies. The females
are slightly larger and have a light-colored, pointed abdomen. The abdomen
of the males will be dark and blunt. The male flies also have dark bristles on
the upper portion of the forelegs, which are known as sex combs.
Sex combs on
male foreleg

Animal Behavior
Mating in Drosophila melanogaster follows a strict behavioral pattern, which generally occurs in
six phases:
1. Orientation: The male consistently positions himself near the female.
2. Wing Vibration: The male produces the equivalent of a “song” by vibrating his wings.
3. Licking of the Female Genitalia
4. Attempted Copulation
5. Copulation
6. Rejection: The female signals that she is no longer receptive by extruding her ovipositor
(egg-laying structure).

Computer 11
OBJECTIVES
• Observe and note general behavior characteristics of pillbugs.
• Hypothesize as to whether pillbugs have adapted to perceive and react to certain
environmental changes.
• Design an experiment to determine how pillbugs respond to environmental changes.
• Examine similarities and differences between male and female Drosophila.
• Observe courtship and mating rituals between male and female Drosophila as an example of a
strict behavioral pattern.
• Hypothesize as to whether fruit flies respond to certain environmental changes.
MATERIALS
Part I Materials Part II Materials
10 pillbugs vial of collected Drosophila males
1% hydrochloric acid solution vial of collected Drosophila virgin females
2% potassium hydroxide solution thermo-anesthetizer
animal behavior tray petri dish
2 filter papers camel’s hair brush
camel’s hair brush magnifier
masking tape
magnifier
PROCEDURE
Part I Behavior of Pillbugs

A. General Observations
1. To become familiar with the organisms, place several pillbugs in the behavior tray and
carefully observe them for at least 10 minutes. In Table 1 in the Analysis section, document
any behaviors you see. Remember to document even the seemingly unimportant behaviors.
Try to document the behaviors observed in chronological order. Note: Do not disturb the
pillbugs; shaking or tipping the tray will cause unnatural behavior in the pillbugs.
2. In Table 2 in the Analysis section, sketch a pillbug. Label any structures that you recognize.
B. Taxis
3. Place one piece of masking tape on either side of the behavior tray and label one side A, the
other B.
4. Place five pillbugs in each chamber of the tray.
5. Every minute for 10 minutes, count the number of pillbugs in each chamber.
6. Record your observations in Table 3 in the Analysis section.
7. Calculate the average number of pillbugs in each chamber in the 10 minute time period. Enter
the results in Table 3.

Animal Behavior
8. Using the data from every group in the class, calculate the class average for number of
pillbugs in each chamber in a 10 minute time period. Enter the results in Table 3.
C. Experiment Formulation
9. Your instructor will assign you one of three environmental factors that affect pillbug
behavior. These environmental conditions elicit different types of pillbug taxis: phototaxis,
chemotaxis, or hydrotaxis.
• Phototaxis: The orientation of an organism in relation to the presence of light. Movement
toward a light source is positive phototaxis; movement away from light is negative
phototaxis.
• Chemotaxis: The orientation of an organism in relation to the presence of a particular
chemical. Chemotaxis is universal in protozoa and most organisms; they avoid most
chemicals and exhibit a positive taxis to some weak acids.
• Hydrotaxis: The orientation of an organism in relation to the presence of water.
10. Formulate a hypothesis regarding environmental preferences and how the pillbugs may react
to different conditions. Enter your hypothesis in the Analysis section.
11. Design an experiment to test the environmental factor you were assigned. Explain your
experimental setup in the Analysis section.
• Phototaxis: To test phototaxis, vary the amount of light the pillbugs are exposed to and
record their responses.
• Chemotaxis: To test chemotaxis, simulate an alkaline or acidic environment—place several
drops of either a weak acid or weak base on a filter paper on one side of the behavior tray.
• To test hydrotaxis, simulate a moist environment—place several drops of water on a filter
paper on one side of the behavior tray.
12. Check with your instructor before proceeding. Once your instructor has approved of your
experiment, collect the data and enter it in Table 4 in the Analysis section. Note: Be sure that
there is only one possible variable present in your experiment. Multiple variables will
invalidate the experiment.
13. Draw a graph to illustrate the data you collected.

14. Using your graph and data from both the preliminary investigation and the experiment you
designed, discuss your general findings and draw a conclusion based on your experiment.
15. Discuss your results with the rest of the class.
Part II Courtship in Drosophila

1. Obtain a vial of virgin female flies and a vial of male flies.
2. While in the vial, observe the flies’ traits, particularly body features that distinguish males
and females. Use a hand magnifier or stereoscope to assist in your observations. Record the
physical features of each in Table 5 in the Analysis section.

Computer 11
3. Anesthetize the flies by placing the vials in a refrigerator or icebath for 10 minutes. After the
flies are anesthetized, gently tap several male and female flies into a Petri dish. Cover the
dish.
4. Observe the flies’ interaction with each other. Use a hand magnifier or stereoscope for
detailed observations. Keep an accurate record of the courtship behavior sequence and
duration of each phase. Note: It would be easiest to work in pairs, with one team member
observing and dictating the behavior and the other team member recording what is observed.
5. Record the courtship behaviors of both the male and female flies in Table 6 in the Analysis
section. Remember to record all observed behaviors, even seemingly insignificant ones.
6. Read the information on the Drosophila courtship rituals below. Note which behaviors you
were able to observe and which behaviors you may have missed.
Male
• Vibrating Wings: Extends one or both wings, rapidly moving them up and down.
• Waving: Holds a wing at a 90° angle from his body, then relaxes it.
• Tapping: Strikes or taps the female with a foreleg.
• Licking: Licks the female’s genitals, on the rear of the abdomen.
• Circling: Postures and circles the female. This is usually done when she is not receptive.
• Stamping: Stamps his foreleg but does not strike the female.
Female
• Extruding: Extends her ovipositor, a tubelike structure, from her genitalia. This is often
done to prevent copulation.
• Decamping: Avoids the male.
• Depressing: Blocks access by pressing her wings down and curving her abdomen down.
• Ignoring: Ignores the male.
7. Repeat the experiment with new flies. Add to the list any behaviors you may have missed the
first time.
8. Pool the class’s data. Develop a series of behaviors that occur in each of the six phases to put
together a pattern of courtship and copulation behaviors. Keep in mind that many behaviors
are innate (inborn), and some are random.
B. Experiment Formulation
The courtship of Drosophila is affected by a number of factors. The female, once fertilized, stays
fertilized for life. Because of this, competition for females is very intense. As in all insects,
pheromones influence a female’s choice of male to mate with; therefore, the introduction of these
pheromones by the male is crucial to his success in convincing a female to mate with him
9. Choose one of the following questions, or devise your own, and design an experiment to find
the answer.
• Will non-virgin females allow mating?
• Will non-virgin males attempt to mate?
• Will males compete for a virgin female?
• Will one male be successful over another, same male in two different matings?
• Will a female allow mating if a male cannot perform the courtship behaviors?

Animal Behavior
10. Formulate a hypothesis regarding behavior under the conditions you are choosing to test.
Enter your hypothesis in the Analysis section.
11. Design an experiment to test the conditions selected. Explain your experimental setup in the
Analysis section.
12. Check with your instructor before proceeding. Once your instructor has approved of your
experiment, collect the data and enter it in Table 4 in the Analysis section. Note: Be sure that
there is only one possible variable present in your experiment. Multiple variables will
invalidate the experiment.
ANALYSIS
Part I Behavior of Pillbugs
Table 1
General Observations of Pillbug Behavior

Computer 11
Table 2
Pillbug Drawing
B. Pillbug Taxis
Table 3
Pillbug Taxis
Time (min) # Pillbugs in Side A # Pillbugs in Side B
10
Avg.
Class avg.

Animal Behavior
C. Experiment Formulation
Hypothesis:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
Design:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
Table 4
Pillbug Taxis with Altered Environmental Conditions
Condition Tested:________________________________
Time # Pillbugs in Side A # Pillbugs in Side B

(min)
10
Conclusion:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
Graph your results as directed by your instructor.

Computer 11
II Courtship in Drosophila
Table 5
Characteristics of male and female Drosophila
Drosophila Characteristics
Male
Female
Table 6
Mating behaviors of male and female Drosophila
Drosophila Characteristics
Male
Female
B. Experiment Formulation
Hypothesis:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
Design:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

Animal Behavior
Drosophila Observations
Male
Female
Conclusion:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
QUESTIONS
1. Below are some structural features of pillbugs. Discuss as a class what advantages you think
they might offer the organism.
Multi-segmented body
Antennae
Color
2. In Part I, B of the experiment, you placed pillbugs in a chamber and acquired data. What do
you think was the purpose of this experiment? What numbers did you expect? Why?
3. In Part I, C, you tested your hypothesis with regards to pillbug behavior and environmental
preference. Did your conclusions agree with your hypothesis? Are you confident with your
conclusions or would you need to test more factors?
4. Your teacher wants you maintain some pillbugs in the classroom and has asked you to set up
a terrarium to keep them in. What would you include in the terrarium to insure they have the
best chance of survival?
5. Would you describe the pillbugs’ response to moisture as kinesis or taxis? Why?

Computer 11
6. In Part II, B of the lab, how did your initial observations compare to the list of Drosophila
courtship behavior? Was there anything you observed the second time that you missed the
first time?
7. Why do you think you were provided with virgin Drosophila females?
8. Why is it necessary for a scientist to have good observational skills and maintain a sense of
objectivity when observing animal behavior?
9. While you were observing the various behaviors throughout the lab, do you think you
remained objective? What did you learn about yourself as a scientist?
10. In this lab you investigated behavior in two different terrestrial organisms. Pick an aquatic
organism and design an experiment to test a behavior of the organism. State what you would
like to test and explain or draw your experimental setup below.
11. As you learned in Part II, B of the experiment, Drosophila courtship and mating follows a
strict set of behaviors. Name something in which you engage that also follows a strict set of
behaviors.

Experiment
Animal Behavior
PRE-LAB PREP
Note: Virgin females will not mate until approximately 12–15 hours after emergence. After
separating virgin female and male flies, try to allow 24 hours of isolation before proceeding with
the courtship observations.
1. Prepare culture vial by placing approximately one tablespoon of medium in the bottom of a
vial. Add an equal amount of water and let it absorb. You may want to add a piece of plastic
mesh to give the flies something to crawl on, but it is not essential. Insert a foam plug in the
vial to prevent any contamination.
2. Prepare two vials for each lab group; label one “Males” and the other “Females”.
3. Place Drosophila wild-type culture in a warm area or in an incubator. Once the pupae emerge,
remove the parental generation from the vial and place them in a vial of 10% alcohol.
4. As the flies begin to emerge, thermally immobilize them by placing the vial in a refrigerator
or ice bath for five to 10 minutes. When the flies are immobilized, place them on a piece of
filter paper in a petri dish. Place the petri dish on the ice pack to keep the flies immobilized.
5. Separate the males from the females, placing three to five males in each “Male” vial and
three to five females in each “Female” vial. The males can be identified by the presence of
the sex combs on their forelegs. Note: The females will remain virgins for only 12 to
15 hours after emerging; be sure to separate the males and females during this time.
6. Prepare an anesthetizer for each lab group by placing each petri dish on an ice pack and place
them in the freezer for at least 24 hours before the lab. Note: Cut an opening in the cover of
each ice pack to allow better contact between the ice pack and the petri dish.
TIPS
1. The Drosophila for this activity can be kept in the original vial for up to a month. They
should be kept at room temperature out of direct sunlight.
2. The pill bugs can be kept in their original jar for 2–3 days in a refrigerator. For longer time,
they should be put in a container with potting soil and some paper towels to hide under. They
can be given potato slices and misted with water every other day.
Ideas for Inquiry

researchable variables can be found in the Procedure.
Advanced Biology with Vernier 11 - 1 T

Experiment 11
1. Answers will vary. Allow students to address topics such as protection, camouflage, chemo-
reception, etc.
2. In part B, there were no changes in the environment. This part of the experiment served as a
control to gather data before testing the environmental conditions. Since there was no
difference, the data should show no preference with fluctuations in the numbers being solely
by chance.
3. Answers will vary. Students may feel confident in their results or may want to test more
factors, such as the degree of acidity or alkalinity, the degree of moisture in one of the
chambers, etc.
4. Students should design a terrarium with slightly acidic soil and a fair degree of moisture.
They might also want to add some cover material for the pillbugs to crawl under.
5. Students should conclude that the reaction is one of taxis. The data should reveal that the
pillbugs favor the moist environment.
6. Answers will vary. Some students may have caught the majority of behaviors. After
observing once, re-reading the behaviors, and observing again, students should observe some
behaviors they may have missed the first time.
7. Once a female Drosophila has mated, it is fertile for life. Virgin females are necessary to
ensure that mating will occur and the behavior can be observed.
8. Scientists must be able to note all behaviors, however seemingly insignificant. A scientist
must also avoid ascribing human traits to the organisms being observed and not have any bias
when trying to determine the purpose for the behaviors displayed.
11 - 2 T Advanced Biology with Vernier

Computer
Dissolved Oxygen in Water

12A
Aquatic life depends upon oxygen dissolved in water, just as organisms on land rely upon oxygen
in the atmosphere. Molecular oxygen is used by organisms in aerobic respiration where energy is
released during the combustion of sugar in the mitochondria. Without sufficient oxygen, they
suffocate. Some organisms, such as salmon, mayflies, and trout, require high concentrations of
oxygen in the water. Other organisms, such as catfish, midge fly larvae, and carp can survive with
much less oxygen.
Oxygen dissolves at the interface between the water and the air or when aquatic autotrophs
release oxygen as a byproduct of photosynthesis. Abiotic factors including temperature and
pressure influence the maximum amount of oxygen that can be dissolved in pure water. Biotic life
also influences the amount of oxygen that is dissolved.
The following table indicates the oxygen and temperature tolerance level of selected animals. The
quality of the water can be assessed with fair accuracy by observing the aquatic animal
populations in a stream. These assessments are based on known dissolved oxygen tolerance. If a
stream has only species that can survive at low oxygen levels, it is expected to have low oxygen
levels.
Table 1
Temperature Range Minimum Dissolved

Animal (°C) Oxygen (mg/L)
Trout 5–20 6.5
Smallmouth bass 5–28 6.5
Caddisfly larvae 10–25 4.0
Mayfly larvae 10–25 4.0
Stonefly larvae 10–25 4.0
Catfish 20–25 2.5
Carp 10–25 2.0
Mosquito 10–25 1.0
Water boatmen 10–25 2.0
OBJECTIVES
• Use a dissolved oxygen probe to measure the concentration of dissolved oxygen in water.
• Study the effect of temperature on the amount of dissolved oxygen in water.
• Predict the effect of water temperature on aquatic life.
Advanced Biology with Vernier © Vernier Software & Technology 12A - 1

Computer 12A
MATERIALS
Vernier computer interface 100 mL beaker
Logger Pro hot and cold water
Vernier Optical DO Probe or one 1-gallon plastic milk container
Dissolved Oxygen Probe Styrofoam cup
Temperature Probe
Additional Materials for Dissolved Oxygen Probe Users
calibration bottle Sodium Sulfite Calibration Solution
DO Electrode Filling Solution 250 mL beaker with distilled water
PROBE PREPARATION
Optical DO Probe Users Only
(Dissolved Oxygen Probe users proceed to the Dissolved Oxygen Probe section)
1. Set the switch on the Optical DO Probe to the mg/L setting. The switch is located on the box
containing the microSD card.
2. Connect the Optical DO Probe to Channel 1 of the Vernier interface. Connect the
Temperature Probe to Channel 2.
3. Prepare the computer for data collection by opening the file “12A Dissolved Oxygen” from
4. You are now ready to collect DO and temperature data. Continue to the Procedure.
Dissolved Oxygen Probe Users Only

1. Prepare the Dissolved Oxygen Probe for use.
a. Remove the blue protective cap.
b. Unscrew the membrane cap from the tip of the probe.
c. Using a pipet, fill the membrane cap with 1 mL of DO Electrode Filling Solution.
d. Carefully thread the membrane cap back onto the electrode.
e. Place the probe into a container of water.
Remove membrane cap Add electrode filling solution Replace membrane cap
Figure 1

2. Connect the Dissolved Oxygen Probe to Channel 1 of the Vernier interface. Connect the
Temperature Probe to Channel 2.
3. Prepare the computer for data collection by opening the file “12A Dissolved Oxygen” from
4. It is necessary to warm up the Dissolved Oxygen Probe before taking readings. To warm up
the probe, leave it connected to the interface, with Logger Pro running, for 10 minutes. The
probe must stay connected at all times to keep it warmed up. If disconnected for more than a
few minutes, it will be necessary to warm up the probe again.
5. You are now ready to calibrate the Dissolved Oxygen Probe.
Zero-Oxygen Calibration Point
a. Choose Calibrate  CH1: Dissolved Oxygen (mg/L)
from the Experiment menu and click .
b. Remove the probe from the water and place the tip of
the probe into the Sodium Sulfite Calibration Solution.
Important: No air bubbles can be trapped below the tip
of the probe or the probe will sense an inaccurate
dissolved oxygen level. If the voltage does not rapidly
decrease, tap the side of the bottle with the probe to
dislodge any bubbles. The readings should be in the
0.2 to 0.5 V range.
c. Enter 0 in the box for Reading 1.
d. When the voltage for Reading 1 stabilizes, click
.
Saturated DO Calibration Point Figure 2
e. Rinse the probe with distilled water and gently blot dry.
f. Unscrew the lid of the calibration bottle provided with the probe. Slide the lid and the
grommet about 2 cm onto the probe body.
Figure 3
g. Add water to the bottle to a depth of about 1 cm and screw the bottle into the cap, as
shown. Keep the probe in this position for about a minute. Important: Do not touch the
membrane or get it wet during this step.
h. Enter the value for Reading 2 using Table 3 (at the end of the document) to determine the
correct saturated dissolved oxygen concentration based on the current barometric pressure

Computer 12A
and temperature. If you do not have the current air pressure, use Table 4 to estimate the air
pressure at your elevation in meters.
i. When the displayed voltage reading for Reading 2 stabilizes (readings should be above
2.0 V), click .
Store Calibration to Probe
• If your instructor directs you to store the calibration, continue with this step, otherwise,
continue to the Procedure.
j. To store the new calibration, select the Storage tab.
k. Click Set Sensor Calibration and then click OK.
PROCEDURE
1. Prepare for data collection by clicking .
2. Obtain two 250 mL beakers. Fill one beaker with ice and
cold water. Fill the second beaker with warm water about
40–50°C.
3. Place approximately 100 mL of cold water and a couple

small pieces of ice, from the beaker filled with ice, into a
clean plastic one-gallon milk container. Seal the container
and vigorously shake the water for a period of 2 minutes.
This will allow the air inside the container to dissolve into
the water sample. Pour the water into the Styrofoam cup.
4. Place the Temperature Probe in the Styrofoam cup as

shown in Figure 4. Place the shaft of the dissolved oxygen
probe into the water. Make sure the silver dot on the side of
the probe is submerged. Note: Dissolved Oxygen Probe
users need to stir gently (avoid hitting the edge of the cup). Figure 4
Optical DO Probe users do not need to stir.
5. Monitor the dissolved oxygen readings in the meter. Give the dissolved oxygen readings
ample time to stabilize (90–120 seconds). At colder temperatures the probe will require a
greater amount of time to stabilize. When the readings have stabilized, click .
6. Remove the probes from the water sample. Note: If you are using a Dissolved Oxygen Probe,
place it back in the beaker of distilled water.
7. Pour the water from the Styrofoam cup back into the milk container. Seal the container and
shake the water vigorously for 1 minute.
8. Repeat Steps 4–7 until the water sample reaches room temperature. When room temperature
has been reached then begin adding about 25 mL of warm water (40–50°C) prior to shaking
the water sample. This will allow you to take warmer water readings. Take dissolved oxygen
readings until the water temperature reaches 35°C.
9. When all readings have been taken click .
10. In Table 2, record the dissolved oxygen and temperature readings from the table.

11. Print the graph of dissolved oxygen vs. temperature. Enter your name(s) and the number of
copies of the graph.
DATA
Table 2
Temperature Dissolved Oxygen

(°C) (mg/L)
QUESTIONS
1. At what temperature was the dissolved oxygen concentration the highest? Lowest?
2. Does your data indicate how the amount of dissolved oxygen in the water is affected by the
temperature of water? Explain.
3. If you analyzed the invertebrates in a stream and found an abundant supply of caddisflies,
mayflies, dragonfly larvae, and trout, what minimum concentration of dissolved oxygen would
be present in the stream? What maximum temperature would you expect the stream to
sustain?
4. Mosquito larvae can tolerate extremely low dissolved oxygen concentrations, yet cannot
survive at temperatures above approximately 25°C. How might you account for dissolved
oxygen concentrations of such a low value at a temperature of 25°C? Explain.
5. Why might trout be found in pools of water shaded by trees and shrubs more commonly than
in water where the trees have been cleared?

Computer 12A
CALIBRATION TABLES
Table 3: 100% Dissolved Oxygen Capacity (mg/L)
770 mm 760 mm 750 mm 740 mm 730 mm 720 mm 710 mm 700 mm 690 mm 680 mm 670 mm 660 mm
0°C 14.76 14.57 14.38 14.19 13.99 13.80 13.61 13.42 13.23 13.04 12.84 12.65
1°C 14.38 14.19 14.00 13.82 13.63 13.44 13.26 13.07 12.88 12.70 12.51 12.32
2°C 14.01 13.82 13.64 13.46 13.28 13.10 12.92 12.73 12.55 12.37 12.19 12.01
3°C 13.65 13.47 13.29 13.12 12.94 12.76 12.59 12.41 12.23 12.05 11.88 11.70
4°C 13.31 13.13 12.96 12.79 12.61 12.44 12.27 12.10 11.92 11.75 11.58 11.40
5°C 12.97 12.81 12.64 12.47 12.30 12.13 11.96 11.80 11.63 11.46 11.29 11.12
6°C 12.66 12.49 12.33 12.16 12.00 11.83 11.67 11.51 11.34 11.18 11.01 10.85
7°C 12.35 12.19 12.03 11.87 11.71 11.55 11.39 11.23 11.07 10.91 10.75 10.59
8°C 12.05 11.90 11.74 11.58 11.43 11.27 11.11 10.96 10.80 10.65 10.49 10.33
9°C 11.77 11.62 11.46 11.31 11.16 11.01 10.85 10.70 10.55 10.39 10.24 10.09
10°C 11.50 11.35 11.20 11.05 10.90 10.75 10.60 10.45 10.30 10.15 10.00 9.86
11°C 11.24 11.09 10.94 10.80 10.65 10.51 10.36 10.21 10.07 9.92 9.78 9.63
12°C 10.98 10.84 10.70 10.56 10.41 10.27 10.13 9.99 9.84 9.70 9.56 9.41
13°C 10.74 10.60 10.46 10.32 10.18 10.04 9.90 9.77 9.63 9.49 9.35 9.21
14°C 10.51 10.37 10.24 10.10 9.96 9.83 9.69 9.55 9.42 9.28 9.14 9.01
15°C 10.29 10.15 10.02 9.88 9.75 9.62 9.48 9.35 9.22 9.08 8.95 8.82
16°C 10.07 9.94 9.81 9.68 9.55 9.42 9.29 9.15 9.02 8.89 8.76 8.63
17°C 9.86 9.74 9.61 9.48 9.35 9.22 9.10 8.97 8.84 8.71 8.58 8.45
18°C 9.67 9.54 9.41 9.29 9.16 9.04 8.91 8.79 8.66 8.54 8.41 8.28
19°C 9.47 9.35 9.23 9.11 8.98 8.86 8.74 8.61 8.49 8.37 8.24 8.12
20°C 9.29 9.17 9.05 8.93 8.81 8.69 8.57 8.45 8.33 8.20 8.08 7.96
21°C 9.11 9.00 8.88 8.76 8.64 8.52 8.40 8.28 8.17 8.05 7.93 7.81
22°C 8.94 8.83 8.71 8.59 8.48 8.36 8.25 8.13 8.01 7.90 7.78 7.67
23°C 8.78 8.66 8.55 8.44 8.32 8.21 8.09 7.98 7.87 7.75 7.64 7.52
24°C 8.62 8.51 8.40 8.28 8.17 8.06 7.95 7.84 7.72 7.61 7.50 7.39
25°C 8.47 8.36 8.25 8.14 8.03 7.92 7.81 7.70 7.59 7.48 7.37 7.26
26°C 8.32 8.21 8.10 7.99 7.89 7.78 7.67 7.56 7.45 7.35 7.24 7.13
27°C 8.17 8.07 7.96 7.86 7.75 7.64 7.54 7.43 7.33 7.22 7.11 7.01
28°C 8.04 7.93 7.83 7.72 7.62 7.51 7.41 7.30 7.20 7.10 6.99 6.89
29°C 7.90 7.80 7.69 7.59 7.49 7.39 7.28 7.18 7.08 6.98 6.87 6.77
30°C 7.77 7.67 7.57 7.47 7.36 7.26 7.16 7.06 6.96 6.86 6.76 6.66
Table 4: Approximate Barometric Pressure at Different Elevations

Elevation Pressure Elevation Pressure Elevation Pressure
(m) (mm Hg) (m) (mm Hg) (m) (mm Hg)
0 760 800 693 1600 628

100 748 900 685 1700 620
200 741 1000 676 1800 612
300 733 1100 669 1900 604
400 725 1200 661 2000 596
500 717 1300 652 2100 588
600 709 1400 643 2200 580
700 701 1500 636 2300 571

Experiment

1. This experiment correlates with AP Biology curriculum framework concepts 4A6 and 4B4.
3. Temperatures of 5, 10, 15, 20, 25, and 30°C are suggested.
4. When students are adding ice into their milk container, warn them to not add more than will
melt while they are shaking the container.
5. There are two types of dissolved oxygen probes: the Dissolved Oxygen Probe and the Vernier
Optical DO Probe. The calibration, use, and storage procedures vary between the two probes.
Before giving the lab to students, you may want to delete preparation instructions for the
probe you are not using. More information about each probe can be found below.

6. The Optical DO Probe requires Logger Pro 3.8.6 or newer. Updates are available at the
Vernier web site.
7. Optical DO Probes are not compatible with EasyLink or Go! Link.
8. The Optical DO Probe does not require calibration. Follow the probe preparation instructions
for the Optical DO Probe.
9. The Optical DO Probe has a switch on the box containing the microSD card which changes
the units from mg/L to % saturation. For this experiment, ensure the switch is set to mg/L
before connecting to the interface.

10. In order to compare with the information in Table 1, the Dissolved Oxygen Probe should be
calibrated. To prepare and calibrate the Dissolved Oxygen Probe, follow the Probe
Preparation instructions for the Dissolved Oxygen Probe in the student instructions.
If you prepare the probes rather than the students, the probe preparation instructions may be
deleted from the student procedure.
11. If you calibrate a Dissolved Oxygen Probe using Logger Pro or LabQuest App, you can store
the calibration directly on the probe (this cannot be done using EasyData). Once the
calibration is stored on the probe, it will be used automatically each time the probe is
connected to an interface. Directions about how to store the calibration are included in the
student version. Note: Due to various factors, such as changes in the characteristics of the
membrane over time, the stored calibration should be updated every few weeks.
12. When setting up the Dissolved Oxygen Probe, remove the blue plastic cap from the end of the
probe. The cap is made of a soft plastic material and easily slides off the probe end.

Experiment 12A
13. In order for the Dissolved Oxygen Probe to warm up and stay polarized, power to the sensor
must be continuous. LabPro, LabQuest, and CBL 2 deliver continuous power once the data-
collection software is started even if the screen goes to sleep. However, EasyLink used with a
TI-84 graphing calculator and the EasyData App stops powering the sensor when the
calculator goes to sleep. The calculator goes to sleep to conserve battery power if no user
interaction is detected for 3 minutes. If power to the sensor is disrupted for more than a few
minutes, the sensor must be warmed up again before calibrating or taking readings. To avoid
having to warm up the sensor again, students must press a button on the calculator every few
minutes to keep the calculator awake.
14. As a time-saving measure, you could instruct students to leave Logger Pro or LabQuest App
running. This will keep power going to the probes. When the next group of students comes in,
they can begin at the procedure. They can skip the probe preparation section because the
initial group of students has completed all of the setup. Have the last group of students for the
day shut everything off and put things away.
15. Between classes store the Dissolved Oxygen Probes in a beaker of distilled water. At the end
of the day be sure to empty out the electrode filling solution in the Dissolved Oxygen Probe
and rinse the inside of the membrane cap with distilled water.
Ideas for Inquiry

• How does water movement affect the rate of oxygen diffusion?
• What happens to dissolved oxygen when the temperature is changed but no stirring occurs?
• How does surface are affect dissolved oxygen?
• How does salt in the water affect dissolved oxygen?
SAMPLE RESULTS
The following data may be different from students’ results. Note that the actual temperatures will
be close to, but not necessarily the same as, the assigned temperatures.
Table 1
Temperature Dissolved oxygen
(°C) (mg/L)
8.96 11.19
14.89 9.66
18.74 8.81
22.71 7.99
33.65 5.71
Dissolved oxygen vs. temperature

Teacher Information Dissolved Oxygen in Water
1. The amount of dissolved oxygen will be highest at the coldest temperature and lowest at the
warmest temperature.
2. As the temperature increases, the amount of dissolved oxygen decreases. The relationship
does not appear to be linear.
3. Since trout were present, the minimum amount of dissolved oxygen would be 6.5 mg/L.
According to these data, fast-moving water could not be warmer than 30–32°C. This
temperature is much higher than trout generally live in, however.
4. Other factors must have caused the low dissolved oxygen levels. Bacteria and other organisms
can lower the dissolved oxygen of water when they respire aerobically.
5. Trout require high dissolved oxygen levels. Since trees shade the water from the sun, they
help keep it cool. Cool water has a higher dissolved oxygen level than warm water and is
preferred by trout.

Computer
Primary Productivity
12B
Oxygen is vital to life. In the atmosphere, oxygen comprises over 20% of the available gases. In
aquatic ecosystems, however, oxygen is scarce. To be useful to aquatic organisms, oxygen must
be in the form of molecular oxygen, O2. The concentration of oxygen in water can be affected by
many physical and biological factors. Respiration by plants and animals reduces oxygen
concentrations, while the photosynthetic activity of plants increases it. In photosynthesis, carbon
is assimilated into the biosphere and oxygen is made available, as follows:
6 H2O + 6 CO2(g) + energy → C6H12O6 + 6O2(g)
The rate of assimilation of carbon in water depends on the type and quantity of plants within the
water. Primary productivity is the measure of this rate of carbon assimilation. As the above
equation indicates, the production of oxygen can be used to monitor the primary productivity of
an aquatic ecosystem. A measure of oxygen production over time provides a means of calculating
the amount of carbon that has been bound in organic compounds during that period of time.
Primary productivity can also be measured by determining the rate of carbon dioxide utilization or
the rate of formation of organic compounds.
Figure 1 The light and dark bottle method of measuring oxygen production
One method of measuring the production of oxygen is the light and dark bottle method. In this
method, a sample of water is placed into two bottles. One bottle is stored in the dark and the
other in a lighted area. Only respiration can occur in the bottle stored in the dark. The decrease in
dissolved oxygen (DO) in the dark bottle over time is a measure of the rate of respiration. Both
photosynthesis and respiration can occur in the bottle exposed to light, however. The difference
between the amount of oxygen produced through photosynthesis and that consumed through
aerobic respiration is the net productivity. The difference in dissolved oxygen over time between
the bottles stored in the light and in the dark is a measure of the total amount of oxygen produced
by photosynthesis. The total amount of oxygen produced is called the gross productivity.
The measurement of the DO concentration of a body of water is often used to determine whether
the biological activities requiring oxygen are occurring and is an important indicator of pollution.
OBJECTIVES
• Measure the rate of respiration in an aquatic environment using a dissolved oxygen probe.
• Determine the net and gross productivity in an aquatic environment.
Advanced Biology with Vernier © Vernier Software & Technology 12B - 1

Computer 12B
MATERIALS
computer shallow pan
Vernier computer interface 7 Vernier Water Quality Bottles or large test tubes
Logger Pro 7 stoppers to fit bottles or test tubes
Vernier Optical DO Probe or Primary Productivity Kit screens (set of 4) or
Dissolved Oxygen Probe 17 pieces of 12 cm × 12 cm (5″ × 5″) plastic
scissors window screen
siphon tube 500 mL pond, lake, seawater, or algal culture
aluminum foil 250 mL beaker*
distilled water DO Electrode Filling Solution*
rubber bands *Needed only if using a Dissolved Oxygen Probe
PRE-LAB PROCEDURE
1. Set the switch on the Optical DO Probe to the mg/L setting. The switch is located on the box
containing the microSD card.
2. Connect the Optical DO Probe to the Vernier interface.
3. Prepare the computer for data collection by opening the file “12B Primary Productivity” from

1. Prepare the Dissolved Oxygen Probe for use.
a. Remove the blue protective cap.
b. Unscrew the membrane cap from the tip of the probe.
c. Using a pipet, fill the membrane cap with 1 mL of DO Electrode Filling Solution.
d. Carefully thread the membrane cap back onto the electrode.
e. Place the probe into a 250 mL beaker containing distilled water.
Remove membrane cap Add electrode filling solution Replace membrane cap
Figure 2
2. Connect the Dissolved Oxygen Probe to the Vernier computer interface.
3. Prepare the computer for data collection by opening the file “12B Primary Productivity” from

4. It is necessary to warm up the Dissolved Oxygen Probe before taking readings. To warm up
the probe, leave it connected to the interface, with Logger Pro running, for 10 minutes. The
probe must stay connected at all times to keep it warmed up. If disconnected for more than a
few minutes, it will be necessary to warm up the probe again.
5. You are now ready to calibrate the Dissolved Oxygen Probe.

• If your instructor directs you to use the default calibration, continue to the Procedure.
• If your instructor directs you to perform a new calibration for the Dissolved Oxygen Probe,
use the calibration procedure provided by your instructor.
PROCEDURE
Day 1
1. Fill seven Water Quality bottles or test tubes with the water sample, ensuring that air does not
mix into the sample.
a. Obtain a siphon tube.
b. Insert the tube into the water sample and fill the tube completely with water.
c. Pinch the tube (or use a tube clamp) to close off the siphon tube.
d. Place one end of the tube in the bottom of a test tube or Water Quality bottle. Keep the
other end in the water sample, well below the surface. Position the test tube or Water
Quality bottle lower than the water sample. Place a shallow pan under the test tube or
Water Quality bottle to collect any water that spills over.
e. Siphon the water into the test tube or Water Quality bottle. Fill the test tube or Water
Quality bottle until it overflows. Ensure the test tube or Water Quality bottle is filled
completely to the top of the rim.
f. Insert a stopper in the test tube or Water Quality bottle. Be sure no air is in the test tube or
Water Quality bottle.
2. The percentage of available natural light for each water sample is listed in Table 1.
Table 1
Sample Number of screens % Light
1 0 Initial
2 0 100%
3 1 65%
4 3 25%
5 5 10%
6 8 2%
7 Aluminum foil Dark
3. Label the samples as follows: dark, initial, 2%, 10%, 25%, 65%, 100%.

Computer 12B
4. Wrap sample 7 with aluminum foil so that it is lightproof. This water sample will remain in the
dark.
5. Apply layers of screen to simulate the amount of natural light available for photosynthesis at
different depths in a body of water.
• If using the Primary Productivity Kit, select the screen number that corresponds to the
number of screens in Table 1.
• If using your own screens, wrap screens around samples according to Table 1. Trim the
screens so each one only wraps around once. Hold the screens in place with rubber bands or
clothespins. The bottoms need not be covered unless they are to be stored on their side.
6. Place the dissolved oxygen probe into sample 1, the initial sample, so that it is submerged half
the depth of the water. Do not agitate the water, or oxygen from the atmosphere will mix into
the water and cause erroneous readings. Note: Dissolved Oxygen Probe users need to gently
stir the sample to allow the water to move past the probe’s tip (if using an Optical DO Probe,
this is not necessary).
7. When the dissolved oxygen reading stabilizes, record the reading in Table 2. Discard the initial
sample. Rinse the dissolved oxygen probe with distilled water. Note: If you are using a
Dissolved Oxygen Probe, place it back in the beaker of distilled water.
8. Place samples 2–7 near the light source, as directed by your instructor.
Day 2
9. Dissolved Oxygen Probe users must allow the probe 10 minutes to warm up. Keep it in the
beaker of distilled water during the warm-up period. Note: The Optical DO Probe does not
require warm-up.
10. Place the probe into the 100% light sample. Note: Dissolved Oxygen Probe users need to
gently stir the sample to allow the water to move past the probe’s tip (if using an Optical DO
Probe, this is not necessary). Do not agitate the water, or oxygen from the atmosphere will
mix into the water and cause erroneous readings.
11. When the dissolved oxygen reading stabilizes, record the reading in Table 2.
12. Repeat Steps 10–11 for the remaining samples.
13. Rinse the probe with distilled water. Note: If you are using a Dissolved Oxygen Probe, place
it back in the beaker of distilled water.

DATA
Table 2
Test tube % Light DO (mg/L)
1 Initial
2 100%
3 65%
4 25%
5 10%
6 2%
7 Dark
PROCESSING THE DATA

1. Determine the number of hours that have passed since the onset of this experiment. Subtract
the DO value in the test tube 1 (the initial DO value) from that of sample 7 (the dark test
tube’s DO value). Divide the DO value by the time in hours. Record the resulting value as the
respiration rate in Table 3.
Respiration rate = (dark DO – initial DO) / time
Table 3
Respiration (mg O2/L/hr)
2. Determine the gross productivity in each test tube. To do this, subtract the DO in sample 7
(the dark test tube’s DO value) from that of sample 2–6 (the light test tubes’ DO value).
Divide each DO value by the length of the experiment in hours. Record each resulting value as
the gross productivity in Table 4.
Gross productivity = (DO of test tube – dark DO) / time
Table 4
Gross productivity Net productivity

Test tube % Light
(mg/L/hr) (mg/L/hr)
2 100%
3 65%
4 25%
5 10%
6 2%

Computer 12B
3. Determine the net productivity in each test tube. To do this, subtract the DO in sample 2–6
(the light test tube’s DO value) from that of sample 1 (the initial DO value). Divide the result
by the length of the experiment in hours. Record each resulting value as the net productivity in
Table 4.
Net Productivity = (DO of test tube – initial DO) / time
4. Prepare a graph of gross productivity and net productivity as a function of light intensity.
Graph both types of productivity on the same piece of graph paper.
QUESTIONS
1. Is there evidence that photosynthetic activity added oxygen to the water? Explain.
2. Is there evidence that aerobic respiration occurred in the water? If so, what kind of organisms
might be responsible for this—autotrophs? Heterotrophs? Explain.
3. What effect did light have on the primary productivity? Explain.
4. Refer to your graph of productivity and light intensity. At what light intensity do you expect
there to be no net productivity? no gross productivity?
5. How would turbidity affect the primary productivity of a pond?
EXTENSIONS
1. Determine the effects of nitrogen and phosphates on primary productivity. Why would the
presence of phosphates and nitrogen, in the form of nitrates and ammonium ions, be important
to an aquatic ecosystem during the spring season? How do they accumulate in the watershed?
What is eutrophication?
2. Measure the dissolved oxygen of a pond at different temperatures. What is the effect of
temperature on the primary productivity of a pond?
3. Calculate the amount of carbon that was fixed in each of the tubes. Use the following
conversion factors to do the calculations.
mg O2/L X 0.698 = mL O2/L
mL O2/L X .536 = mg carbon fixed/L

Experiment

1. This experiment correlates with AP Biology curriculum framework concepts 4A2 and 4A6.
3. This experiment requires two periods to complete. There will be time during the second
period to discuss the experiment and to begin the follow-up activities. Results will be best if
student use the same set of equipment to make measurements each day. You can leave the
probes set up at the end of each day so that when students collect data, they can skip some of
the Pre-Lab Procedure.
4. If time permits, the first part of this lab can be completed during a field trip.
5. The Vernier Primary Productivity Kit (order code: PPK) is recommended for this lab and
includes appropriate screens, cut to size, and Water Quality bottles. Alternately, bottles or test
tubes that can form an airtight seal must be used. Air should not be in any of the tubes while
they are stored overnight.
6. If you are not using the Primary Productivity Kit, you can use pieces of plastic window
screen. Plastic window screen is available from most hardware stores; it is much easier to use
than metal screen.
7. The samples should be water-tight when they are stored overnight. If using test tubes,
consider having students lay the tubes horizontally on a table and lower a fluorescent light
about 3–6 cm above the tubes. This provides ample light without heating the tubes. If the
tubes are stored vertically, non-motile algae may sink to the bottom of the tube and be
shielded from the light.
8. Bottles or test tubes that can form an airtight seal must be used. Air should not be in any of
the tubes while they are stored overnight.
9. The test tubes should be water-tight when they are stored overnight. Lay the tubes
horizontally on a table and lower a fluorescent light about 3–6 cm above the tubes. This
provides ample light without heating the tubes. If the tubes are stored vertically, non-motile
algae may sink to the bottom of the tube and be shielded from the light.
10. Vernier sells two types of dissolved oxygen probes: the Dissolved Oxygen Probe and the
Vernier Optical DO Probe. The calibration, use, and storage procedures vary between the two
probes. Before giving the lab to students, you may want to delete preparation instructions for
the probe you are not using. More information about each probe can be found below
Vernier Optical DO Probe Users Only

11. The Optical DO Probe requires Logger Pro 3.8.6 or newer. Updates are available at the
Vernier web site.
12. Optical DO Probes are not compatible with EasyLink or Go! Link.

Experiment 12B
13. The Optical DO Probe does not require calibration. Follow the probe preparation instructions
for the Optical DO Probe.
14. The Optical DO Probe has a switch on the box containing the microSD card which changes
the units from mg/L to % saturation. For this experiment, ensure the switch is set to mg/L
before connecting to the interface.

15. Since students are looking at relative change in DO, calibration is not necessary. If you want
to calibrate the Dissolved Oxygen Probes for better absolute dissolved oxygen readings,
follow the calibration instructions for the Dissolved Oxygen Probe in the student instructions
of Experiment 12A, Dissolved Oxygen in Water. The instructions from Experiment 12A can
be added to this experiment using the files on the CD that accompanies this book. Be sure to
include copies of the calibration tables found at the end of the student version of the
experiment.
16. In order for the Dissolved Oxygen Probe to warm up and stay polarized, power to the probe
must be continuous. LabPro, LabQuest, and CBL 2 deliver continuous power once the data-
collection software is started even if the screen goes to sleep. However, EasyLink used with a
TI-84 graphing calculator and the EasyData App stops powering the sensor when the
calculator goes to sleep. The calculator goes to sleep to conserve battery power if no user
interaction is detected for 3 minutes. If power to the sensor is disrupted for more than a few
minutes, the sensor must be warmed up again before calibrating or taking readings. To avoid
having to warm up the sensor again, students must press a button on the calculator every few
minutes to keep the calculator awake.
17. As a time-saving measure, instruct the students to leave Logger Pro running at the end of the
lab period. This will keep power going to the probes. When the next group of students comes
in, they can begin at the Procedure section. They can skip the pre-lab procedure because the
initial group of students has completed all of the setup.
18. When setting up the Dissolved Oxygen Probe, be sure to remove the blue plastic cap from the
end of the probe. The cap is made of a soft plastic material and easily slides off the probe.
Ideas for Inquiry

• How do different color lights affect net productivity?
• How does the initial concentration of Chlorella culture affect productivity?
• How is productivity affected by other aquatic plants such as duckweed?
• How does the addition of CO2 affect productivity?

Teacher Information Primary Productivity
SAMPLE RESULTS
The following data may be different from students’ results. This water sample was taken over a
25-hour period.
Table 2
Test tube % Light DO (mg/L)
1 Initial 7.5
2 100% 7.8
3 65% 7.6
4 25% 6.8
5 10% 6.7
6 2% 6.5
7 Dark 6.5
Table 3
Respiration (mg/L/hr) –0.04
Table 4
Gross productivity Net productivity

Test tube % Light
(mg/L/hr) (mg/L/hr)
2 100% 0.052 –0.012
3 65% 0.044 –0.004
4 25% 0.012 0.028
5 10% 0.008 0.032
6 2% 0 0.04
1. There is evidence that photosynthetic activity added oxygen to the water. The gross primary
productivity was positive and greater than zero. The gross primary productivity is a
measurement of the total amount of oxygen produced in the water sample, regardless of the
amount of aerobic respiration that occurs. The net primary productivity may or may not be a
positive value, depending on the BOD due to heterotrophs. If so, this does not mean that
photosynthesis did not occur—just that the rate of photosynthesis was less than the rate of
aerobic respiration.
2. There is evidence that aerobic respiration occurred in the water, as the respiration rate was
negative and non-zero. The dark bottle had less oxygen after one day than the bottle had

Experiment 12B
initially. Both autotrophs and heterotrophs may be responsible for removing oxygen,
depending upon the types of organisms in the water sample.
3. Light was necessary for the assimilation of carbon into the ecosystem within the water sample,
since light is necessary for photosynthesis.
4. Student answers will vary. According to the sample data, there would be no net productivity
at 70% light and no gross productivity at 0% light.
5. The higher the turbidity of the water, the less clear it is. Less light reaches the plants going
through photosynthesis, and less oxygen is produced. This results in a decrease in primary
productivity.

Computer
The Visible Spectra of

13
Plant Pigments
Plants contain many different molecules directly or indirectly involved with photosynthesis, which
may also impart color to the plant. The mixture of chlorophyll molecules found in spinach, for
example, absorbs several wavelengths of visible light, with distinct absorbance peaks in the blue
range (400–500 nm) and in the yellow-red range (600–700 nm). The combination of visible light
that is not absorbed appears green to the human eye. Chlorophyll contains a porphyrin ring in its
structure with a magnesium ion in the center. The porphyrin ring accounts for much of the
molecule’s light absorbance. Chlorophyll is found in the thylakoid plate of a plant chloroplast.
Carotenoids, accessory pigments produced in chromoplasts, are associated with many colors
observed in vegetation. There are hundreds of different types of carotenoids. Carrots get their
color, which is often orange but is not restricted to orange, from carotene. And carotene is not so
much a specific compound as a family name for several compounds that also go by the name
terpene.
Another type of carotenoid phyto-pigment is called anthocyanin. The purplish color of a red
cabbage and the rusty red of the flesh of a blood orange are a result of the presence of
anthocyanins, which also have the curious property of changing color with changes in pH.
Anthocyanins absorb UV light, which is used by plants to perform two important functions: to
attract insects, which augment pollination, and as a “sunscreen” to protect the other parts of the
plant cells such as DNA from harmful UV radiation.
In this experiment you will extract pigments from spinach and carrots and measure their visible
absorbance spectra with a Spectrometer. While you wait for the extracts to develop, you will
measure the absorbance of blue and yellow food-colored water samples, which will provide an
analogy to the absorbance of the plant pigment extracts.
OBJECTIVES
• Measure and analyze the visible light absorbance spectra of pigments from spinach and
carrots.
• Measure and analyze blue and yellow food coloring to compare with the plant pigments.

Computer 13
MATERIALS
computer carrot slices or shavings
Logger Pro 70% isopropanol (IPA)
Spectrometer acetone or petroleum ether
cuvettes yellow and blue food colored solutions
two 10 mL graduated cylinders distilled water
funnel plastic Beral pipets
filter paper ring stand and ring
three small beakers balance, ± 0.1 g accuracy
13 × 100 mm glass test tube, or 125 mL Erlenmeyer flask and stopper
glass cuvette mortar and pestle
fresh spinach
PROCEDURE
2. Connect a Spectrometer to the USB port of your computer.
3. Start Logger Pro. If it is already running, choose New from the File menu.

a. Prepare a blank by filling an empty cuvette 3/4 full with distilled water.
b. Choose Calibrate  Spectrometer from the Experiment menu.
c. When the warmup period is complete, place the blank in the Spectrometer. Make sure to
align the cuvette so that the clear sides are facing the light source of the Spectrometer.
d. Click Finish Calibration, and then click .
Part I Prepare the Plant Pigment Extracts
5. Measure out 0.5 g of fresh spinach. Tear the spinach into tiny pieces and grind them with a
mortar and pestle. Add 20 mL of 70% isopropanol (IPA) and transfer the mixture to a small
beaker. Allow the mixture to sit.
6. Measure out 0.5 g of carrot slices (or shavings) and place them in a 125 mL Erlenmeyer flask.
Add 20 mL of either acetone or petroleum ether to the flask and stopper it. CAUTION:
Handle the acetone, or petroleum ether, with care. The fumes may irritate your nasal
passages. It is best to use these liquids in a hood.
Part II The Absorbance of Food Coloring

7. Conduct a full spectrum analysis of the blue food coloring.
a. Empty the blank cuvette and rinse it twice with small amounts of the blue food coloring
mixture. Fill the cuvette 3/4 full with the blue liquid and place it in the Spectrometer. Align
the cuvette so the clear sides are facing the light source of the Spectrometer.
b. Click . A full spectrum graph of the blue food coloring sample will be displayed.
c. Click . Examine the graph, noting the peak or peaks of very high absorbance or
other distinguishing features.
8. Choose Store Latest Run from the Experiment menu to store your data.

The Visible Spectra of Plant Pigments
9. Repeat Steps 7 and 8 with the yellow food coloring sample. Remember to store the data.
10. Mix equal amounts of the blue and yellow solutions into a small beaker. Repeat Steps 7 and 8
with the mixture.
11. Display a graph of any of the three plots.

a. Choose Graph Options from the Options menu.
b. In the Graph Options dialog box, select the Axes Options tab.
c. Select Absorbance for the run you want plotted and de-select runs you do not want to
display.
d. Click .
12. Print or save your experiment file as directed.
Part III Measure the Absorbance of the Plant Pigment Samples

13. Prepare a purified sample of your spinach extract. Use a funnel and filter to slowly pour the
IPA/spinach extract into a clean beaker.
14. Calibrate the Spectrometer. You will calibrate the Spectrometer with isopropanol because
your solvent in the spinach extract is isopropanol, not water.
a. Choose New from the File menu.
b. Prepare a blank by filling an empty cuvette 3/4 full with isopropanol.
c. Choose Calibrate  Spectrometer from the Experiment menu.
d. When the warmup period is complete, place the blank in the Spectrometer. Make sure to
e. Click Finish Calibration, and then click .
15. Measure the absorbance spectrum of the spinach extract.
a. Pour out the isopropanol from the cuvette, rinse, and fill it 3/4 full with the spinach extract.
b. Place the cuvette in the Spectrometer.
c. Click to see a plot of the absorbance spectrum for the spinach extract.
d. Click .
16. Examine the graph, noting the absorbance peak ranges for chlorophyll described in the
introductory remarks. If any of the peak absorbance values are greater than 1.5, dilute your
sample to bring the peaks down to a more reasonable level. Repeat data collection.
17. Write down your observations of the graph. Print or save your experiment file as directed.

Computer 13
18. Calibrate the Spectrometer with a different solvent for testing the carrot extract.
b. Choose Calibrate  Spectrometer from the Experiment menu.
c. Obtain a 13 × 100 mm glass test tube. Mark the test tube so that you can align it in the
Spectrometer the same way each time you use it. Fill the test tube ~1/2 full with the
solvent, acetone or petroleum ether that you used with the carrot slices. This test tube of
solvent will serve as your blank.
d. When the warmup period is complete, place the test tube in the Spectrometer, being careful
to line it up with the mark. Make sure to align the test tube so that the clear sides are facing
the light source of the spectrometer.
e. Click Finish Calibration, and then click .
19. Measure the absorbance spectrum of the carrot extract.
a. Pour out the solvent from the test tube, rinse, and fill it ~1/2 full with the carrot extract.
b. Place the test tube in the Spectrometer, being careful to line it up with the mark.
c. Click to see a plot of the absorbance spectrum for the carrot extract.
d. Click .
20. Examine the graph. If any of the peak absorbance values are greater than 1.5, dilute your
sample to bring the peaks down to a more reasonable level. Repeat data collection.
21. Record your observations of the graph. Print or save your experiment file as directed.
DATA TABLE
Trial Sample Peaks or unique features of the spectrum
1 Blue
2 Yellow
3 Mixture
4 Spinach Extract
5 Carrot Extract

The Visible Spectra of Plant Pigments
DATA ANALYSIS
1. Describe, in detail, the spectrum of each food coloring sample. With the mixture of blue and
yellow food coloring, can you clearly distinguish the characteristics of each coloring? Explain.
2. Consult a reliable resource to identify the major absorbance peaks of chlorophyll a and
chlorophyll b. Examine the absorbance vs. wavelength graph for your spinach extract. Does
your graph clearly show these absorbance peaks? Are there other peaks on your graph that are
not characteristic of chlorophyll? If so, speculate about what caused these peaks.
3. Consult a reliable resource to identify the major absorbance peaks of α-carotene and
β-carotene. Examine the absorbance vs. wavelength graph for your carrot extract. Does your
graph clearly show these absorbance peaks? Are there other peaks on your graph that are not
characteristic of carotene? If so, speculate about what caused these peaks.
4. How did your tests of the absorbance of the blue and yellow food colored solutions compare
with the tests of the spinach and carrot extracts?
EXTENSIONS
1. Your spinach extract contains chlorophyll, which is a fluorescent molecule. Fluorescent
molecules can absorb light of one wavelength and then reemit light at a new and longer
wavelength of light. As you have seen in this exercise, chlorophyll absorbs light in the violet
and blue regions of the spectra. If you were to shine a violet or blue light through a sample of
spinach extract, you would see the solution turn red in color. The “Long-Wave UV Pen
Light” from Bio-Rad Laboratories, Inc. (Catalog # 166-0530EDU) can be used for this
purpose. Fill a new cuvette with 1 mL of 70% IPA. Add 1 mL of the spinach extract to the
cuvette. Shine the light from the Long-Wave UV Pen Light through the cuvette. Does the
solution in the light path turn red in color?
2. Fluorescence spectroscopy is a method that is used to quantify fluorescent compounds in

solution. In fluorescence spectroscopy, a sample can be “excited” with a chosen wavelength of
light and the resulting light that is emitted from the sample can be measured and quantified.
The SpectroVis Plus from Vernier Software & Technology can be used for this purpose.
Follow the directions below to measure the fluorescence of the spinach extract after it has
been diluted 50% with 70% IPA.
File menu.
b. Place the cuvette containing the diluted spinach extract into the cuvette slot of the
Spectrometer.
c. Choose Change Units ►Spectrometer from the Experiment menu and select Fluorescence
405 nm.
d. Choose Set up Sensors ►Spectrometer from the Experiment menu and change the sample
time to 150 ms.
e. Click . A full spectrum graph of the solution will be displayed. Note that one area of
the graph contains a peak at approximately 675 nm. This peak is from chlorophyll. Click
.
f. Adjust the sample time to increase or decrease the size of the peak. If the peak intensity is
above 1, decrease the sample time by 10 ms and collect a new fluorescence spectrum.
Continue to decrease the sample time until the fluorescent peak is fully visible. If the peak

Computer 13
is below 0.3, increase the sample time by 10 ms. Collect a new fluorescence spectrum.
Increase the sample time until the peak amplitude for chlorophyll is above 0.8.
e. Print or save your experiment file as directed

Experiment
The Visible Spectra of
Plant Pigments
collection with a Spectrometer. It can be conducted using a Vernier Spectrometer or any other
visible Spectrometer supported by Logger Pro and LabQuest.
2. Prepare the food coloring solutions by adding two drops of food coloring per 250 mL of
water.
3. While this lab specifies spinach and carrots as the plants to test, most any green leafy plant or
vegetable will work equally well. Make sure that the plant or vegetable is fresh. Your
students can extract the anthocyanins in red cabbage or blood oranges with 70% isopropanol.
Another source of carotenoids is red bell pepper, extracting with acetone.
4. The masses of spinach and carrot given in the student procedure should produce a suitably
concentrated mixture. However, it is wise to try this procedure yourself with the plants of
choice to confirm that the amount of plant and volume of solvent work well. This will
prevent your students from repeatedly diluting their samples to bring the absorbance
measurements down to an acceptable range [(absorbance max) < ~1.5].
5. Acetone or petroleum ether will permanently damage plastic cuvettes. Thus, your students
must use a glass cuvette or a 13 × 100 mm glass test tube when they are testing carotenes
from a carrot or other vegetable. The student procedure is written for a glass test tube because
it is more readily available and less expensive than a glass cuvette.
6. It is best for your students to handle the acetone, or petroleum ether, in a hood. If your room
does not have a hood, circulate the room air as much as possible without causing a
windstorm.
7. A 13 × 100 mm glass test tube will fit nicely into a spectrometer if the set screws are
loosened so that the tips of the set screws are flush with the inside wall of the Vernier
Spectrometer’s cuvette holder. You will need a small diameter, flathead screwdriver to turn
the set screws. The Vernier SpectroVis holds the test tube with no adjustment.
8. It is very important for your students to align the test tube in the cuvette holder the same way
for calibration as for testing the carrot extract. If they mark the test tube in some way and use
the mark for alignment in the cuvette holder, their absorbance measurements will be
optimally precise.
9. The graphs on the next page are examples of the absorbance spectra your students will see,
but small differences are likely. Your students’ graphs may vary depending on how long they
allow the mixtures to sit before they measure absorbance, as well as the solvent they use.

Experiment 13
SAMPLE DATA
Sample absorbance spectrum of food coloring solutions
Sample absorbance spectrum of chlorophyll from spinach leaves

Teacher Information The Visible Spectra of Plant Pigments
Sample absorbance spectrum of carotene
Trial Food Coloring Peaks or unique features of the spectrum
1 Blue The plot has one large, wide peak at about 630 nm
and a very small peak at about 410 nm.
2 Yellow The plot shows a wide “hump” of absorbance in the

400–440 nm range with a peak at about 420 nm.
The mixture looks green to the naked eye and

3 Mixture shows the combined absorbance peaks of the blue
and yellow food coloring.
There are 3-4 peaks in the blue region (~412, 435,

4 Spinach Extract 460, and 466 nm) and one peak in the red region at
about 665 nm.
The plot has two distinct peaks at about 450 nm and

5 Carrot Extract 475 nm. There is also a “shoulder” of absorbance at
about 420 nm–430 nm.
1. Answers will vary. Students should provide a fairly detailed description of the absorbance
spectrum of the food coloring samples, specifying the regions of peak absorbance. Students
should also recognize that, while the mixture of blue and yellow food coloring is a green
color to the naked eye, it’s absorbance spectrum is a combination of the characteristics of
blue and yellow rather than a unique plot with different peaks.
2. Answers may vary, depending on the source your students use. A common answer will
describe the absorbance peaks of chlorophyll α as ~420 nm and ~660 nm, and chlorophyll β
as ~435 nm and 643 nm. In most cases your students’ graphs will not have sufficient
precision to distinguish between the two types of chlorophyll.

Experiment 13
3. Answers may vary, depending on two factors: (1) the resource cited; and (2) the solvent used.
The two common carotenes, α and β, have three absorbance peaks in the range: 420 nm to
480 nm. The peaks of α carotene are: 420 nm, 440 nm, and 470 nm. The peaks of β carotene
are: 425 nm, 450 nm, and 480 nm. As with chlorophyll, the two common forms of carotene
are very difficult to distinguish from each other on an absorbance spectrum graph.
4. Answers will vary. Students should note that the peak absorbances for the food coloring
mixture (which is a green color) fall in similar wavelength regions as the spinach extract. The
carrot extract compares reasonably well with the yellow food coloring. Students may also
point out that the wavelengths of light absorbed by a particular substance contributes directly
to its color as viewed by the naked eye.

Computer
Determination of 14
Chlorophyll in Olive Oil
Olive oil is made by pressing or extracting the rich oil from the olive fruit. It seems like a simple
matter to press the olives and collect the oil, but many oil extraction processes exist for the many
different types of olives grown around the world. To complicate things further, there are also
various grades of olive oil, and carefully selected groups of officials meet to define and redefine
the grading of olive oil. To help make our experiment a more scientific and less political
exercise, we will winnow our investigation of olive oil down to a manageable few variables.
After processing, olive oil comes in three common grades: extra virgin, regular, and light. Extra
virgin olive oil is considered the highest quality. It is the first pressing from freshly prepared
olives. It has a greenish-yellow tint and a distinctively fruity aroma because of the high levels of
volatile materials extracted from the fruit. Regular olive oil is collected with the help of a warm
water slurry to increase yield, squeezing every last drop of oil out of the olives. It is pale yellow
in color, with a slight aroma, because it contains fewer volatile compounds. Light olive oil is
very light in color and has virtually no aroma because it has been processed under pressure. This
removes most of the chlorophyll and volatile compounds. Light olive oil is commonly used for
frying because it does not affect the taste of fried foods, and it is relatively inexpensive.
The visible light absorbance spectrum of chlorophyll gives interesting results. The chemistry of
chlorophyll (some references site four types: a, b, c, and d) creates absorbance peaks in the
400–500 nm range and in the 600–700 nm range. The combination of visible light that is not
absorbed appears green to the human eye, but different sources of chlorophylls will have
different ratios of these peaks, which create various shades of green. The ability of chlorophyll to
soak up light energy across a wide swath of the visible range helps power photosynthesis at
optimum efficiency in plants.
In this experiment, you will have two primary goals. First, you will analyze the various grades of
olive oil to determine the absorbance peaks that are present and the relative amount of
chlorophyll found in each grade. You will use a Spectrometer to measure the absorbance of the
olive oil samples over the visible light spectrum. You will then test an unknown sample of olive
oil and grade it as extra virgin, regular, or light.
OBJECTIVES
• Measure and analyze the visible light absorbance spectra of three standard olive oils: extra
virgin, regular, and light.
• Measure the absorbance spectrum of an “unknown” olive oil sample.
• Identify the unknown olive oil as one of the three standard types.

Computer 14
MATERIALS
computer olive oil of unknown grade
Logger Pro five cuvettes and lids
Spectrometer plastic Beral pipets
samples of three olive oil standards: distilled water
extra virgin, regular and light isopropyl alcohol
PROCEDURE
2. Connect the Spectrometer to the USB port of your computer.
3. Start Logger Pro. If it is already running, choose New from the File menu.
4. Obtain small volumes of the three standard and one unknown olive oils. Transfer enough of
one olive oil sample to fill a cuvette 3/4 full. Place a lid on the cuvette and mark the lid.
Prepare all of your samples in this way so that you have four cuvettes of olive oil with
labeled lids.

a. Prepare a blank by filling an empty cuvette 3/4 full with distilled water.
b. Choose Calibrate Spectrometer from the Experiment menu.
c. When the warmup period is complete, place the blank in the Spectrometer. Make sure to
d. Click Finish Calibration, and then click .
Part I Comparing Three Grades Of Olive Oil and Identifying an Unknown

For Part I of this experiment, you will calibrate the Spectrometer with distilled water. Your goals
are: (1) to compare the absorbance spectra of the different grades of olive oil; and (2) to identify
the grade of an unknown sample of olive oil.
6. Conduct a full spectrum analysis of an olive oil sample.
a. Place one of the olive oil samples in the Spectrometer.
b. Click . A full spectrum graph of the olive oil will be displayed. Review the graph to
identify the peak absorbance values. Click to complete the analysis.
7. To save your data, choose Store Latest Run from the Experiment menu.
8. Repeat Steps 6–7 with the remaining olive oil standard samples.
9. Repeat Step 6 with the unknown. Note: Do not store the last run.
10. Examine the plots of the olive oil samples. Before continuing with data collection, answer the
Part I Data Analysis questions. Print or save your experiment file, if instructed to do so.
11. Rinse and clean the cuvettes and other oil-bearing containers with isopropyl alcohol.

Determination of Chlorophyll in Olive Oil
Part II Comparing the Chlorophyll Concentration of Regular and Extra Virgin Olive Oil
In Part II, you will use the light grade of olive oil to calibrate the Spectrometer and presume that
light olive oil contains no chlorophyll. Next, you will compare the chlorophyll content of the
regular grade with the extra virgin grade.
12. Set up a new file and calibrate the Spectrometer using light olive oil.
b. Prepare a blank by filling an empty cuvette 3/4 full with light olive oil.
c. Choose Calibrate Spectrometer from the Experiment menu.
d. When the warmup period is complete, place the light olive oil blank in the Spectrometer.
Make sure to align the cuvette so that the clear sides are facing the light source of the
Spectrometer
e. Click “Finish Calibration”, and then click .
13. Measure the absorbance spectrum of regular and extra virgin olive oil.
a. Remove the cuvette of light olive oil from the Spectrometer and replace it with the cuvette
of regular olive oil.
b. Click . A full spectrum graph of the regular olive oil will be displayed. Note the
slight difference in the plot as a result of using the light olive oil as the calibration blank.
Click .
c. To save your data, choose Store Latest Run from the Experiment menu.
d. Measure the absorbance spectrum of the extra virgin grade in the same way.
14. Print or save your experiment file as directed.
DATA ANALYSIS
Part I Comparing Three Grades Of Olive Oil and Identifying an Unknown
1. Describe the graph of each of the standard olive oil
solutions. Emphasize the differences between each
grade of olive oil, identifying the absorbance peaks
and other distinguishing features.
2. Compare the absorbance spectra of the three grades

of olive oil with the sample graph in Figure 1. What
evidence is there that regular and extra virgin olive
oil contain chlorophyll while the light grade of olive
oil does not?
3. Identify your unknown olive oil as extra virgin,

regular, or light. Explain your choice. Figure 1
Part II Comparing the Chlorophyll Concentration of Regular and Extra Virgin Olive Oil
4. Which grade of olive oil, regular or extra virgin, contains the greater amount of chlorophyll?
Use your absorbance spectrum graphs to speculate about how much more chlorophyll one
grade contains compared to the other.

Computer 14
EXTENSIONS
1. Chlorophyll is a fluorescent molecule. Fluorescent molecules can absorb light of one
wavelength and then reemit light at a new and longer wavelength of light. As you have seen
in this exercise, chlorophyll absorbs light in the violet and blue regions of the spectra. If you
were to shine a violet or blue light through a sample of extra virgin olive oil, you would see
the oil turn red in color. The intensity of the red color is an indication of how much
chlorophyll is in the olive oil. The “Long-Wave UV Pen Light” from Bio-Rad Laboratories,
Inc. (Catalog # 166-0530EDU) can be used for this purpose. Shine the light from the
Long-Wave UV Pen Light through a cuvette containing extra virgin olive oil. Does the
sample that is hit by the light turn red in color? Repeat this test for regular olive oil, light
olive oil, and your unknown. Could you use this method to determine if a sample of olive oil
is really extra virgin olive oil? Could you use this method to determine the grade of any
sample of olive oil?
2. Fluorescence spectroscopy is another method that can be used to determine the quality of
olive oil. In fluorescence spectroscopy, a sample can be “excited” with a chosen wavelength
of light and the resulting fluorescence from the sample can be measured and quantified. The
SpectroVis Plus from Vernier Software & Technology can be used for this purpose. Follow
the directions below to measure the fluorescence of all of your olive oil samples using the
SpectroVis Plus.
File menu.
b. Place the cuvette containing the extra virgin olive oil into the cuvette slot of the
Spectrometer.
c. Choose Change Units ►Spectrometer from the Experiment menu and select Fluorescence
405 nm.
d. Choose Set up Sensors ►Spectrometer from the Experiment menu and change the sample
time to 150 ms.
e. Click . A full spectrum graph of the fluorescence of the oil will be displayed. Note
that one area of the graph contains a peak at approximately 675 nm. This peak is from
chlorophyll. Click .
f. Adjust the sample time to increase or decrease the size of the fluorescent peak. If the peak
intensity is above 1, decrease the sample time by 10 ms and collect a new fluorescent
spectrum. Continue to decrease the sample time until the peak is fully visible. If the
fluorescent peak is below 0.3, increase the sample time by 10 ms and collect a new
fluorescent spectrum. Continue to increase the sample time until the peak fluorescent
amplitude for the chlorophyll is above 0.8.
g. Once you have a nice peak, store your data by choosing Store Latest Run from the
Experiment menu.
h. Collect full spectrum graphs from the remaining olive oil samples. Do not adjust the
sample time.
i. Compare the fluorescent spectra of the three grades of olive oil. The peak that is visible at
approximately 675 nm is from chlorophyll. Which sample has the largest peak in this
region?
j. Using the fluorescence from the known olive oil samples as your standards, determine the
quality of your unknown olive oil sample.
k. Compare your results using fluorescent spectroscopy to your results using traditional
spectroscopy. Is one method better than the other? If so, please explain why.
l. Print or save your experiment file as directed.

Experiment
Determination of Chlorophyll
in Olive Oil
collection with a spectrophotometer. It can be conducted using a Vernier Spectrometer or any
other visible spectrophotometer supported by Logger Pro and LabQuest.
2. There can be a wide variety of types and grades of olive oil available in a grocery store. The
International Olive Oil Council defines several grades of olive oil, and the United States
Department of Agriculture has its own grading standards. Your students will achieve best
results if they test one grade labeled “Light” or “Extra Light”, one regular grade and a third
that is labeled “Extra Virgin”. Regular olive oil may not have a specified grade.
3. If you encourage your students to bring samples of olive oil from home, expect the various
grades of regular and extra virgin to contain different amounts of chlorophyll. This will be
evident by the height of the absorbance peaks on the spectrum plots. This can present an
interesting possibility for an extended research project for your students.
4. The cuvette should be ~3/4 full to get good absorbance measurements and allow enough
room to seal the cuvette with a plastic cap. It is important to seal the samples to avoid time
consuming clean up of any spilled oils.
5. Because this lab is qualitative in nature, and olive oil can be difficult to clean from a cuvette,
students can prepare their samples in different cuvettes and still achieve good results.
6. Any type of olive oil can be used as the unknown.
7. The example graph of chlorophyll in the student procedure is the absorbance spectrum of a
sample of ~0.5 g of spinach leaves ground up and mixed with ~20 mL of isopropanol.
8. An interesting extension for this lab is to extract carotene from extra virgin olive oil. A
simple method is to add equal volumes of olive oil and acetone to a test tube. Stopper the test
tube and gently invert it a few times to mix the liquids. The acetone layer will be the top
layer. Your students can measure the absorbance spectrum of the extracted carotene and
compare it to the carotene extracted from carrots. Make sure your students use glass test tubes
or glass cuvettes when using acetone as the solvent.
9. You may make this experiment quantitative by supplying your students with a standard
solution of chlorophyll of a specific concentration. The students can prepare a few serial
dilutions of the chlorophyll standard and develop a Beer’s law plot of absorbance vs.
concentration at one or two wavelengths. Any of the absorbance peaks between 400 nm and
500 nm are suitable for this analysis.
Ideas for Inquiry

• Many plant oils contain compounds other than chlorophyll that are fluorescent. Vitamins E,
A, and even some of the B vitamins are fluorescent when illuminated with the proper

Experiment 14
wavelength of light. The SpectroVis Plus from Vernier Software & Technology can be used
to investigate the fluorescent spectra of various plant oils. Students can collect
representative fluorescent spectra from different oils and will see that each spectrum is
unique. Peanut, sunflower, and sesame oil are relatively inexpensive oils that exhibit
different fluorescent spectra. Nut oils such as almond, hazelnut, and walnut oil also have
unique fluorescent spectra. Use the general directions below to collect fluorescent spectra
from different plant oils.
File menu.
b. Place the cuvette containing a given plant oil into the cuvette slot of the Spectrometer.
c. Choose Change Units ►Spectrometer from the Experiment menu and select
Fluorescence 405 nm.
d. Choose Set up Sensors ►Spectrometer from the Experiment menu and change the
sample time to 500 ms.
e. Click . A full spectrum graph of the oil will be displayed. Store your data by
choosing Store Latest Run from the Experiment menu.
f. Collect fluorescent spectra from other oils of interest.
g. Print or save your experiment file as directed.

Teacher Information Determination of Chlorophyll in Olive Oil
SAMPLE DATA
Comparing three grades of olive oil using water as a blank
This unknown most closely matches extra virgin olive oil

Experiment 14
Comparing olive oils using light grade as a blank
1. Answers will vary. Students should describe the light grade as having no distinct absorbance
peaks and an absorbance maximum near 400 nm that gradually decreases through ~500 nm.
Regular grade will have overall greater absorbance than the light grade. Regular grade may
have a recognized absorbance peak at ~400 nm, with smaller secondary peaks at about
450 nm and 480 nm. Some students’ samples will also show a small peak at ~670 nm. The
extra virgin grade will have the greatest overall absorbance, with its peak at 410 –420 nm.
Secondary peaks appear at ~450 nm, ~480 nm, and ~670 nm.
2. Answers will vary. Students should note that the absorbance spectrum of the extra virgin
olive oil is a very close match to the chlorophyll graph. Regular olive oil makes a less strong
case, but it does possess peaks in the same regions as chlorophyll.
3. Answers will vary, based on the unknown the student has chosen. In the sample data above,
the absorbance spectrum of the unknown most closely matched the extra virgin grade.
4. Answers will vary. In all cases, however, it should be clearly evident that extra virgin grade
olive oil contains more chlorophyll than regular grade. The light grade of olive oil may
contain a tiny amount of chlorophyll, but it is generally considered negligible and accounts
for the use of the light grade as a calibration blank. Students may estimate the relative
amounts of chlorophyll by comparing the absorbance values at a specific wavelength. For
example, in the sample graphs it can be seen that at 454 nm the absorbance of the regular
grade was 0.225 and the absorbance of the extra virgin grade was 0.990, and at 482 nm the
absorbance values were 0.180 and 0.820 respectively. Thus, in relative terms, the extra virgin
grade contained about 4.5 times more chlorophyll than the regular grade.

Computer
15
Enzyme Analysis using Tyrosinase
Enzymes are molecules that regulate the chemical reactions that occur in all living organisms.
Almost all enzymes are globular proteins that act as catalysts, substances that speed up chemical
reactions. Enzymes catalyze reactions by reducing the activation energy for a specific reaction to
occur and yet are neither destroyed nor altered during this process. At the molecular level,
enzymes catalyze these reactions by briefly binding to the substrate or reactants to form an
enzyme-substrate complex. The reaction takes place while the substrate is bound to the enzyme,
converting the substrate to the new product. The new product is then released from the enzyme
substrate complex and the enzyme is then free to bind with more substrate.
Enzyme + Substrate → Enzyme-Substrate Complex → Enzyme + Product
Based on this model, the rate at which the product can be produced depends on the amount of
enzyme and substrate that are present during the reaction. If there is excess substrate and a small
amount of enzyme in solution, the reaction rate, or velocity of the reaction, will increase with the
amount of enzyme in the solution. In this case, all of the enzyme molecules are busy catalyzing
reactions even though there is still plenty of substrate that can be turned into product. The
velocity of the reaction can only increase if the concentration of enzyme is increased. Put another
way, the velocity of the reaction should increase in direct proportion to the concentration of
enzyme in solution.
In a case where the enzyme

concentration is kept constant and the
concentration of substrate is increased,
the velocity of the reaction will
increase rapidly until half of the
enzyme becomes saturated with
substrate. At this point, the velocity of
the reaction will not increase as
rapidly. Eventually, the velocity of the
reaction will approach a constant rate
even when the substrate concentration
is increased. When the reaction rate
ceases to increase, the maximum
velocity or Vmax for the reaction has
been reached. A diagram of this case
is presented in Figure 1. Fifty percent
or half of the maximum velocity is Figure 1
referred to as ½Vmax. The substrate
concentration that coincides with ½Vmax is called the Km or Michaelis-Menten constant. At this
concentration, half of the enzyme molecules in solution are bound to the substrate. Biochemists
use these parameters to characterize enzymes and how enzymes react to different substrates. For
example, the Km is inversely related to enzyme binding affinity for a given substrate. In other
words, the smaller the Km, the greater the binding affinity an enzyme has for a substrate.
This exercise is designed to introduce you to the quantitative analysis of enzymes using the
enzyme tyrosinase. Tyrosinase is an enzyme that is involved in melanin synthesis, which gives

Computer 15
skin its color. Tyrosinase is also involved in the browning of fruits, tubers, and fungi that have
been damaged. As shown in Figure 2, in the presence of oxygen (O2), tyrosinase (E) catalyzes the
hydroxylation of tyrosine into the compound 3,4-dihydroxyphenylalanine, or DOPA for short.
Tyrosinase then catalyzes DOPA into dopaquinone which spontaneously converts into
dopachrome. Dopachrome will eventually be turned into melanin. Dopachrome is a colored
compound with a peak absorbance at 475 nm that can be monitored using a Spectrometer or
Colorimeter.
Figure 2
The first step in this series of reactions is the rate-limiting step for the entire process. This means
that tyrosinase is much better at converting DOPA to dopaquinone than it is at converting
tyrosine to DOPA. Knowing which step is the slowest in a complex biochemical process can be
medically important. People with Parkinson’s disease have low levels of dopamine in their brain.
Dopamine is synthesized from tyrosine; the first step involves converting tyrosine to DOPA,
which is also the rate-limiting step for the entire process. As a result, DOPA is given to people
with Parkinson’s disease to increase their natural production of dopamine. This, in turn,
alleviates the symptoms of the disease.
OBJECTIVES
• Observe and compare the reaction rate of two substrates, tyrosine and DOPA.
• Determine the effect of increasing enzyme concentration on the reaction rate of an enzyme
at a given substrate concentration.
• Determine the effect of increasing substrate concentration on the reaction rate of an enzyme
at a given enzyme concentration.
• Estimate the parameters, Vmax, ½Vmax, and Km for your enzyme extract.

MATERIALS
computer 20 mM DOPA solution
Vernier computer interface* 1 mM DOPA solution
Logger Pro 1 mM tyrosine solution
Colorimeter or Spectrometer four 15 mL centrifuge tubes
12 plastic cuvettes with caps 4 mL enzyme extract (on ice)
20-200 µL micropipette** 200 µL micropipette tips (1 box)
100-1000 µL micropipette** 1000 µL micropipette tips (1 box)
0.1M phosphate buffer, pH 6.8
*No interface required if using a Spectrometer.
**Appropriate graduated transfer pipettes (1 and 5 mL) may be substituted.
PROCEDURE
2. Use a 15 mL centrifuge tube to obtain 10 mL of 0.1 M phosphate buffer, pH 6.8. Use another
15 mL centrifuge tube to obtain 10 mL of 20 mM DOPA solution.
3. Prepare a blank by filling a cuvette with 2 mL of buffer. To correctly use cuvettes, remember:
4. Use a USB cable to connect the Spectrometer to your computer. Choose New from the File
menu.

a. Place the blank cuvette into the cuvette slot of the Spectrometer.
b. Choose Calibrate ►Spectrometer from the Experiment menu. The calibration dialog box
will display the message: “Waiting 90 seconds for lamp to warm up.” After 90 seconds,
the message will change to “Warmup complete.”
c. Click Finish Calibration. When the calibration has finished, click .
6. Determine the optimum wavelength for examining the absorbance of dopachrome and set up
the data-collection mode.
a. Empty the blank cuvette. Fill the cuvette with 2 mL of the 20 mM DOPA solution. Add
100 µL of enzyme extract to the cuvette. Cap the cuvette and gently invert it twice. Let the
cuvette sit for two minutes, and then place the cuvette into the Spectrometer.
of the graph contains a peak absorbance. Click .
c. To store your data, choose Store Latest Run from the Experiment menu.
d. To set up the data-collection mode and select a wavelength for analysis, click Configure
Spectrometer Data Collection, .

Computer 15
e. Select Abs vs. Time as the Collection Mode. The maximum absorbance wavelength
(λ max) will be selected. Verify that the maximum absorbance is close to 475 nm. Click
. Remove the cuvette from the Spectrometer and dispose of the solution as directed.
f. Proceed to Step 7.

by opening the file “15a Enzyme Analysis” from the Advanced Biology with Vernier folder of
Logger Pro.
5. Open the Colorimeter lid, insert the blank, and close the lid.
6. To calibrate the Colorimeter, press the < or > button on the Colorimeter to select the
wavelength of 470 nm (Blue). Press the CAL button until the red LED begins to flash and then
release the CAL button. When the LED stops flashing, the calibration is complete. Remove
the cuvette from the Colorimeter and proceed to Step 7.

Part I Comparison of two substrates
7. Obtain 3 cuvettes. Label the cap of Cuvette 1 with a C. Label the cap of Cuvette 2 with a D.
Label the cap of Cuvette 3 with a T.
8. Fill Cuvette 1 with 2 mL of 0.1 M phosphate buffer, pH 6.8. Fill Cuvette 2 with 2 mL of
1 mM DOPA solution. Fill Cuvette 3 with 2 mL of 1 mM tyrosise solution.
9. Do this quickly! Add 200 µL of enzyme extract to Cuvette 1. Cap the cuvette and gently
invert the cuvette twice. Place it in the device (close the lid if using a Colorimeter).
Click . Absorbance data will be collected for 200 s. Discard the cuvette contents as
directed by your instructor at the end of the run.
11. Repeat Steps 9–10 for Cuvette 2 and Step 9 Cuvette 3 (do not store the data for Cuvette 3).
12. Select the initial linear region of your data on the graph. This should correspond to the first
60 seconds of data.
13. Click Linear Fit, . Check all three runs of data. Click and a best-fit linear regression
line will be displayed for each run selected.
14. Record the value of the slope, m, for each run in Table 1.
15. (Optional) Print or choose Save As from the File menu to save your data.
Part II Increasing enzyme concentration

16. Choose Clear All Data from the Data menu, and close any remaining analysis objects.
17. Prepare 15 mL of 5 mM DOPA solution. Use the 20 mM DOPA solution as a stock solution
and add the appropriate amount of buffer to a 15 mL centrifuge tube. Ask your instructor if
you have questions about how to prepare this solution.

18. Obtain 5 cuvettes. Fill each cuvette with 2 mL of 5 mM DOPA solution.

19. Do Steps 19 and 20 quickly! Add 200 µL of enzyme extract to the first cuvette.
20. Cap the cuvette and gently invert it twice. Place it in the device (close the lid if using a
Colorimeter). Click . Absorbance data will be collected for 200 seconds. Discard the
cuvette contents as directed by your instructor at the end of the run.
22. Collect data for Cuvettes 2–5. Remember to add the extract and then quickly move to
Step 20.
• Add 150 µL of the enzyme extract to Cuvette 2. Repeat Steps 20–21.
• Add 0 µL of the enzyme extract to Cuvette 5. Repeat Step 20 (do not store the data for the
last run).
23. Select the initial linear region on the graph. This should correspond to the first 60 seconds of
the record. Click Linear Fit, ,and check all runs. Click and a best-fit linear
regression line will be shown for each run selected.
24. Record the value of the slope, m, for each of the five runs in Table 2.
Part III Increasing substrate concentration

26. Choose Clear All Data from the Data menu.
27. Obtain 15 mL of 20 mM DOPA solution in a centrifuge tube.
28. Obtain 6 cuvettes. Fill Cuvette 1 with 2 mL of 20 mM DOPA solution.
29. Fill Cuvettes 2–6 with 2 mL of 15, 10, 5, 2.5 and 1 mM DOPA respectively. Add the
appropriate amount of 20 mM DOPA to the cuvette and then dilute with the proper amount
of buffer. Ask your instructor if you have any questions about how to prepare these solutions.
30. Do this quickly! Add 100 µL of enzyme extract to the first cuvette. Cap the cuvette and
gently invert the cuvette two times. Place it in the device (close the lid if using a
Colorimeter). Click . Absorbance data will be collected for two minutes. Discard the
32. Repeat Steps 30–31 to collect data for each cuvette (Note: Do not store your data for the last
cuvette).
33. Select the initial linear region of your data on the graph. This should correspond to the first
60 seconds of the record. Click Linear Fit, ,and check all runs. Click . A best-fit
linear regression line will be shown for each run selected.
34. Record the value of the slope, m, for each of the six substrate concentrations in Table 3.

Computer 15
DATA
Table 1
Substrate Rate (Δ abs/second)
Buffer
1 mM DOPA
1 mM Tyrosine
% Difference in reaction rate = Rate DOPA/Rate Tyrosine x 100

% = ______________________
Table 2
Enyzme Enzyme conc. Rate

(µL) (relative) (Δ abs/second)
200 4
150 3
100 2
50 1
0 0
Slope, m, from Linear Fit = _________________________
Table 3
Substrate concentration Rate

(mM) (Δ abs/second)
20
15
10
2.5
Estimated Vmax Estimated ½Vmax Estimated Km

PROCESSING THE DATA

1. Calculate the percent difference in the rate of dopachrome production for DOPA and
tyrosine. Divide the slope for DOPA as a substrate by the slope for tyrosine as a substrate.
Multiply this number by 100 to get the percent difference. Record this value in Table 1.
2. Create a bar graph of your data in Logger Pro.

a. Open the file “15b Enzyme Analysis” from the Advanced Biology with Vernier folder of
Logger Pro.
b. In the table on Page 1, enter the reaction rate (slope) for buffer, DOPA, and tyrosine in
Data Set 1.
c. Click Next Page, , to go to Page 2 and change the scale if necessary.

3. Create a graph of your data in Logger Pro.
a. In the Data Set for Part II on Page 1, enter the reaction rate you observed for 0, 50, 100,
150 and 200 µL of enzyme extract in the space provided.
b. Go to Page 3 and change the scale if necessary.
c. Click the Linear Regression button, . Record the slope value in Table 2.

4. Create a graph of your data in Logger Pro.
a. In the Data Set for Part III on Page 1, enter the reaction rate you observed for each
substrate concentration.
b. Go to Page 4 and change the scale if necessary.
c. Click Curve Fit, .
d. Choose Natural Exponent (y = A*exp(–Cx)+B) as the General Equation. Click .
e. A best-fit curve will be displayed on the graph. The curve should match up well with the
points. If the curve has a good fit with the data points, click .
5. The slope that you observed for 20 mM DOPA will be used as an estimate of Vmax. Record
this value into the space provided in Table 3.
6. Multiply the slope you observed at 20 mM DOPA by 0.5. This value will be your estimate of
½Vmax. Record this value in Table 3.
7. Choose Interpolate from the Analyze menu. Find the point on your graph that corresponds to,
or is closest to, the estimated value for ½Vmax. Record the corresponding concentration in
mM in the space provided in Table 3. This is your estimate of the Km of your enzyme extract.
8. (Optional) Save your file and print copies of your graphs.

Computer 15
QUESTIONS
1. Was the change in absorbance faster for DOPA or tyrosine? How many times faster was the
change in absorbance for DOPA?
2. Do your data indicate that the conversion of tyrosine to DOPA is the rate limiting step in the
production of dopachrome? Why?

3. How does changing the concentration of enzyme affect the change in absorbance?
4. Is the change in absorbance proportional to the amount of enzyme in solution? Is the trend
linear for your data set?
5. Use your results to predict what the rate of dopachrome production would be is you had used
25 µL of enzyme extract. Predict what the rate would be for 400 µL of enzyme extract.

6. Do your data for this exercise resemble Figure 1? Is 20 mM of DOPA above or below Vmax
for your sample of enzyme?
7. Were you able to estimate ½Vmax and the Km for your enzyme extract?
8. What would happen to the Vmax and Km if you repeated this exercise with twice the
concentration of enzyme? What would happen to these parameters if you cut the enzyme
concentration in half?
EXTENSIONS
1. Calculate the rate of dopachrome production for each run.
Reaction rate = (Δ abs/s)/(3700 M-1 cm-1) x 1 cm
2. Calculate the specific enzyme activity of your enzyme extract. Specific enzyme activity can
be defined as the reaction rate of the extract for a known concentration of substrate divided
by the amount of protein found in 1 mL of the extract. The “Got Protein? Kit” from Bio-Rad
Laboratories Inc. (Catalog # 166-2900EDU) can be used for this purpose.
3. Using the data from Part III of this exercise, construct a Lineweaver–Burke plot and calculate
the apparent Km, Vmax, and ½Vmax of tyrosinase from your extract. A Lineweaver-Burk plot is
a double reciprocal plot of the data in Part III of this exercise. To construct a Lineweaver-
Burk plot take the inverse of the slope and substrate concentrations in Part III. Plot the
inverse of the slope on the y-axis and the inverse of the substrate concentrations on the
x-axis. Your data should now be linear. Perform a linear fit of the data. The y-intercept of this
line is equivalent to 1/Vmax and the x-intercept is equivalent to –1/Km. Do the following to
perform a Lineweaver-Burk pot using Logger Pro.
a. Open the file “15b Enzyme Analysis” from the Advanced Biology with Vernier folder of
Logger Pro.
b. Make sure that you have entered data for Part III on Page 3 on the file.

c. Go to Page 5 and change the scale if necessary. A double reciprocal plot of your data
should now be visible.
d. Verify that the scale of the x-axis is a negative value so you can see the x-intercept.
e. Click the Linear Regression button, . Record the slope and y-intercept. Calculate the
inverse of the y-intercept to get the apparent Vmax.
f. Multiply Vmax by 0.5 to get ½Vmax.
g. Remember that Xi = –b/m, where b is the y-intercept and m is the slope. Find the
x-intercept using this formula. Take the inverse of the x-intercept and multiply by –1 to
get the apparent Km.
h. Compare the calculated values to your estimated values in Part III of this exercise.
4. Construct a Lineweaver-Burk plot using the reaction rate for dopachrome production. See
Extension 1.
5. The substrate concentrations in all of your experiments are off by a small amount. This is
because you have added a small volume of enzyme extract to each cuvette. This small
volume is diluting the initial substrate concentrations. Go through all of these exercises and
correct for this source of error.

Experiment
1. For best results, we recommend doing this experiment with a Spectrometer. You can get
results using a Colorimeter. However, the absorbance values may not be as high as shown in
the Sample Data below because the wavelength does not match the peak absorbance for the
reaction. Note: Calculator users must perform this activity with a Colorimeter. EasyData does
not support the use of Spectrometers.
Logger Pro (computers), and EasyData (calculators), can be found on the CD that
3. The Logger Pro experiment files for this activity are located in the Advanced Biology with
Vernier folder of Logger Pro 3.8.2 and newer. If your computer is too old to upgrade to
Logger Pro 3.8.2, contact Vernier Technical Support for the experiment files.
4. This experiment can be conducted in a standard college lab period of three hours. For shorter
laboratory periods it may take a single group 2–3 lab periods to complete this exercise. A
good breaking point is after the completion of Part II. If time is limited, different groups can
be assigned each part of the exercise and the data can be shared. High school instructors may
want to perform Part I as a demonstration in class and then allow each group to conduct
Parts II and III during a separate laboratory period.
5. To prepare 0.1 M sodium phosphate buffer stock solutions:

a. Add 26.81 g of Na2HPO4•7H2O to distilled water to make a total volume of 1 L. Label this
stock solution A.
b. Add 13.80 g of NaH2PO4•H2O to distilled water to make a total volume of 1 L. Label this
stock solution B.
c. Store in capped containers at 4ºC. Stock solutions are stable for weeks at this temperature.
6. To prepare 0.1 M sodium phosphate buffer:
a. Combine the two stock solutions until the pH is 6.8. Add about 208 mL of solution A to
192 mL of solution B to obtain 400 mL of buffer with a pH of 6.8.
b. Store in a capped container at 4ºC. Phosphate buffer can be stored for a week at this
temperature.
7. To prepare 20 mM DOPA (throughout the lab, DL-DOPA is referred to as DOPA):
a. Add 0.394 g DOPA to 100 mL of phosphate buffer.
b. Stir using a magnetic stirrer at low speed. The DOPA will dissolve in 5–10 minutes.
c. DOPA can be made a few days ahead of time. For storage, divide the DOPA into
20–25 mL aliquots and store in capped bottles at 4ºC for up to 72 hours. Discard if the
solution turns black or brownish.
8. To prepare 1 mM DOPA:
a. Add 5 mL of 20 mM DOPA to 95 mL of phosphate buffer.
b. Store at 4ºC in a capped bottle for up to 72 hours. Discard if the solution turns black or
brownish.

Experiment 15
9. To prepare 1 mM tyrosine:
a. Add 0.018 g of tyrosine to 100 mL of buffer phosphate buffer. If a suitable balance is not
available, add 0.1 g tyrosine to 500 mL of buffer.
b. Stir using a magnetic stirrer at low speed. The tyrosine will dissolve in approximately
1 hour.
c. Store at 4ºC in a capped bottle for up to 72 hours.
10. To prepare the enzyme extract:
a. Peel a potato and cut into small pieces.
b. Place 100 g of potato in a blender. Add 200 mL of ice-cold phosphate buffer.
c. Blend for 5 minutes using 5 second bursts. Wait 5 seconds between bursts.
d. Filter through four layers of cheesecloth into a pre-chilled container on ice.
e. Fill microcentrifuge tubes with filtrate and centrifuge at 6000 RPM for 5–10 minutes. If
possible, place the centrifuge in a cold room or refrigerator during this process.
f. Carefully remove the centrifuge tubes and verify that starch pellets are visible at the
bottom of the filtrate.
g. Transfer the supernatant to a new microcentrifuge tube using a pipette. Be careful not to
disturb the starch pellet at the base of the tube.
h. Cap and label the tubes with an E and place on ice. This is the enzyme extract.
i. Use immediately or store at 4ºC for up to 2 hours before use.
j. The extract can be stored at –20ºC in a frostless freezer for a period of 4–5 days. The
enzyme activity will be reduced, but should be sufficient to complete this exercise.
11. Enzyme extract previously frozen at –20ºC should give good results for Parts II and III of the
exercise. For Part I, use fresh enzyme extract to see a significant rate of dopachrome
production when tyrosine is used as a substrate.
12. The activity of the enzyme extract will need to be tested before it is used by the students. To
determine the activity of the extract:
a. Follow the student instructions for Part III, using 20, 15, 10 and 5 mM DOPA.
b. To get good Vmax data, the initial slope for 20 and 15 mM DOPA should be close to the
same value (see sample data below).
c. The initial slopes for 5, 10, and 15 mM DOPA should also be significantly different (see
sample data).
d. If good Vmax data is not observed, the enzyme activity is probably too high. Repeat the test
and decrease the volume of enzyme in the cuvette by 25%.
e. Repeat until the appropriate relationship is observed.
f. Use this new enzyme volume for Part III of the exercise.
g. Adjust the enzyme concentrations for Parts I and II accordingly.
13. For Part II, students should add 2.5 mL of 20 mM DOPA to 7.5 mL phosphate buffer to make
10 mL of 5 mM DOPA solution.

14. To make the dilution series for Part III, students can use the following equation to solve for
the proper volume of stock solution and buffer that should be added to each cuvette.
C1V1 = C2V2
• V2 is 2 mL. This is the final volume of cuvette.
• C1 is 20 mM. This is the concentration of the stock solution.
• C2 is the desired final concentration in mM.
• Solving for V1 will provide the amount of stock solution to be added to the cuvette.
• Subtract V1 from V2 to get the amount of buffer that needs to be added to the cuvette.
15. For the dilution series in Part III, the correct volume of stock solution and buffer for each
cuvette is provided in the table below.
Cuvette Stock solution of DOPA Buffer (mL) Final conc.

(mL) (mM)
1 2.0 0.0 20
2 1.5 0.5 15
3 1.0 1.0 10
4 0.5 1.5 5
5 0.25 1.75 2.5
6 0.1 1.9 1
16. We recommend using Logger Pro for performing the Processing the Data procedure, whether
you use computers, LabQuest, or calculators to collect data. It is possible to complete the
Processing the Data procedure using LabQuest, however it requires a multi-step process of
entering data values. If you would like to complete the Processing the Data procedure using
LabQuest, student instructions are available on the CD that came with this book. It is not
possible to conduct the analysis using calculators.
17. Make sure that only the initial rate is used when calculating the slope of each run. This
typically corresponds with the first 60 seconds of data collected (see sample data).
Ideas for Inquiry
• Compare the substrate catechol to DOPA. Tyrosinase is also known as catechol oxidase.
Tyrosinase will oxidize the compound catechol to ortho-quinone, which spontaneously
converts to a colored product with a peak absorbance at 540 nm. Repeat Part III of this
exercise using catechol as a substrate.
• Compare stereoisomers of tyrosine or DOPA as substrates for this enzyme. Some enzymes are
very specific for a given substrate and will only react with the stereoisomer of a given
compound. Repeat Part I of this exercise and compare D to L-DOPA.
• Compare the activity of tyrosinase from different sources. Tyrosinase can be found in bananas,
mushrooms, apples, yams and other fruits, tubers, and vegetables. Prepare enzyme extracts
from different sources and compare the activity of tyrosinase from each source. Use the “Got
Protein? Kit” from Bio-Rad (Catalog # 166-2900EDU) to control for different amounts of
protein that may be found in each source. Instructions for how to prepare the enzyme extract
can be found above.

Experiment 15
HAZARD ALERTS
3,4-dihydroxyphenylalanine (DL-DOPA): Respiratory, skin and eye irritant; moderately toxic by
ingestion. HMIS hazard rating: 2–Moderately hazardous. Wear gloves and eye protection and
appropriate dust mask (N95 or equivalent).
The hazard information reference is: Sigma-Aldrich, MSDS for product D9503, (800) 325-5832,
www.sigmaaldrich.com.
Tyrosine (DL-TYROSINE): Possible respiratory, skin and eye irritant; possibly toxic by
ingestion. Hazard code: 1–Possibly hazardous. Wear gloves and eye protection and appropriate
dust mask (N95 or equivalent).
The hazard information reference is: Flinn Scientific, Inc., Chemical and Biological Catalog
Reference Manual, (800) 452-1261, www.flinnsci.com.
SAMPLE DATA AND GRAPHS

Table 1
Rate
(Δ abs/s)
Substrate
Buffer (Control) 0.00010
1 mM DOPA 0.00201
1 mM Tyrosine 0.00028
% Difference in reaction rate = Approximately 720%
Table 2
Enzyme volume Enzyme concentration Rate

(µL) (relative) (Δ abs/s)
200 4 0.0044
150 3 0.0034
100 2 0.0023
50 1 0.0009
0 0 0.0001
Slope from linear fit = 0.001160 abs/s

Table 3
Substrate concentration Slope

(mM) (Δ abs/s)
20 0.00320
15 0.00310
10 0.00280
5 0.00200
2.5 0.00130
1 0.00064
Estimate of Vmax Estimate of ½Vmax Estimate of Km
0.00320 Δ abs/s 0.00160 Δ abs/s 3.4 mM
Comparison of tyrosine and DOPA as substrates for tyrosinase
Logger Pro bar graph comparing the rate of enzyme activity for tyrosine and DOPA

Experiment 15
The effect of increasing enzyme concentration on the rate of enzyme activity
Plot demonstrating that enzyme activity is proportional to enzyme concentration
The effect of increasing DOPA concentration on the rate of enzyme activity

Plot demonstrating that enzyme activity approaches Vmax at 20 mM DOPA
1. The rate of dopachrome production should be much faster for DOPA than tyrosine. Results
will vary depending on the quality of the enzyme extract, but the rate for 1 mM DOPA should
be at least five times that of 1 mM Tyrosine.
2. When tyrosine is used as the substrate, dopachrome production is very slow or not apparent.
The same concentration of DOPA is converted to dopachrome at a much faster rate. This
suggests that the conversion of tyrosine to DOPA is the rate-limiting step in the production of
dopachrome.
3. As the enzyme concentration is increased the rate of the reaction should also increase. The
slope for each run should get steeper (see sample data).
4. The change in absorbance should be roughly proportional to the volume of enzyme extract
used. The trend should be linear (see sample data).
5. The rate of dopachrome production for 25 µL of enzyme extract should be 50% of the rate for
50 µL of extract. The rate for 400 µL of enzyme extract should be eight times the rate for
50 µL of the extract.

Experiment 15

6. A graph of the data from this exercise should resemble Figure 1 (see example data). The Vmax
should occur between 15 and 20 mM DOPA. If 20 mM DOPA has the greatest slope, then
Vmax was reached at or above 20 mM DOPA. If the slope for 20 mM DOPA is equal to or
smaller than the slope for 15 mM DOPA, then Vmax was reached between these two
concentrations.
7. Students should be able to estimate the ½Vmax and the Km of the enzyme extract? The rate
observed at 50% of the estimated Vmax is the estimate of ½Vmax. The Km is just the
concentration of DOPA that corresponds to ½Vmax.
8. The Vmax and ½Vmax will decrease if the enzyme concentration is lowered and will increase if
the enzyme concentration is raised. The Km should be independent of the enzyme
concentration.
SAMPLE DATA FOR EXTENSIONS
Reaction rate as a function of substrate concentration (reaction rate

was calculated from dopachrome extinction coefficient)
Lineweaver-Burke plot for tryosinase enzyme extract

Computer
Introduction to Neurotransmitters
16
using AChE
Neurons, the cells of the brain, communicate with each other and the rest of the body by
releasing neurotransmitters. Neurotransmitters are small chemicals that bind to receptors on
other neurons, cells, or tissues of the body. When a neurotransmitter binds to a receptor, a
cellular response is produced in the target cell. If enough target cells are activated, a
physiological response is produced in the body. If the neurotransmitter produces an increase in a
physiological response, we refer to it as an excitatory neurotransmitter. If the neurotransmitter
produces a decrease in a physiological response, we refer to the neurotransmitter as inhibitory.
The physiological effect produced by a neurotransmitter is terminated, in large part, by the action
of enzymes that break down the neurotransmitter.
The neurotransmitter acetylcholine (ACh) is an excellent example of a neurotransmitter that can

be either excitatory or inhibitory. ACh is one of the primary neurotransmitters of the peripheral
nervous system. The skeletal muscles of your body and the cardiac muscles of your heart are all
controlled by neurons that release ACh. Your brain makes your skeletal muscles move by
activating neurons that release ACh on your muscle cells. In contrast, your heart is inhibited by
neurons that release ACh. If these neurons become more active, and release more ACh, your
heart slows down. If these neurons become less active, and release less ACh, your heart will
speed up. Skeletal and cardiac muscles cells also contain an enzyme called acetylcholinesterase
(AChE). This enzyme rapidly breaks down ACh into the compounds acetate and choline,
terminating the action of the neurotransmitter (see Figure 1).
Figure 1
Acetylcholine is also a very important neurotransmitter in the central nervous system. In
Alzheimer’s disease, neurons in the brain that release ACh die. The death of these neurons
decreases the level of ACh in the brain. This decrease in ACh is thought to cause some of the
symptoms of Alzheimer’s disease. The drug tacrine is used to treat Alzheimer’s disease. Tacrine
inhibits the activity of AChE (see Figure 1). This causes an increase in the level of ACh in the
brain, and alleviates some of the symptoms of Alzheimer’s disease. However, tacrine also
inhibits AChE found in skeletal and cardiac muscles, and can produce some unwanted side
effects.

Computer 16
This exercise is designed to introduce you to the pharmacology of neurotransmitters. It is very

difficult to study neurotransmitters directly. In many cases, the activity of an enzyme that
produces or breaks down a neurotransmitter is used instead. A simple method for assaying the
activity of AChE is the Ellman method. The compounds acetylthiocholine iodide (ACTHi) and
dithiobisnitrobenzoate (DTNB) are added to a solution containing AChE. AChE breaks ACTHi
down into acetate and thiocholine. Thiocholine then reacts with DTNB to form a compound
called 5-thio-2-nitrobenzoate (TNB). TNB is a yellow-colored compound with a peak
absorbance at 412 nm, which can be monitored using a Spectrometer or Colorimeter.
Your instructor has homogenized heart tissue and filtered the solution through cheesecloth. A
centrifuge was then used to isolate different fractions from the original homogenate. The nuclear
fraction should contain cell nuclei and other remnants of cardiac cells. The supernatant should
contain components from the extracellular fluid, membrane and intracellular contents of the
cells. In the first part of this activity, you will use the Ellman method to determine which fraction
has the greatest amount of AChE activity.
In the second part, you will create a dose-response curve for the compound tacrine. A dose
response curve is a graph that shows how increasing concentrations of a compound change a
biochemical or physiological process. A dose-response curve for an inhibitor is shown in
Figure 2. The graph has been normalized to the activity of a control that does not contain the
inhibitor. As the concentration
of the inhibitor increases, the
activity decreases. The
concentration that inhibits 50%
of the activity of the control is
called the IC50. This is a
parameter that pharmacologists
use to classify different
compounds. Note that the
shape of the dose-response
curve is shaped like a backward
letter S. However, the center of
the dose-response curve, which
contains the IC50, is usually
linear. You will use the linear
portion of the dose-response
graph to estimate the IC50 for
your data.
Figure 2
OBJECTIVES
• Observe the reaction rate of acetylcholinesterase (AChE) found in heart tissue.
• Compare the reaction rate of AChE from different fractions of heart tissue.
• Observe the effect that the compound tacrine has on the reaction rate of AChE.
• Generate a dose-response curve for the compound tacrine.
• Estimate the effective IC50 of tacrine on heart AChE.

Introduction to Neurotransmitters using AChE
MATERIALS
computer 0.1 M phosphate buffer, pH 7.9
Vernier computer interface* 2 mL 10 mM DNTB
Logger Pro 2 mL 100 mM ACTHi
Colorimeter or Spectrometer 2 mL 1 mM Tacrine
4 plastic cuvettes with caps 2 mL whole extract (on ice)
20–200 µL micropipette** 2 mL supernatant (on ice)
100–1000 µL micropipette** 2 mL nuclear fraction (on ice)
200 µL micropipette tips (1 box) 2 mL dH20 (on ice)
1000 µL micropipette tips (1 box) eight 15 mL centrifuge tubes
*No interface is required if using a Spectrometer.
**Appropriate graduated transfer pipettes (1 mL and 5 mL) may be substituted.
PROCEDURE
1. Obtain and wear goggles and gloves.
2. Use a 15 mL centrifuge tube to obtain 10 mL of 0.1 M phosphate buffer, pH 7.9.
3. Prepare a blank by filling a cuvette with 2 mL of phosphate buffer. To correctly use cuvettes,
remember:
4. Add the following to the blank: 100 µL from the nuclear fraction, 100 µL of DTNB solution
and 100 µL dH20.

menu.

c. Click Finish Calibration and allow the calibration to finish. Click .
7. Determine the optimum wavelength for examining the absorbance and set up the data-
collection mode.
a. Empty the blank cuvette. Fill a new cuvette with 2 mL of phosphate buffer.
b. Add 100 µL of whole extract, 100 µL of DNTB solution, and 100 µL of acetylthiocholine
iodide solution (ACTHi) to the cuvette.

Computer 16
c. Cap the cuvette and gently invert the cuvette three times. Let the cuvette sit for 5 minutes
and then place the cuvette into the Spectrometer.
d. Click . A full spectrum graph of the solution will be displayed. Note that one area
e. Store your data by choosing Store Latest Run from the Experiment menu.
f. To set up the data collection mode and select a wavelength for analysis, click Configure
g. Select Abs vs. Time as the Collection Mode. The wavelength of the maximum absorbance
(λ max) will be selected. Verify that the maximum absorbance is close to 412 nm. Click
.
h. Choose Data Collection from the Experiment menu. Change the data-collection length to
5 minutes. Change the data-collection rate to 30 samples/minute. Click .
i. Remove the cuvette from the Spectrometer and dispose of the solution as directed and
proceed to Step 8.

by opening the file “16a Neurotransmitters” from the Advanced Biology with Vernier folder
of Logger Pro.
wavelength of 430 nm (Blue). Press the CAL button until the red LED begins to flash and
then release the CAL button. When the LED stops flashing, the calibration is complete.
Remove the cuvette from the Colorimeter and proceed to Step 8.

Part I Comparison of heart acetylcholinesterase activity from different fractions
8. Obtain 4 cuvettes and label the caps.
Cuvette 1 = N
Cuvette 2 = S
Cuvette 3 = W
Cuvette 4 = C
9. Fill Cuvettes 1–4 with 2 mL of 0.1M phosphate buffer.
10. Do this quickly! Add 100 µL of whole extract, 100 µL of DNTB solution, and 100 µL of
acetylthiocholine iodide solution to Cuvette 1.
11. Cap the cuvette and gently invert the cuvette three times. Place it in the device (close the lid
if using a Colorimeter). Click . Absorbance data will be collected for 5 minutes.
Discard the cuvette contents as directed by your instructor at the end of the run.
12. Store your data by choosing Store Latest Run from the Experiment menu.
13. Do this quickly! Add 100 µL of supernatant, 100 µL of DNTB solution, and 100 µL of
ACTHi to Cuvette 2.

14. Repeat Steps 11–12.
15. Do this quickly! Add 100 µL from the nuclear fraction, 100 µL of DNTB solution, and
100 µL of ACTHi to Cuvette 3.
17. Do this quickly! Add 100 µL from the nuclear fraction, 100 µL of DNTB solution, and
100 µL of dH20 to Cuvette 4.
19. On the graph, select the most linear region of all data, typically between minutes 1 and 4.
20. Click Linear Fit, . Select all runs and click . A best-fit linear regression line will be
shown for each run selected.
21. Record the value of the rate (slope), m, for each run in Table 1.

23. Choose Clear All Data from the Data menu.
24. Using 1 mM tacrine as a stock, create 10 mL of the following solutions: 1×10-5, 1×10-7,
1×10-9, 1×10-11, and 1×10-13 M tacrine in 0.1 M phosphate buffer. The best way to accomplish
this is to perform a set of serial dilutions. Ask your instructor if you have questions about
how to prepare these solutions.
25. Obtain 6 cuvetttes with caps.

a. Label the cap of Cuvette 1 with a C and add 2 mL of 0.1M phosphate buffer.
b. Label the cap of Cuvette 2 with a –13 and add 2 ml of 1×10-13 M tacrine.
c. Label the cap of Cuvette 3 with a –11 and add 2 ml of 1×10-11 M tacrine.
d. Label the cap of Cuvette 4 with a –9 and add 2 ml of 1×10-9 M tacrine.
e. Label the cap of Cuvette 5 with a –7 and add 2 ml of 1×10-7 tacrine.
f. Label the cap of Cuvette 6 with a –5 and add 2 ml of 1×10-5 tacrine.
26. Select Cuvette 1.
27. Do this quickly! Add 100 µL of whole filtrate, 100 µL of DNTB solution and 100 µL of
ACTHi.
28. Cap the cuvette and gently invert it three times. Place it in the device (close the lid if using a
Colorimeter). Click . Absorbance data will be collected for 5 minutes. Discard the
29. Store your data by choosing Store Latest Run from the Experiment menu.
30. Repeat Steps 27–29 for Cuvettes 2–6.

Computer 16
31. Select all of the data on the graph. Click the Linear Fit button, . Select the correct runs and
click . A best-fit linear regression line will be shown for each run selected.
32. Record the value of the rate, m, for each of the 6 runs in Table 2.
DATA
Table 1
Rate
Source Percent difference
(∆ abs/min)
Whole filtrate 100%
Supernatant
Nuclear fraction
Control
Part II Effect of tacrine on heart acetylcholinesterase activity
Table 2
Tacrine concentration Rate Normalized activity

(M) (∆ abs/min) (%)
Control (no tacrine)
1x10-13
1x10-11
1x10-9
1x10-7
1x10-5
Est. IC50
_____________________
PROCESSING THE DATA

Part I Comparison of heart acetylcholineseterase activity from different cellular fractions
1. Calculate the percent difference in activity for each sample. The rate for the whole filtrate is
considered 100%. Divide the rate for each fraction by the rate of the whole filtrate. Multiply
this number by 100 to get the percent difference from the filtrate. Record this value in the
space provide in Table 1.

2. Create a bar graph of your raw data using Logger Pro.

a. Open the file “16b Neurotransmitters” from the Advanced Biology with Vernier folder of
Logger Pro.
b. Enter the slopes you observed for each fraction in rows 1–4 of the Reaction Rate column.

3. Calculate the normalized activity rate for each concentration of tacrine. The rate for the
control in Table 2 is considered 100%. Take the rate observed for each tacrine concentration
and divide it by rate observed for the control. Multiply this number by 100 to get the
normalized activity rate for each concentration. Record the normalized response for each
tacrine concentration in the space provide in Table 2.
4. Create a dose-response graph of your data using Logger Pro.
a. Click Next Page, , to go to the next page of the file.
b. Enter the percent activity that you calculated for each tacrine concentration in rows 1–6 of
the Normalized Activity column. These values are located in Table 2.
c. Drag your cursor across the central, linear portion of the data. The curve fit from this
portion will be used to estimate IC50 for your data.
d. Click Curve Fit, .
e. Choose Base-10 Logarithm (y = A*log(B*x)) as the General Equation. Click .
f. A best-fit curve for the selected data points will be displayed on the graph. Click .
5. Choose Interpolate from the Analyze menu and find the point on your graph that corresponds
to, or is closest to, a normalized activity rate of 50%. Record the corresponding concentration
using scientific notation in the space provided in Table 2. This is your estimate of the IC50 of
tacrine for your heart tissue extract.
6. (Optional) Save your file. Insert a Text Annotation that corresponds to your estimate of the
IC50 and print copies of your graphs.
QUESTIONS
1. Was the change in absorbance faster for the nuclear fraction or the supernatant? Which
fraction contains the greatest amount of acetylcholinesterase based on your data?
2. How many times faster was the change in absorbance for the whole filtrate when compared
to the supernatant and the nuclear fraction? If you add the rates for the supernatant and the
nuclear fraction together do they equal the rate observed for the whole filtrate.
Part II. Effect of tacrine on heart acetylcholinesterase activity
3. How does increasing the concentration of tacrine affect the change in absorbance?
4. Were you able to estimate the IC50 of tacrine for your sample? How does the IC50 of your
sample compare to data for other students.
5. If tacrine was given to a person, can you explain what would happen to levels of
acetylcholine in the heart, skeletal muscles and brain? Can you explain how this would
happen at the cellular or molecular level?

Computer 16
EXTENSIONS
1. Calculate the reaction rate of TNB production for each run using the formula below. This
value corresponds to the actual enzymatic rate of the acetylcholinesterase in the heart extract.
Reaction rate = (∆ abs/min)/(1415 M-1 cm-1) x 1 cm.
2. Calculate the specific acetylcholinesterase activity of the heart extract for each part of this
exercise. Specific enzyme activity can be defined as the reaction rate of the extract for a
known concentration of substrate divided by the amount of protein found in 1 mL of the
extract. The whole filtrate was prepared at a concentration of 100 mg tissue/mL buffer. Use
the “Got Protein? Kit” from Bio-Rad Laboratories Inc. (Catalog # 166-2900EDU) to
determine the actual protein concentration of the whole filtrate and each fraction.
3. Calculate the IC50 of tacrine from your data and compare this value to your estimate of the
IC50. Take the log of the tacrine concentrations from Part II (located in Table 2) and record
these values. Follow the instructions below to create a new dose-response curve using
Logger Pro.
a. Open a new, blank file in Logger Pro or, if you have a file that was created while doing
Experiment 16, choose Add Page from the Page menu and choose New Data Set and
Graph as the Starting Contents. Click .
b. In the data table, double-click the heading, Data Set, and rename the data set Dose
Response for Tacrine.
c. Double-click the heading of the X column.
d. Enter Log Concentration as the Name and Log Conc as the Short Name. Leave Units
blank. Click .
e. In the Log Concentration column, enter the log of the concentration of tacrine values that
you calculated above.
f. Double-click the heading of the Y column.
g. Enter Normalized Activity as the Name, % Act as the Short Name, and % as the Units.
Click .
h. In rows 1–5 of the Normalized Activity column, enter the percent activity that you
calculated for each tacrine concentration. These values are located in Table 2.
i. Autoscale the graph.
j. Click and drag on the graph to select the portion of your data that is now linear.
k. Click Linear Regression, . Using the linear regression formula, y = mx + b, solve for x
where y = 50%. Record the value for x.
l. Calculate the anitlog, 10x, to determine the calculated value for IC50.

4. Estimate the IC50 of tacrine from your data using the Four Parameter Log Model and
compare this estimate to your original estimate of the IC50. Take the log of the tacrine
concentrations from Part II and record these values. Follow the instructions below to create a
new dose-response curve using Logger Pro.
a. Open a new, blank file in Logger Pro or, if you have a file that was created previously for
this activity, choose Add Page from the Page menu and choose New Data Set and Graph
as the Starting Contents. Click .
b. In the data table, double-click the heading, Data Set, and rename the data set Dose
Response for Tacrine.
c. Double-click the heading of the X column.
d. Enter Log Concentration as the Name and Log Conc as the Short Name. Leave Units
blank. Click .
e. Enter the log of the concentration of tacrine used for each run in rows 1-5 of the Log
Concentration column. These values are located in Table 2.
f. Double-click the heading of the Y column.
g. Enter Normalized Activity as the Name, % Act as the Short Name, and % as the Units.
Click .
h. Enter the percent activity that you calculated for each tacrine concentration in rows 1–5 of
the Normalized Activity column. These values are located in Table 2.
i. Autoscale the graph.
j. Click Curve Fit, , and then click the Define Function button.
k. Enter a+(b–a)/(1+10^((x–c) x d)) as f(x) and Four Parameter Log Model as the
Description. Click .
l. Click . A best-fit curve will be displayed on the graph. The curve should match up
well with the points. If the curve has a good fit with the data points, then click .
m. Choose Interpolate from the Analyze menu and find the point on your graph that
corresponds to, or is closest to, a normalized activity rate of 50%. This is your estimate of
the IC50 of tacrine for your heart tissue extract.
5. Repeat your dose-response curve for tacrine using a larger number of concentrations. Start at
1×10-15 M tacrine and proceed in 10X steps to 1×10-3 M tacrine. Repeat the experiment
3-5 times. Make sure you also run the appropriate number of controls. Calculate the average
response for each concentration. Convert each average response to an actual reaction rate.
See Extension 1. Normalize your data, plot a new dose-response curve and estimate or
calculate the IC50 of tacrine using the instructions in Extension 3.
6. Design an experiment that will determine the Km of acetylcholinesterase from your heart
tissue homogenate. Ask your instructor how to proceed.

Experiment
Introduction to Neurotransmitters
using AChE
1. For best results, we recommend doing this activity with a Spectrometer. You can get results
using a Colorimeter, however, the absorbance values may not be as high as shown in the
Sample Data below because the wavelength does not match the peak absorbance for the
reaction. Note: Calculator users must perform this activity with a Colorimeter. EasyData does
not support the use of Spectrometers.
3. The Logger Pro experiment files for this activity are located in the Advanced Biology with
Vernier folder of Logger Pro 3.8.2 and newer. If your computer is too old to upgrade to
Logger Pro 3.8.2, contact Vernier Technical Support for the experiment files.
4. This experiment can be conducted in a standard college lab period of 3 hours. For shorter
laboratory periods, it may take a single group 2 lab periods to complete this exercise. A good
breaking point is after the completion of Part I. Alternatively, high school instructors can
modify the lab so that students assay the whole filtrate and then compare the activity of a
single tacrine concentration (1x10-6 M).
5. To prepare 0.1 M sodium phosphate buffer stock solutions:

a. Add 26.81 g of Na2HPO4•7H2O to distilled water to make a total volume of 1 L. Label this
stock solution A.
b. Add 13.80 g of NaH2PO4•H2O to distilled water to make a total volume of 1 L. Label this
stock solution B.
c. Store in capped containers at 4ºC. Stock solutions are stable for weeks at this temperature.
6. To prepare 0.1 M sodium phosphate buffer, pH 7.9:
a. Combine the two stock solutions until the pH is 8. About 16 mL of solution B should be
added to 384 mL of solution A to obtain 400 mL of buffer with a pH of 8.
temperature.
7. To prepare 0.1 M sodium phosphate buffer, pH 7.4 for DNTB:
a. Combine the two stock solutions until the pH is 7.4. About 2 mL of solution B should be
added to 10 mL of solution A to obtain 12 mL of buffer with a pH of 7.4.
temperature.
8. To prepare 100 mM acetylthiocholine iodide (ACTHi):
a. Add 0.289 g ACTHi to 10 mL of dH20.
b. Stir using a magnetic stirrer at low speed. The ACTHi will dissolve in 5–10 minutes.

Experiment 16
c. ACTHi can be made up several weeks ahead of time. For storage, divide the ACTHi into
2 mL aliquots in capped centrifuge tubes and store in an opaque container at 4ºC.
d. Each group will need 1–2 aliquots of ACTHi to complete this exercise.
9. To prepare 10 mM DTNB:
a. Add 0.04 g DTNB and 0.015 g sodium bicarbonate to 10 mL of 0.1 M phosphate buffer,
pH 7.4.
b. Stir using a magnetic stirrer at low speed. The DTNB will dissolve in 5–10 minutes.
c. DTNB can be made up 5 days ahead of time. For storage, divide the DTNB into 2 mL
aliquots in capped centrifuge tubes and store in an opaque container at 4ºC.
d. Each group will need 1–2 aliquots of DTNB to complete this exercise.
10. To prepare 1 mM tacrine hydrochloride:
a. Add 0.117 g of tacrine hydrochloride to 5 mL of dH20. This will be a 0.1 M solution of
tacrine.
b. Stir using a magnetic stirrer at low speed. The tacrine will dissolve in 5–10 minutes.
c. Add 100 µL of 0.1M tacrine to 9.9 mL of 0.1M phosphate buffer, pH 7.9. This is now a
1 mM or 1x10-3 M solution of tacrine.
c. The tacrine can be made up 12–24 hrs ahead of time. For storage, divide the 1x10-3 M
tacrine into two 5 mL aliquots. Store in a capped container at 4ºC.
11. To prepare the heart tissue homogenate:
a. Obtain fresh chicken hearts from a grocery store.
b. Cut a single chicken heart into small pieces.
c. Place 10 g of heart tissue in a blender. Add 100 mL of ice cold 0.1 M phosphate buffer.
d. Blend for 3 minutes using 5 second bursts. Wait 5 seconds between bursts.
e. Filter through four layers of cheesecloth into a pre-chilled container on ice.
f. Use immediately or store at 4ºC in a capped container for up to 2 hrs before use.
g. Aliquot the whole filtrate into microcentrifuge tubes using a pipette. Label with a W and
place on ice. This is the whole filtrate.
h. Each group will need 1–2 aliquots of whole filtrate to complete this exercise.
12. To prepare the heart tissue fractions:
a. Fill microcentrifuge tubes with whole filtrate and centrifuge at 6000 RPM for 5 minutes. If
possible, place the centrifuge in a cold room or refrigerator during this process.
b. Carefully remove the centrifuge tubes and verify that pellets are visible at the bottom of
the filtrate.
c. Transfer the supernatant to a new microcentrifuge tube using a pipette. Be careful not to
disturb the pellet at the base of the tube.
d. Cap and label the tubes with an S and place on ice. This is the supernatant.
e. Transfer 1 mL of ice cold 0.1 M phosphate buffer into the microcentrifuge tube with the
pellet and vortex the tube to re-suspend the pellet in the buffer.
f. Label with an N and place on ice. This is the nuclear fraction.
g. Each group will need 1–2 aliquots of supernatant and the nuclear fraction to complete this
exercise.

Introduction to Neurostransmitters using ACHe
13. Use fresh hearts if possible for homogenization. Fresh chicken hearts can be stored at 4ºC for
12–24 hr before homogenization. Chicken hearts frozen at –20ºC will have a reduced rate of
enzymatic activity, but should give decent results for this exercise.
14. The activity of the filtrate will need to be tested before it is used by the students. To
determine the activity of the filtrate:
a. Follow the student instructions in Part I to determine the activity of the whole filtrate.
b. Verify that a significant change in absorbance (at least 0.75 abs/min) is observed over a
5 minute period (see example data).
c. If a significant increase in absorbance is not observed, repeat the test using 200 µL of all
reactants. Keep buffer volume at 2 mL.
d. If a significant increase is observed, adjust the filtrate concentrations for Parts I and II
accordingly.
15. The tacrine will need to be tested before it is used by the students. To determine the activity
of the tacrine on the whole filtrate:
a. Prepare 5 mL of 1x10-9 M tacrine hyrdrochloride in 0.1M phosphate buffer.
b. Follow the student instructions for Part II to determine the inhibitory action of 1x10-9 M
tacrine on the whole filtrate.
c. To get good IC50 data, the normalized response for 1x10-9 M tacrine should be
significantly lower than or close to 50% of the activity of the whole filtrate (see sample
data).
16. To make the dilution series for Part II, students can use the following equation to solve for
the proper volume of stock solution and buffer that should be added to each 15 mL centrifuge
tube to make 10 mL of the desired concentration of tacrine. See the next step for instructions
on how to perform this step using serial dilution.
C1V1 = C2V2
• V2 is 10 mL. This is the final volume that is desired.
• C1 is the molar concentration of the stock solution.
• C2 is the desired final molar concentration.
• Solving for V1 will provide the amount of stock solution that should be added to the
15 mL centrifuge tube after the addition of the appropriate amount of buffer.
• Subtract V1 from V2 to get the amount of buffer that needs to be added to the tube.

Experiment 16
17. For the dilution series in Part II, the simplest way to create the required dilution series is by
performing a set of serial dilutions. For example, if the stock solution is at 1x10-3 M and is
diluted in buffer by a factor of 100, the resulting solution will be at 1x10-5 M. If this resulting
solution is then diluted by another factor of 100, the resulting solution will be 1x10-7. Use the
table below to create the proper dilution series. Students can verify that the concentrations are
correct by using the formula above.
Concentration of Amount of Final concentration
tacrine to add tacrine to add Buffer of tacrine
(M) (µL) (mL) (M)
-3 -5
1x10 100 µL 9.9 1x10
-5 -7
1x10 100 µL 9.9 1x10
-7 -9
1x10 100 µL 9.9 1x10
-9 -11
1x10 100 µL 9.9 1x10
-11 -13
1x10 100 µL 9.9 1x10
18. We recommend using Logger Pro for performing the Processing the Data procedure, whether
you use computers, LabQuest, or calculators to collect data. It is possible to complete the
Processing the Data procedure using LabQuest, however it requires a multi-step process of
entering data values. If you would like to complete the Processing the Data procedure using
LabQuest, student instructions are available on the CD that came with this book. It is not
possible to conduct the analysis using calculators.
Ideas for Inquiry

• Compare the activity of heart tissue to skeletal muscle and/or brain tissue. To do this, tissue
samples will need to come from the same source and will need to be prepared at the same
tissue concentration.
• Compare different methods of filtration. The whole filtrate that you used was filtered through
four layers of cheesecloth. Compare the activity of different fractions that have been filtered
through 1–3 layers of cheesecloth. You will need to make up new heart tissue homogenates
and you will need to use a minicentrifuge to prepare the fractions.
• The gene that codes for acetylcholineserase in muscle tissue can be regulated by the activity of
the muscle. Compare the acetylcholinesterase activity of muscle from farm raised fish and
wild caught fish.
• Investigate the action of common pesticides on acetylcholinesterase. Many common
insecticides and pesticides are acetylcholinesterase inhibitors. Parathion and malathion are two
examples. Design your own experiment that determines the IC50 of a common pesticide.
IMPORTANT: Take necessary safety precautions.
• Design and conduct an environmental study. Many common insecticides break down rapidly
and are not detectable in the environment after a short period of time. However, the effects of
insecticides on invertebrates and fish can still be detected in brain, muscle, and heart tissue
long after an acute exposure to an insecticide. Design your own experiment that determines if
fish or other invertebrates in a local stream or body of water have been exposed to pesticide.
You will need to have a body of water or stream that will serve as your control.

HAZARD ALERTS
Acetylthiocholine iodide: Hazard code T, Toxic. Respiratory, skin and eye irritant; May be
harmful if inhaled, toxic by ingestion, harmful if absorbed through skin. Wear gloves and eye
protection and appropriate full face particle respirator (N99 or equivalent).
9-Amino-1,2,3,4-tetrahydroacridine (Tacirne hydrochloride): Hazard code Xn, Harmful.
Respiratory, skin and eye irritant; Toxic by ingestion and inhalation. Wear gloves and eye
protection and appropriate dust mask (N95 or equivalent).
5,5′-Dithiobis(2-nitrobenzoic acid), (DTNB), (Ellman’s Reagent): Hazard code Xi, Irritant.
Respiratory, skin and eye irritant; May be toxic by ingestion, inhalation, absorption through skin.
Wear gloves and eye protection and appropriate dust mask (N95 or equivalent).
The hazard information reference is: Sigma-Aldrich, MSDS for product D218200, 800-325-
5832, www.sigmaaldrich.com.

Part I Comparison of heart acetylcholineseterase activity from different cellular fractions
Table 1
Source Slope (Δ abs/min) Percent difference
Whole filtrate 0.154 100%
Supernatant 0.111 72%
Nuclear fraction 0.083 54%
Control 0.005 0%
Table 2
Tacrine concentration Slope Normalized activity

(M) (Δ abs/min) (%)
Control 0.154 100

-13
1 x10 0.151 97.4
-11
1x10 0.114 73.5
-9
1x10 0.063 41.0
-7
1x10 0.029 18.7
-5
1x10 0.014 9.03
Est. IC50
-9
6.5 x 10

Experiment 16
Raw data for whole filtrate, supernatant, and nuclear fraction
Bar chart comparing enzyme activity for each fraction
Raw data demonstrating that tacrine inhibits AChE activity

Plot demonstrating that tacrine inhibition of AChE is dose-dependent (the

estimate of the IC50 for tacrine is the visible above the Auto Fit label)
1. The whole filtrate should have the greatest rate of activity and the nuclear fraction should
have the slowest rate. The supernatant should be slower than the whole filtrate, but faster
than the nuclear fraction. The fraction with the greatest slope is the fraction with the greatest
amount of active acetylcholinesterase.
2. Results will vary, but the supernatant should be close to or greater than 50% of the activity of
the whole filtrate and the nuclear fraction should be close to or less than 50% of the activity
of the whole filtrate. Adding the slopes of the supernatant and the nuclear fraction together
may or may not equal the slope for the whole filtrate.

3. The slope for each run should decrease as the concentration of tacrine is increased.
4. Students should be able to estimate an IC50 for tacrine using their data. IC50 values will vary,
but they should be between 1x10-9 and 1x10-10 M. Different groups will likely come up with
varying estimates, but all of the IC50 values should be within this range.
5. If tacrine was given to a person, levels of the neurotransmitter acetylcholine would increase
in the blood, heart, muscles, and brain. Tacrine inhibits the enzyme acetylcholinestase. If
acetylcholinesterase is inhibited, the neurotransmitter acetylcholine will not be broken down
as rapidly in the tissues mentioned above.

Experiment 16
Dose response curve using the log of the tacrine concentration. Note different IC50 value
Classic dose-response curve using the four parameter log model. Note different IC50 value

Computer
Macromolecules:
17
Experiments with Protein
This exercised is designed to introduce you to the study of macromolecules. Proteins, DNA,
RNA, and polysaccharides such as starch, glycogen, and cellulose are all macromolecules.
Macromolecules are formed by connecting many smaller molecules together. The individual
components of a macromolecule are referred to as monomers. Proteins are composed of
monomers called amino acids. All amino acids have a carboxyl group, an amino group, and a
central or alpha carbon. The central carbon of each amino acid contains a side chain that is often
referred to as an R group. Amino acids form polymers when the carboxyl group and amino group
of two amino acids form a peptide bond as shown in Figure 1. Water and a dipeptide are formed
in the reaction. More amino acids can be added to the carboxyl group of this dipeptide until a
polypeptide is formed.
Figure 1
There are 20 different amino acids that are found in proteins, and each one has a different
R-group. These side chains are very important because they impart each amino acid with
different characteristics. Amino acids can be characterized as polar, nonpolar, or charged.
Charged amino acids are further characterized as acid or basic. Uncharged amino acids can be
considered neutral. Three different amino acids are shown in Figure 2. Aspartic acid is acidic,
lysine is basic, and alanine is
neutral. The sequence of amino
acids that make up a polypeptide
are referred to as the primary
structure of the protein. The
primary structure determines
how the protein will fold, which
will determine its function. The
shapes within a polypeptide are
referred to as the secondary
structure. The three dimensional
structure of an entire
polypeptide is referred to as its
tertiary structure. Figure 2
In this exercise you will use the
Bradford assay to determine the protein content of two samples. The Bradford assays is an
extremely sensitive assay for protein. The Bradford reagent contains a dye called Coomassie
G-250 that can interact with the R-group of specific amino acids. One of your samples is milk.
The dominant protein in milk is called casein, which is composed of 224 amino acids. Thirteen

Computer 17
of these amino acids react with the dye in the Bradford reagent. These amino acids include
one tryptophan, four arginines, four tyrosines, and four histidines.
When the dye in the Bradford reagent interacts with these specific amino acids it turns the
solution blue. The greater the concentration of protein in solution the deeper the color will be. If
a set of known protein concentrations are allowed to react with a known concentration of
Bradford reagent, we can measure the absorbance of the resulting solutions to create a standard
curve. When a graph of absorbance vs. concentration is plotted for the standard solutions, a
direct relationship should result, as shown in Figure 3. The direct relationship between
absorbance and concentration for a solution is known as Beer’s law. To determine the protein
concentration of an unknown solution, we can measure its absorbance and see where it falls on
the standard curve. Because the relationship is linear, we could also calculate the protein
concentration using the formula for the standard curve.
In the second part of this

exercise, you will compare the
Bradford reagent to the Biuret
Reagent. The Biuret reagent is
also used to detect proteins in
solution, but it is not nearly as
sensitive as the Bradford assay.
The Biuret reagent will turn a
purplish color in the presence of
protein. The Biuret reagent
contains copper ions that can
interact with strands of protein
molecules. These complexes
form between the peptide bonds
of different polypeptide chains.
As a result, the intensity of the
color change will be directly
related to the amount of protein
in solution. Figure 3
OBJECTIVES
• Create a standard protein curve using the Bradford assay.
• Determine the protein concentration of milk and a high protein drink.
• Determine if the Bradford assay can detect both proteins and amino acids.
• Determine if the Biuret assay can detect both proteins and amino acids.

Macromolecules: Experiments with Protein
MATERIALS
computer six 15 mL centrifuge tubes
Vernier computer interface 1% tryptophan solution
Logger Pro 1% nonfat milk protein solution
Colorimeter or Spectrometer+ Biuret reagent mixture
twenty 1.5 mL cuvettes with caps* Quick Start Bradford Reagent
20–200 µL micropipette** Phosphate buffered saline (PBS)
100–1000 µL micropipette** Bovine γ-globulin standard set
200 µL micropipette tips (1 box) milk (lowfat or nonfat)
1000 µL micropipette tips (1 box) high protein drink
two 1.5 mL microtubes
+ No interface is required if using a Spectrometer.
* If using a Colorimeter, use 3 mL cuvettes supplied by Vernier and double the volume of all reagents.
** Appropriate graduated transfer pipettes (1 and 5 mL) may be substituted.
PROCEDURE
Part I Determination of protein content in different samples using the Bradford Assay
1. Obtain and wear goggles and gloves.
2. Obtain three 15 mL centrifuge tubes.

• Label one tube PB and add 10 mL of PBS.
• Label the other two tubes BR and add 15 mL of Quick Start Bradford Reagent to each.
3. Obtain two 1.5 mL microtubes.

• Label one tube M and add 980 µL of PBS. Then add 20 µL of milk.
• Label the other tube HP and add 980 µL of PBS. Then add 20 µL of high protein drink.
4. Obtain seven empty cuvettes with caps and a set of Bovine γ-globulin standards to create a
new set of protein standards.
a. Fill each cuvette with 1 mL of Bradford reagent.
b. Label one cuvette 2.0 and add 20 µL of solution from the 2.0 mg/mL standard. Cap the
cuvette and gently invert the cuvette three times.
c. Label the next cuvette 1.5 and add 20 µL of solution from the 1.5 mg/mL standard. Cap
the cuvette and gently invert the cuvette three times.
d. Label the next cuvette 1.0 and add 20 µL of solution from the 1 mg/mL standard. Cap the
e. Label the next cuvette 0.75 and add 20 µL of solution from the 0.75 mg/mL standard. Cap
f. Label the next cuvette 0.5 and add 20 µL of solution from the 0.5 mg/mL standard. Cap
g. Label the next cuvette 0.25 and add 20 µL of solution from the 0.25 mg/mL standard. Cap
h. Label the next cuvette 0.125 and add 20 µL of solution from the 0.125 mg/mL standard.
Cap the cuvette and gently invert the cuvette three times.

Computer 17
5. Obtain two empty cuvettes with caps and the microtubes labeled with M and HP.
b. Label one cuvette with an M and add 20 µL from microtube M. Cap the cuvette and
gently invert the cuvette three times.
c. Label the other cuvette HP and add 20 µL from microtube HP. Cap the cuvette and gently
invert the cuvette three times.
6. Prepare a blank by filling an empty cuvette with 1 mL of Bradford reagent and 20 µL of
PBS. Label it B. To correctly use cuvettes, remember:

menu.

c. Click Finish Calibration and allow the calibration to finish. Click .
9. Determine the optimum wavelength for examining the absorbance of the Bradford reagent
when it is bound to protein and set up the data-collection mode.
a. Remove the blank cuvette. Gently invert Cuvette M twice and then place it into the
Spectrometer.
c. Store the data by choosing Store Latest Run from the Experiment menu.
d. To set up the data-collection mode and select a wavelength for analysis, click Configure
e. Select Abs vs. Concentration as the Set Collection Mode. The wavelength of maximum
absorbance (λ max) will be selected. Verify that the maximum absorbance is close to
595 nm.
f. Enter Protein Concentration as the Column Name. Enter Pr. Conc. as the Short Name.
Enter mg/mL as the Units.
g. Click . Remove the cuvette from the Spectrometer and proceed to Step 10.

by opening the file “17 Macromolecules” from the Advanced Biology with Vernier folder of
Logger Pro.

wavelength of 635 nm (Red). Press the CAL button until the red LED begins to flash and then
release the CAL button. When the LED stops flashing, the calibration is complete. Remove
the cuvette from the Colorimeter and proceed to Step 10.

10. You are now ready to collect absorbance data for the seven protein standards. Click .
Obtain the cuvette labeled 2.0. Wipe the outside with a tissue and place it in the device (close
the lid if using a Colorimeter). Wait for the absorbance value displayed on the screen to
stabilize, then click . Enter 2.00 as the concentration, and then press ENTER or click
. The data pair you just collected will now be plotted on the graph. Remove the
cuvette from the device.
11. Obtain the cuvette labeled 1.5. Wipe the outside and place it in the device. When the
absorbance value stabilizes, click , enter 1.50, and press ENTER.
12. Repeat the Step 11 procedure for the remaining protein standards. When you have finished
with the 0.125 mg/mL standard solution, click .
13. In Table 1, record the absorbance values.
14. Examine the graph of absorbance vs. concentration. To see if the curve represents a direct
relationship between these two variables, click Linear Fit, . A best-fit linear regression line
will be shown for your data points. This line should pass near or through the data points.
15. In Table 1, record the equation of this line in the space provided.
16. You are now ready to collect absorbance data for your unknowns. Obtain the cuvette
labeled M. Wipe the outside of the cuvette and place it in the device (close the lid if using a
Colorimeter). When the displayed absorbance value stabilizes, record the value in Table 2.
Important: The reading on the screen is live, so it is not necessary to click to read
the absorbance value.
17. Choose Interpolation Calculator from the Analyze menu.
18. Check that the absorbance value that you recorded is displayed. If it is not, enter the
absorbance value in the correct space. The protein concentration in mg/mL will be displayed.
Record the concentration value in Table 2. Click . A point on the graph will be
displayed for this protein sample.
19. Obtain the cuvette labeled with a HP. Wipe the outside of the cuvette and place it in the
device. When the displayed absorbance value stabilizes, record the value in the Data and
Calculations table. Important: The reading on the screen is live, so it is not necessary to
click to read the absorbance value.
20. Repeat Steps 17–18, then proceed to Step 21.
Part II Comparison of Bradford and Biuret Reagents
21. Obtain another 15 mL centrifuge tube and label the tube with BI. Add 10 mL of the Biuret
Reagent.

Computer 17
22. Obtain two more 15 mL centrifuge tubes.

• Label one tube NFM and add 4.0 mL of 1% non-fat milk protein solution.
• Label the other tube TRP and add 4.0 mL of 1% tryptophan solution.
23. Obtain three empty cuvettes with caps.
b. Label the first cuvette BR T and add 100 µL of 1% tryptophan solution. Cap the cuvette
and gently invert the cuvette three times.
c. Label the next cuvette BR M and add 100 µL of 1% non-fat milk protein solution. Cap
d. Label the next cuvette BR C and add 100 µL of PBS, the control.
24. Obtain three more empty cuvettes with caps.
a. Fill each cuvette with 1 mL of Biuret reagent.
b. Label the first cuvette BI T and add 500 µL of 1% tryptophan solution. Cap the cuvette
and gently invert the cuvette three times.
c. Label the next cuvette BI M and add 500 µL of 1% non-fat milk protein solution. Cap the
d. Label the next cuvette BI C and add 100 µL of PBS, the control.
DATA AND CALCULATIONS

Table 1
Protein Concentration
Absorbance
(mg/mL)
2.00
1.50
1.00
0.750
0.500
0.250
0.125
Linear Fit for Standard Curve
_________________________

Table 2
Protein conc. Actual conc. Concentration

Samples Absorbance (mg/mL) (mg/mL) from label
(mg/mL)
Milk
High Protein
Calculated protein concentrations from linear fit
Milk
High Protein
Table 3
Sample Bradford reagent Biuret reagent
Tryptophan
Non-Fat milk protein
Control
PROCESSING THE DATA

1. Determine the actual protein concentration of your samples. Remember that you diluted your
original milk and high-protein samples by a factor of 50 before conducting the Bradford
assay. Multiply the protein concentration that you observed for each sample by 50 to get the
actual protein concentration of your sample. Record this value in the space provided in
Table 2.
2. Obtain the published protein values for each sample from your instructor. These values are
typically found on the nutrition labels of the milk or protein drink container. Convert the
published values to mg/mL of protein. To do this, divide the amount of protein per serving
by the volume of each serving. You may need to convert from fluid ounces to mL. Write
these values down in the space provided in Table 2.
3. Calculate the actual protein concentration of each sample using the formula for the standard
curve. Use the absorbance that you observed for each sample as your y value and then solve
for x. Multiply the resulting value by 50 to get the actual protein concentration. Write these
two numbers down in the space provide in Table 2.
Part II Comparison of the Bradford and Biuret Reagents
4. Examine the cuvettes that contain the Bradford reagent from Part II. Did the cuvette contents
turn a blue color? Write down your observations in the space provided in Table 3.
5. Examine the cuvettes that contain the Biuret reagent from Part II. Did the cuvette contents
turn a purplish color? Write down your observations in the space provided in Table 3.

Computer 17
QUESTIONS
1. Compare the protein values that you observed for each sample to the published protein
values. Are the values close? If they are not, can you think of any reason why they would be
different?
2. Compare your observed protein values to the protein values that you calculated using the
formula for the standard curve. Are the values different or are they the same? Is there any
reason why they should be different?

3. Did the Bradford reagent turn a blue color in the presence of non-fat milk protein? If it did,
can you explain why it would do this? What about the 1% solution of the amino acid
tryptophan? Did the Bradford reagent react with this amino acid even though it is not a
protein? Can you explain why this would happen?
4. Did the Biuret reagent turn a purplish color in the presence of non-fat milk protein? If it did,
can you explain why it would do this? What about the 1% solution of the amino acid
tryptophan? Did the Biuret reagent react with this amino acid even though it is not a protein?
Can you explain why this would or would not happen?
5. How are the results from this part of the exercise related to the primary and/or secondary
structure of a given protein?
EXTENSIONS
1. Serial dilution is the typical method that is used to create a standard curve. Follow the
instructions below to create a new standard curve and then repeat Part I of this exercise.
Note: The new protein standards do not include a 1.5 mg/mL standard.
a. Obtain the 2.0 mg/mL solution from the bovine γ-globulin standard set.
b. Obtain four empty microtubes and one microtube with 1 mL of PBS.
c. Add 100 µL of PBS to each empty microtube.
d. Label the first tube 1.0 and add 100 µL of the 2.0 mg/mL γ-globulin standard. Cap and
invert the tube three times.
e. Label the next tube 0.5 and add 100 µL from the tube labeled 1.0. Cap and invert the tube
three times.
f. Label the next tube 0.25 and add 100 µL from the tube labeled 0.5. Cap and invert the
tube three times.
g. Label the next tube 0.125 and add 100 µL from the tube labeled 0.25. Cap and invert three
times.
2. The dominant protein in milk is called casein and it is composed of 224 amino acids.
Thirteen of these amino acids react with the dye in the Bradford reagent to turn the solution
blue. Your standard curve is based on the protein bovine γ-globulin, which is not composed
of the same number and/or ratio of amino acids. Non-fat milk powder does contain the
protein casein. Use the non-fat milk protein solution from Part II to create a new standard
curve. Your current stock of non-fat milk protein solution is at 10 mg/mL (1%). Dilute the
stock by a factor of 5 in PBS to get a 2 mg/mL solution. Repeat the steps in Extension 1 to

generate your set of protein standards. Then repeat Part I of this exercise and compare your
results.
3. Compare the Biuret reagent to the Bradford reagent. To do this, repeat Part I of this exercise
but use the Biuret reagent instead of the Bradford reagent. You will need to create 1 mL of
each protein standard to create a standard curve. Add 500 µL of each protein standard to an
appropriately labeled cuvette and then add 1 mL of Biuret reagent. Measure the absorbance
of each cuvette at 540 nm to create your standard curve. Determine the protein concentration
of your samples by adding 500 µL of each diluted sample to a cuvette and then add 1 mL of
Biuret reagent. Measure the absorbance of each cuvette at 540 nm. Then use the formula for
the standard curve or the interpolation calculator to determine the protein concentration of
your samples. Compare your results using this method to results you obtained using the
Bradford assay.
4. Design an experiment that quantifies the qualitative differences you observed in Part II of
this exercise. You can start by repeating Part II of this exercise. Then measure each cuvette at
the proper absorbance. For the Bradford reagent measure the absorbance at 595 nm. For the
Biuret regent measure the absorbance at 540 nm.
5. The Bradford reagent is a very sensitive method for determining protein concentrations.
Design an experiment to determine the sensitivity of this method. You can start by creating a
set of protein standards that are in the 1–10 µg/mL range. Create 1 mL of each standard. Start
by determining if you can use the same ratio of Bradford reagent to protein standard to create
a standard curve in this new range. If you cannot, try increasing the volume of protein
solution that you add to each cuvette.
6. Amino acids are classified as polar, non-polar, basic or acidic. This classification is based on
their R-groups or side chains. Most polar and non-polar amino acids are considered neutral.
We can easily determine if an amino acid is basic, acid or neutral by using Logger Pro, a pH
Sensor, some distilled water, and a magnetic stirrer. You can start with the amino acids
arginine, tyrosine, and aspartic acid.
a. Place a pH Sensor in 250 mL of distilled water. Make sure that the magnetic stir bar is
turning at a moderate speed.
b. Collect pH data using Logger Pro and a Vernier computer interface.
c. After a few seconds have elapsed, add a very small amount of aspartic acid to the distilled
water.
d. Record the pH for at least one minute.
e. Dispose of the distilled water and then repeat the experiment for tyrosine and arginine.
f. Determine if each amino acid is basic, acidic or neutral.
g. Compare your results with the description of each amino acid in a textbook. Do your data
support what is in your textbook?

Experiment
Macromolecules:
Introduction to Protein
1. For best results we recommend doing this activity with a Spectrometer. You can get results
using a Colorimeter. However, the absorbance values may not be as high as shown in the
Sample Data below because the data-collection wavelength does not match the peak
absorbance for the reaction. Note: Calculator users must perform this activity with a
Colorimeter. EasyData does not support the use of Spectrometers.
3. The Logger Pro experiment files for this activity are located in the Advance Biology with
Vernier folder of Logger Pro 3.8.2 and newer.
4. This experiment can be conducted in a standard college lab period of three hours. For shorter
lab periods, it may take a single group two periods to complete this exercise. A good breaking
point is after the completion of Part I. Alternatively, high school instructors may want to give
a basic introduction to protein structure and function and then perform Part II as a
demonstration.
5. The instructions provided assume that you have purchased the “Got Protein? Kit” from Bio-
Rad Laboratories Inc. (Catalog # 166-2900EDU).
Additional notes if products were purchased through Bio-Rad:
• If you are using Colorimeters and purchased the kit from Bio-Rad, you will not be able to
use the cuvettes that came from Bio-Rad because they are too large. Cuvettes that will work
with Colorimeters, which are slightly more narrow at the bottom, are available from
Vernier (order code: CUV). Simply double the volume of all reagents in the student section
of this activity to obtain accurate readings.
• If you are using Spectrometers and purchased the kit from Bio-Rad, cuvette lids are
available from Vernier (order code: CUV-LID).
6. To prepare the Bradford Reagent:
a. Remove the Quick Start Bradford reagent from proper storage (4ºC) just before use.
b. Invert the bottle several times and then pour 20 mL of reagent into a beaker on ice. This
should be enough for 1–2 groups.
c. You can also choose to aliquot the reagent into 15 mL centrifuge tubes. In this case, give a
centrifuge tube of Bradford reagent to each group when the lab starts.
7. To prepare the 1x PBS solution:
a. Remove the 10x PBS solution from proper storage (4ºC).
b. Add 10 mL of 10x PBS solution to 90 mL distilled water.
c. Store in a capped container at 4ºC. Phosphate buffer can be stored for a week at this
temperature.

Experiment 17
8. To prepare the protein standards:

a. The protein standards come in seven pre-mixed and labeled microtubes.
b. To avoid contamination, aliquot 40 µL of each standard into a separate microtube.
c. Provide each group with a full set of protein standard aliquots.
9. To prepare the Biuret reagent:
a. Fill a beaker with 200 mL of distilled water. Stir using a magnetic stirrer at low speed.
Add the following compounds in the order specified.
• 0.6 g of cupric sulphate
• 0.68 g of potassium sodium tartrate
• 1.0 g of potassium iodide
• 4.4 g sodium hydroxide
b. The solution will go from an opaque yellow-green to deep blue when the sodium
hydroxide is added.
c. When the sodium hydroxide is completely dissolved and the solution has cleared, aliquot
the solution into capped containers and store at room temperature.
d. The Biuret reagent can be made 24–48 hrs ahead of time.
10. To prepare the 1% tryptophan:
a. Add 0.2 g of tryptophan to 20 mL of PBS.
b. Stir using a magnetic stirrer at low speed until completely dissolved.
c. Store in a capped container at 4ºC for up to 24 hours.
11. To prepare the 1% non-fat milk protein solution:
a. Obtain non-fat milk powder from a grocery store.
b. Most non-fat milk powders will contain at least 0.33 g protein/g powder.
c. Assume that the nutrition label is correct and create a 1% solution of non-fat milk protein
in PBS. For example, if the non-fat milk powder contains 0.33 g of protein/g of powder,
then 3 g of powder dissolved in 100 mL of PBS would create a 1% solution of non-fat
milk protein.
d. 10 mL of 1% non-fat protein solution should be more than enough for an entire class.
e. Store at 4ºC in a capped container for up to 1 week before use.
12. Low-fat, non-fat, or Vitamin D milk can be used as the milk sample for this exercise.
13. Any soy or dairy-based high-protein drink with 10 g or more of protein/serving will work.
The Odwalla® high protein drink, Vanilla Super Protein, has been tested and works well.
Ideas for Inquiry

• The Bradford reagent interacts with the R-groups of specific amino acids. Casein contains
thirteen amino acids that interact with the reagent to turn the solution blue. These amino acids
include one tryptophan, four arginines, four tyrosines, and four histidines. Use the Bradford
reagent to compare a solution of 1% tryptophan to a 1% solution of non-fat milk protein.
Would you expect the intensity of the 1% tryptophan to be 1/13 the intensity of the non-fat
milk protein solution? What do your results suggest?

Macromolecules: Introduction to Proteins
• The dye in the Bradford reagent is called Coomassie G-250 and primarily interacts with the R-
group of the basic amino acid ariginine that is found in many proteins. Coomassie G-250
contains two negatively charged sulphate groups that can form electrostatic attractions with
the negatively charged nitrogen found on arginines. Coomassie G-250 also contains six
aromatic rings. Aromatic amino acids like tyrosine and tryptophan can form intermolecular
attractions with the aromatic rings found in Coomassie G-250 through π-electron stacking.
Design an experiment that compares the ability of Coomassie G-250s to interact with the
amino acids tryptophan and arginine. Prepare a 0.2 M solution of each amino acid in PBS.
You can start by adding 20 µL of amino acid solution to a cuvette containing 1 mL of
Bradford reagent. Measure the absorbance of the two different amino acid solutions at
595 nm. Are the absorbance values the same or are they different? Does the data support the
fact that arginine forms a stronger bond with Coomassie G-250 than tryptophan? If it does not,
are there other variables that you need to control for?
• Design an experiment to determine the protein concentration of something other than milk.
Soy milk, beans, or a small piece of salmon steak might be a good place to start. If you are
using beans or a small sample of tissue, weigh the tissue and then homogenize it in buffer
using a blender. Filter the homogenate through cheesecloth and use the resulting filtrate to
perform the Bradford assay.
• The longer a given protein, the greater the color change should be with the Biuret reagent.
Design an experiment to test this assumption. You can start by comparing milk samples which
contain casein to the same concentration of bovine γ-globulin. Use the basic protocol in Part II
for the Biuret assay. You may need to adjust the ratio of reagent to protein solution to get a
decent color change. Make sure you can quantify your results using a Spectrometer or
Colorimeter. You will need to do some research to determine if casein is significantly longer
or shorter than bovine γ-globulin. Determine if your results support what you have found in
the published literature.
HAZARD ALERTS
Quick-Start Bradford Reagent: Contains methanol and phosphoric acid. Hazard code Xn,
Harmful. Eye irritant; may be harmful if inhaled, toxic by ingestion, harmful if absorbed through
skin. Wear gloves and eye protection. The hazard information reference is: Bio-Rad Laboratories
Inc, MSDS for product 5000205, 800-4BIORAD, www.bio-rad.com
Cupric sulphate: Skin, respiratory and eye irritant; moderately toxic by ingestion and inhalation.
Hazard code: 2–Somewhat hazardous. Wear gloves and eye protection and appropriate dust mask
(N95 or equivalent).
Potassium sodium tartrate: Possible respiratory, skin and eye irritant; possibly toxic by ingestion.
Hazard code: 0–Low Hazard. Wear gloves and eye protection and appropriate dust mask (N95 or
equivalent).
Potassium iodide: Possible respiratory, skin and eye irritant; Toxic by ingestion. Hazard code: 0–
Low hazard. Wear gloves and eye protection and appropriate dust mask (N95 or equivalent).
Sodium hyrdroxide: Severe respiratory, skin and eye irritant; Very toxic by ingestion and
inhalation, extremely corrosive to body tissues and skin. Hazard code: 3–Very hazardous. Wear
gloves and eye protection and appropriate dust mask (N95 or equivalent).
The hazard information reference for the above chemicals is: Flinn Scientific, Inc., Chemical and
Biological Catalog Reference Manual, (800) 452-1261, www.flinnsci.com, unless otherwise
noted.

Experiment 17

Table 1
Protein concentration
Absorbance
(mg/mL)
2.000 1.179
1.500 0.912
1.000 0.759
0.750 0.595
0.500 0.317
0.250 0.219
0.125 0.115
Linear fit for standard curve

Abs = 0.5661x + 0.0899
Table 2
Protein conc. Actual conc Concentration from

Samples Abs
(mg/mL) (mg/mL) label (mg/mL)
Milk 0.606 0.912 45.6 38.0
High Protein 0.849 1.341 67 66.6
Calculated protein concentrations from linear fit
Milk 49 mg/mL
High Protein 70 mg/mL
Table 3
Sample Bradford reagent Biuret reagent
Tryptophan Blue No color change
Non-Fat Milk Protein Blue Purple
Control No color change No color change

Data for protein analysis using the Bradford Assay
1. The observed protein values should be close to the published values. They may not match
exactly, but they should be within 3 mg/mL of the published protein concentrations. One
reason why they may not match exactly is that the published values represent the average
protein concentration from multiple tests. If the class data is averaged, the data may be closer
to the published values. User or student error is another issue. Most food testing will be fully
automated or will be performed by a professional. A very small error in pipette volume may
produce a large error in the observed protein value.
2. Calculated values for protein concentration using the formula from the standard curve should
be close to the values reported by the interpolation calculator. They may be different if
students are rounding numbers up or down.

3. The Bradford reagent should turn blue color in the presence of non-fat milk protein. Non-fat
milk protein contains casein which will bind to the dye in the Bradford reagent. The 1%
solution of tryptophan should also turn the Bradford reagent blue, but it may not be as dark.
The aromatic side chain on tryptophan can interact with the dye in the Bradford reagent and
can turn the solution blue.
4. The Biuret reagent will turn a purplish color in the presence of non-fat milk protein. The
Biuret reagent interacts with strands of protein molecules to form copper complexes. In
contrast, the Biuret reagent should not turn a purplish color in the presence of the amino acid
tryptophan. Tryptophan is an amino acid, not a protein, so the Biuret reagent cannot react
with it.
5. Primary structure refers to the sequence of amino acids that make up a given protein. The
Bradford reagent reacts with specific side chains on amino acids. As a result, the ability of

Experiment 17
this dye to change a solution blue will depend on the presence or absence of these specific
amino acids and is thus independent of the secondary or tertiary structure of the protein. The
Biuret reagent forms copper complexes between peptides bonds. A single copper ion will
form a bond between four peptide bonds; two adjacent peptide bonds and two peptide bonds
that are on another polypeptide. As a result, this reagent will only react with complete
proteins.
Results from a Bradford Assay using serial dilution of BSA
Results from a Bradford Assay using serial dilution of non-fat milk protein

Graph demonstrating that the Bradford Reagent can detect amino acids
Graph demonstrating the characteristics of three amino acids using a pH Sensor

Appendix
Using the Electronic Resources A

The electronic resources, accessed through your account at www.vernier.com, contain the
following:
Student
EasyData–Contains the calculator instructions for each of the experiments in this book
that are supported by EasyData.
LabQuest–Contains the LabQuest app instructions for each of the student experiments in
this book that are supported by LabQuest App. Also includes steps for performing the
Processing the Data section for Experiments 15 and 16 using LabQuest.
Logger Pro–Contains the computer instructions for each of the student experiments in
this book.
Teacher– Contains the teacher information pages for each experiment in this book.
Using the Advanced Biology with Vernier Word-Processing Files
All file names begin with the experiment number, followed by an abbreviation of the title;
e.g., 01A Membrane Diffusion is the file name used for Experiment 1A, Diffusion through
Membranes. This provides a way for you to edit the experiments to match your lab situation, your
equipment, or your style of teaching. The files contain all figures, text, and tables in the same
format as printed in Advanced Biology with Vernier.
Advanced Biology with Vernier A-1

Appendix
Using TI Connect: Loading EasyData and B

Capturing Calculator Screen Images
This appendix gives an overview of loading and updating the EasyData application on your
TI-83 Plus or TI-84 Plus graphing calculators. It also describes how to download calculator
screen images, such as graphs, to a computer.
EasyData is part of the bundle of APPS that come preloaded on all new TI-83 Plus, TI-84 Plus
and TI-84 Plus Silver Edition graphing calculators. To do the activities in this book, you will
need to use version 2.0 or newer. To check to see if you have the correct version of EasyData on
your calculator, press and scroll through the list of applications. With EasyData
highlighted, press to launch the App and note the version number on the introductory
screen. If your graphing calculator does not contain an appropriate version of EasyData, you can
download the latest version for free from the Vernier web site www.vernier.com/easy.html and
use TI Connect to transfer it to your graphing calculator.
Before loading EasyData onto your calculator, make sure you have a recent version of the
calculator operating system. If you are using a TI-84 Plus calculator, you will need operating
system version 2.30 or newer. If you are using a TI-83 Plus calculator, you will need operating
system version 1.18 or newer. Operating system updates can be downloaded from the Texas
Instruments web site at education.ti.com.
Loading EasyData
TI Connect for Windows and Macintosh is simple and easy to use. First, be sure you have the
TI Connect software installed on your computer. If you do not, you can download this software
for free from the Texas Instruments web site at www.education.ti.com/ticonnect. When you have
downloaded the EasyData App onto your computer, follow the instructions below to transfer the
EasyData app to your graphing calculator.
Windows Computers running Windows 98, NT 4.x, 2000 or ME, and XP
1. Connect the TI-GRAPH LINK cable or the TI Connectivity cable to the serial or USB port of
your computer and to the port at the bottom edge of the TI-83 Plus graphing calculator.
If you are using the TI-84 Plus or TI-84 Plus Silver Edition, connect the TI unit-to-computer
cable to the USB port of your computer and to the USB port at the top edge of your graphing
calculator.
2. Start the TI Connect software on your computer. Within TI

Connect, click on Device Explorer. You may be asked to
identify which computer port your cable is plugged into.
Advanced Biology with Vernier B-1

Appendix B
3. The software will identify the attached calculator and call up a

window showing the calculator’s contents.
4. To load EasyData onto a TI graphing calculator, simply drag the

EasyData file from wherever you have it saved on your computer
to the Device Explorer window, and it will copy onto your
graphing calculator.
5. The EasyData App should now be loaded into your calculator.

To confirm this, press on the calculator to display the
loaded applications.
Macintosh Computers running Mac OS X 10.2 (Jaguar), 10.3 (Panther), and 10.4 (Tiger).
1. Connect the TI-GRAPH LINK cable, or the TI Connectivity cable to the USB port of your
computer and to the port at the bottom edge of the TI-83 Plus, or TI-83 Plus Silver Edition
If you are using the TI-84 Plus or TI-84 Plus Silver Edition graphing calculator, connect the
TI unit-to-computer cable to the USB port of your computer and to the USB port at the top
edge of your graphing calculator.
2. Turn the calculator on. On the computer, start TI Connect and

double-click on TI Device Explorer.
3. The program will identify the attached calculator and call up a

window displaying the calculator’s contents.
4. To load EasyData onto a TI graphing calculator, simply drag the

EasyData file from wherever you have it saved on your computer
to the Device Explorer window, and it will copy onto your
5. The EasyData App should now be loaded into your calculator. To confirm this, press
on the calculator to display the loaded applications.
B-2 Advanced Biology with Vernier

Using TI Connect
Capturing a Calculator Screen Image

Many experiments in Advanced Biology with Vernier contain an optional step that involves
printing the graph displayed on the calculator screen. To capture and print a TI-83 Plus calculator
screen image, you need a TI-GRAPH LINK or TI Connectivity cable and the TI Connect software.
If you are using the TI-84 Plus or TI-84 Plus Silver Edition graphing calculator, connect the TI
unit-to-computer cable to the USB port of your computer and to the USB port at the top edge of
your graphing calculator.
Before doing an experiment that requires printing, you may want to show your students how to
print graphs. The process described below produces screen images directly from the calculator,
such as seen here.
A graph of force vs. time as displayed in the EasyData application
Windows Computers running Windows 98, NT 4.x, 2000 or ME, XP, and 7
1. On your calculator, display the screen you want to capture.
2. Connect the TI-GRAPH LINK cable, or the TI Connectivity cable to the serial or USB port of
your computer and to the port at the bottom edge of the TI-83 Plus graphing calculator.
calculator.
3. Start the TI Connect software on your computer. Within

TI Connect, click ScreenCapture.
4. The captured screen will appear in TI Screen Capture window, and

it can now be printed or saved to a file. To capture an additional
screen, select Get Screen from the Tools menu.
Advanced Biology with Vernier B-3

Appendix B
Macintosh Computers running Mac OS X 10.2 (Jaguar), 10.3 (Panther), and 10.4 (Tiger).
1. On your calculator, display the screen you want to capture.
2. Connect the TI-GRAPH LINK cable to the USB port of your computer and to the port at the
bottom edge of the TI-83 Plus graphing calculator.
calculator.
3. Start the TI Connect software on your computer. Within TI

Connect, double-click TI Device Explorer.
4. The TI Device Explorer window will appear displaying the

contents of the calculator’s memory.
5. Click the “Screen Capture” toolbar control. The calculator’s screen

image will appear in a separate window. The captured screen can now
be printed or saved to a file.
B-4 Advanced Biology with Vernier

Appendix
Using Logger Pro to Transfer Data C

to a Computer
You may elect to use the Vernier Logger Pro program to transfer data from LabQuest or
TI graphing calculator to a computer. Logger Pro has many graphing features, such as labels and
units for axes, autoscaling, and modification of axes. Printed graphs will have a better resolution
and appearance than printed screens of the LabQuest or TI graphing calculator display. Data
tables can be displayed and printed with side-by-side columns and headings. Logger Pro also
provides advanced data-analysis features, such as curve fitting, statistical analysis, and calculated
spreadsheet columns.
The directions below are for the latest version of Logger Pro (version 3.6 or newer).
Transferring data from LabQuest

If you are not prompted to retrieve the data from LabQuest, Logger Pro can open files saved in
the LabQuest application.
1. Connect LabQuest to your computer with a USB cable.
2. Start Logger Pro on your computer.
3. Choose LabQuest Browser ► Open… from the File menu.
4. You’ll see a standard file selection dialog showing the files available on your LabQuest.
Select the file name you want, and click Open. Logger Pro will open the LabQuest file,
displaying any data, graphs, and notes.
Transferring data from a TI graphing calculator

Logger Pro software has an option for importing data lists from all of the TI graphing calculators.
1. If you are using the TI-83 Plus or TI-83 Plus Silver Edition graphing calculator, connect the
TI-GRAPH LINK cable or the TI Connectivity cable to the serial or USB port of your computer
and to the port at the bottom edge of the calculator.
calculator.
2. Turn on the calculator.
3. Start Logger Pro on your computer.
4. Choose Import from►TI Device from the File menu. A dialog box appears with directions
for importing data.
5. From the pull-down menu, choose the USB port or serial port (COM 1-4 on a PC, modem or
printer port on a Macintosh) to which the TI-GRAPH LINK cable is connected.1
1
If you are using a PC serial cable, identify whether it is a gray or black cable.
Advanced Biology with Vernier C-1

Appendix C
6. Click on the Scan for Calculator button. The calculator model you are using should now be
identified, and you should see a message, “Ready to Import.”
7. Select the lists that you wish to import by clicking on each of them. (To select more than one
list on a Macintosh, hold down the Command and Shift keys while you click.)
8. Click OK to send the lists to the computer. The lists will appear in columns in the data table
in Logger Pro. They will be labeled with the simple list names from the calculator. If you
want to rename them or add units, double-click on the heading in the data table and enter new
labels and units.
9. Click the Refresh Catalog button if you have connected a new interface or calculator to the
computer.
C-2 Advanced Biology with Vernier

Appendix
Safety Information D
Chemical Hazard Information
The reference source for the chemical hazard information in this book is the 2002 edition of
Flinn Scientific’s Chemical & Biological Catalog Reference Manual. Flinn Scientific, Inc. is an
acknowledged leader in the areas of chemical supply, apparatus and laboratory equipment supply,
and chemical safety. Flinn’s Chemical & Biological Catalog Reference Manual is an outstanding
reference to be used as you order chemicals, store chemicals, mix solutions, use chemicals in you
classroom, and dispose of chemicals. Most of the chemicals and the equipment used in Advanced
Biology with Vernier are available from this catalog. We strongly urge you to obtain and use a
current copy of the above mentioned publication by contacting Flinn Scientific at the address
below:
Flinn Scientific, Inc.

P.O. Box 219
Batavia, Illinois 60510
Telephone (800) 452-1261
www.flinnsci.com
The Flinn hazard code is used in the teacher information section of many experiments in
Advanced Biology with Vernier to describe any possible hazards associated with the chemical
reagents used. The Flinn hazard code (A–D) is defined as follows:
A. Extremely Hazardous. This category includes, but is not limited to, concentrated acids,
severely toxic, severely corrosive, unstable and /or explosive chemicals.
B. Hazardous. This category includes, but is not limited to, chemicals that are toxic/poisons,
corrosive, contain heavy metals, and/or are alleged/proven carcinogens.
C. Somewhat Hazardous. This category includes, but is not limited to, chemicals that are highly
flammable/combustible, moderately toxic and/or oxidants.
D. Relatively Non-Hazardous. This category includes, but is not limited to, chemicals that are
irritants and/or allergens.
Advanced Biology with Vernier D-1

Appendix
Vernier Products for Advanced Biology E

All software and laboratory interfacing hardware required for the experiments contained in this
book are available from Vernier Software & Technology and can be found in this appendix.
LabQuest
Vernier LabQuest provides a portable and versatile data-collection device for any class studying
biology. It can be used as a computer interface, as a stand-alone device, or in the field. It has
built-in graphing and analysis software and a vivid color touch screen. It is compatible with
existing Vernier sensors. It has a rechargeable, high-capacity internal battery. It also has a built-in
temperature sensor and microphone.
LabQuest Mini
The Vernier LabQuest Mini is a low-cost data-collection interface that connects to the USB port
of a computer and has five sensor ports.
LabPro
Vernier LabPro offers another option for data collection in biology. A wide variety of Vernier
probes and sensors can be connected to each of the four analog ports and two sonic/digital ports.
LabPro is connected to a computer using a serial or USB port or to a TI graphing calculator.
Data-Collection Software for Advanced Biology

Computer
Logger Pro is the data-collection software for collecting data on a computer. Logger Pro
software now comes with a free site license for both Windows and Macintosh, so you only need
to order one copy of Logger Pro for your school or college department.
Software Macintosh and Windows CD

Logger Pro (order code: LP)
LabQuest
LabQuest App is the data-collection application used to collect data when using LabQuest as a
standalone device.
Calculator
The EasyData App controls the data gathering process, and makes data analysis easier after
experiments are completed. See Appendix B for information on transferring the program to your
calculator.
Advanced Biology with Vernier E-1

Appendix E
Vernier Products for Advanced Biology
Item Order Code

Vernier LabQuest LABQ
Vernier LabQuest Mini LQ-MINI
Vernier LabPro interface LABPRO
Blood Pressure Sensor BPS-BTA
Blue Digital Bioimaging System BL-DBS
White Digital Bioimaging System WHT-DBS
CO2 Gas Sensor CO2-BTA
Colorimeter COL-BTA
Conductivity Probe CON-BTA
Dissolved Oxygen Probe DO-BTA
Optical DO Probe ODO-BTA
Gas Pressure Sensor GPS-BTA
Hand Grip Heart Rate Monitor HGH-BTA
O2 Gas Sensor O2-BTA
ProScope BD-HRB
SpectroVis Plus SVIS-PL
Stainless Steel Temperature Probe TMP-BTA
Advanced Biology with Vernier lab manual BIO-A
Vernier Sensors for Advanced Biology

Blood Pressure The Blood Pressure Sensor is a designed to measure human blood
Sensor pressure. It can be used to measure systolic and diastolic blood
pressure utilizing the oscillometric technique. The sensor includes a
standard adult size adjustable cuff (27 cm to 39 cm), pump bulb and
pressure transducer.
Blue Digital The Blue Digital Bioimaging System allows you to illuminate your
Bioimaging System gels, capture the image digitally on your computer, and analyze the
data using Logger Pro software. The Blue Digital Bioimaging System
includes the BlueView™ Transilluminator, Imaging Hood, ProScope
HR digital USB camera with 1-10X lens, and a ProScope Stand.
E-2 Advanced Biology with Vernier

Vernier Products for Advanced Biology
White Digital The White Digital Bioimaging System allows you to illuminate your
Bioimaging System gels, capture the image digitally on your computer, and analyze the
data using Logger Pro software. The White Digital Bioimaging
System includes the White Light Transilluminator, Imaging Hood,
ProScope HR digital USB camera with 1-10X lens, and a ProScope
Stand.
CO2 Gas Sensor The CO2 Gas Sensor measures gaseous carbon dioxide levels It has
two settings: low range (0–10,000 ppm) and high range
(0–100,000 ppm). This probe is great for measuring changes in CO2
levels during plant photosynthesis and respiration. With this sensor,
you can easily monitor changes in CO2 levels occurring in respiration
of organisms as small as crickets or beans! A chamber with probe
attachment is included for running controlled experiments with small
plants and animals.
Colorimeter The four-wavelength (430 nm, 470 nm, 565 nm, and 635 nm) Vernier
Colorimeter allows you to study the light absorption of various
solutions. It is great for Beer’s law experiments, determining the
concentration of unknown solutions, or studying changes in
concentration vs. time. Fifteen 3.5 mL cuvettes are included.
Conductivity Probe This probe is great for environmental testing for salinity, total
dissolved solids (TDS), or conductivity in water samples. Biology
students can use it to investigate the difference between ionic and
molecular compounds, strong and weak acids, salinity, or ionic
compounds that yield different ratios of ions. The Conductivity Probe
can monitor concentration or conductivity at three different sensitivity
settings: 0–200 µS/cm, 0–2000 µS/cm, and 0–20,000 µS/cm.
Dissolved Oxygen Use the Dissolved Oxygen Probe to determine the concentration of
Probe oxygen in aqueous solutions in the range of 0 –14 mg/L (ppm). It has
built-in temperature compensation and a fast response time. This probe
is great for water quality, biology, or ecology. Included with the probe
is a zero-oxygen solution, two membrane caps, a 100% calibration
bottle, and electrode filling solution. Replacement membranes are
available (order code MEM).
Optical DO The Vernier Optical DO Probe uses luminescent technology to provide

Probe fast, easy, and accurate results, making it a terrific choice for biology,
ecology, or environmental science courses. Students can measure
dissolved oxygen concentration in surface water or to perform a wide
variety of experiments to determine changes in dissolved oxygen
levels, one of the primary indicators of the quality of an aquatic
environment.
Advanced Biology with Vernier E-3

Appendix E
Gas Pressure Sensor The Gas Pressure Sensor can be used for a variety of experiments in
biology where gases, such as oxygen and carbon dioxide, are either
produced or consumed in a reaction. The pressure range is 0 to
2.1 atm (0 to 210 kPa). It comes with a variety of pressure-sensor
accessories, including a syringe, plastic tubing with two Luer-lock
connectors, two rubber stoppers with Luer-lock adapters, and one
two-way valve.
Hand Grip Heart Rate The Hand-Grip Heart Rate Monitor is ideal for determining a person’s
Monitor heart rate while mobile or stationary. With this sensor, heart rate can
be monitored during, as well as after exercise. The sensor consists of
wireless hand grips and a receiver module that plugs into any of our
data-collection devices. The hand grips sense the electrical signals
generated by the heart, much like an EKG. For each pulse detected, a
signal is transmitted to the receiver module, and the individual’s pulse
rate is calculated. The Hand-Grip Heart Rate Monitor includes one
transmitter and one receiver.
O2 Gas Sensor The O2 Gas Sensor measures oxygen concentration in air. Many of the
experiments currently performed using the CO2 Gas Sensor can be
performed or complemented using the O2 Gas Sensor. Due to its wide
measurement range (0–27% oxygen by volume), it can also be used to
monitor oxygen concentration during human respiration.
ProScope The ProScope HR (high resolution) is a USB handheld digital

microscope designed for use with your computer. It uses a high-
quality CMOS sensor and universal lens mount.
SpectroVis Plus The Vernier SpectroVis Plus is a fully functioning visible light
Spectrometer spectrophotometer that also offers flourescence. Range: 380–950 nm,
fluorescence excitation centered at 405 nm or 500 nm
Stainless Steel The Stainless Steel Temperature Probe is an accurate, durable, and
Temperature Probe inexpensive sensor for measuring temperature. Range: –40°C to
+135°C
E-4 Advanced Biology with Vernier

Appendix
Equipment and Supplies F

A list of equipment and supplies for all the experiments is given below. The amounts listed are
for a class of up to 30 students working in groups of two, three, or four students in a classroom
equipped with eight computers. The materials have been divided into nonconsumables,
consumables, and chemicals. Most consumables and chemicals will need to be replaced each
year. Most nonconsumable materials may be used many years without replacement. Some
substitutions can be made.
Nonconsumables
Item Amount Experiment
aprons or lab coats class set all
balance 2 1B, 13, 15
basting bulb 8 1B
beaker, 100 mL 24 5 (all methods), 12A, 13
beaker, 250 mL 16 1B, 2 (O2), 2 (Gas Pressure), 4B, 12A, 12B
beaker, 400 mL 8 1A, 2 (O2)
beaker, 600 mL 24 1B, 2 (Gas Pressure), 4B, 6B (opt. 2)
beaker, 1 L 16 5 (Gas Pressure), 6A
BioChamber 250 8 5 (CO2 and O2)
blender 1 15, 16
bottle, Nalgene, 250 mL 8 2 (O2), 5 (CO2), 5 (O2)
bottle, spray 2 9
brush, camel’s hair 8 7, 11
Bunsen burner 8 3
clamp, dialysis tubing 16 1A, 1B
clamp, plastic tubing 16 1B, 9
clamp, utility 16 1A, 1B, 5 (Gas Pressure), 9
clock or stopwatch 8 4B, 5 (Gas Pressure), 6A, 6B (opt. 2)
clothespins 8 1B, 3, 9
coin 8 4A, 8
cup, large, Styrofoam® 8 1B, 6A, 6B (opt. 2), 12A
* Available from Bio-Rad Laboratories, Inc. See Appendix G for more information.
Advanced Biology with Vernier F-1

Appendix F
cuvette, Colorimeter or Spectrometer, 200 4B, 13, 14, 15, 16, 17

with lids
dissecting scope 8 7
drosophila chromosome, prepared slide 1 7
electrophoresis chamber, horizontal* 1 6B (both options)
Erlenmeyer flask, 125 mL, with stopper 8 13
Erlenmeyer flask, 500 mL 1 6B (both options)
fan 2 9
floodlight, 100 watt 8 4B, 9
forceps 8 5 (Gas Pressure), 7
funnel 8 13
glass beads 250 5 (Gas Pressure)
goggles class set all (except 7, 10A, 10B)
graduated cylinder, 10 mL 16 2 (O2), 2 (Gas Pressure), 13
graduated cylinder, 25 mL 8 1B
graduated cylinder, 50 mL 8 4A
graduated cylinder, 100 mL 8 5 (Gas Pressure)
graduated cylinder, 500 mL 1 6A, 6B (both options)
heater, small electric 2 9
magnifying glass 8 11
microcentrifuge* 1 6B (both options), 15
microcentrifuge tube racks* 8 6A, 6B (both options), 15
micropipette, 10 µL* 8 6A
micropipette, 2–20 µL* 8 6B (both options)
micropipette, 20–200 µL* 8 6B (both options), 15, 16, 17
micropipette, 100–1000 µL* 8 6B (opt. 2), 15, 16, 17
micropipette rack* 8 6B (both options)
microscope, compound 8 3, 7
microscope, dissecting 8 3, 7, 11
microscope slides 72 3, 7
F-2 Advanced Biology with Vernier

Equipment and Supplies
microscope slide, whitefish mitosis 4 3
microscope slide coverslips 1 box 3, 7
microscope slide, onion mitosis 4 3
microwave 1 6A, 6B (both options)
milk container, 1 gallon 8 12A
mini centrifuge* 1 6B (both options)
mini incubation oven* 1 6A
mortar and pestle 8 13
pan, shallow, plastic or metal 8 6A, 12B
pencil 8 1B, 4A
permanent marker 8 6A, 6B (both options)
pipet pump (or pipet bulb) 8 4B
pipet, 5 mL 15 3, 4B, 7
plastic tubing w/Luer-lock fitting (comes

16 1B, 2 (Gas Pressure), 5 (Gas Pressure)
with Gas Pressure Sensor)
plastic window screen, 12 cm X 12 cm 136 12B
razor blade, knife or scalpel 8 3, 9
ring stand 8 1A, 1B, 5 (Gas Pressure), 9, 13
ring for ring stand 8 13
rocking platform* 1 6B (both options)
rubber bands, medium 120 12B
rubber stopper assembly 16 1B, 2 (Gas Pressure), 5 (Gas Pressure)
ruler, metric 8 4A, 6B (both options), 9
scalpels 8 1A, 3, 12B
scissors 2 1A, 4A, 12B
siphon tube, 1.5 m 2 12B
sordaria demonstration plate 1 3
stepping stool, 45 cm (18 inches) high 4 10A


Appendix F
stirring rod; glass, plastic, or wood 16 1A
stopper, cork 8 4A
stopper with plastic connector (comes

18 1B, 2 (Gas Pressure), 5 (Gas Pressure)
with Gas Pressure Sensor)
syringe, plastic (comes with Gas

8 1B, 9
Pressure Sensor)
teasing needles 8 7
test tube rack 16 1A, 1B, 2 (all methods), 5 (Gas Pressure)
test tube, 10 × 100 mm 32 4B
test tube, 13 × 100 mm, glass 8 13
test tube, 16 × 100 mm, with stoppers 32 1B
test tube, 18 × 150 mm 40 2 (all methods), 4B, 5 (Gas Pressure)
test tube, 25 × 150 mm with screw top 64 12B
thermo-anesthetizer 8 7, 11
thermometer 8 2 (all methods), 5 (all methods), 6A, 6B (both

options),
tray, for monitoring animal behavior 8 11
water bath, temperature controlled 1 6A
Consumables
aceto-orcein stain 100 mL 7
agar powder, LB nutrient 1 container 6A
agarose 25 g 6B (both options)
algal culture 1 12B
aluminum foil 1 roll 4B, 11, 12B
beral pipets 100 1A, 9, 13, 14
beral pipets, 1 mL graduated 200 2 (all methods), 4
bleach 10 mL 6A
carrots bunch 13

centrifuge tube, 15 mL with cap 180 1B, 15, 16, 17
cheesecloth 2 packages 15, 16
chicken heart 1 16
chromatography paper 8 pcs 4A
cotton, absorbent 1 bag 5 (Gas Pressure)
cotton, non-absorbent 1 bag 5 (Gas Pressure)
culture vial of wild-type drosophila 1 7
culture vial of monohybrid cross 1 7
culture vial of dihybrid cross (optional) 1 7
culture vial of sex-linked cross (optional) 1 7
dialysis tubing, 2.5 cm × 15 cm segments 1 roll 1A, 1B
distilled water 10 L 1A, 4B, 6A, 6B (opt. 2), 12B, 13, 14, 16, 17
drosophila culture vials, wild type and non 30 7, 11
filter paper 2 boxes 11, 13
fly morgue 8 7
food coloring, blue and yellow 1 bottle ea. 13
gel support film* 1 box 6B (both options)
high protein drink 1 bottle 17
ice 5 bags 2 (all methods), 4B, 5 (all methods), 6A,

6B (opt. 2), 10A, 15, 16
index cards 100 8
inoculating loop 8 3
inoculating loop, sterile, disposable 80 6A
lab mat 1 roll 6A, 6B (both options)
masking tape 1 roll 11
milk, 1% nonfat, powder 1 box 17
milk samples, lowfat or nonfat 10 mL 17
nitrile gloves 1 box 6A, 6B (both options)
olive oil, light, regular, and extra virgin 300 mL of 14

each
onion root tip 8 3


Appendix F
onion root tip 8 3
paper towels 1 roll 1B, 3, 10A
Parafilm 1 roll 1B
peas (garden) 3 oz. 5 (all methods)
permanent marker 8 6A, 6B (both options)
petri dishes, 60 mm X 15 mm* 40 6A
petri dishes, 100 mm X 15 mm 33 7, 11
pH paper box 11
Piccolyte II 10 mL 7
pillbugs 100 11
pipet, transfer plastic* 500 6A
pipet, tips, 2–200 µL* 13 boxes 6B (both options), 15, 16, 17
pipet, tips, 100–1000 µL* 13 boxes 15, 16, 17
plant cuttings 8 9
plastic bag, gallon, with twist tie 4 9
plastic gloves box 3, 7
pond water 7L 12B
potato 1 15
PTC paper 100 strips 8
spinach, fresh 4 bunches 4A, 4B, 13
tape, laboratory 1 roll 6B (both options)
tape, masking 1 roll 9
test tubes, EZ micro, 1.5 mL* box 6B (opt. 2), 17
test tubes, EZ micro, 2.0 mL* box 6A, 6B (both options)
tissues, lint free 80 4B
yeast 6 pkgs 2 (all methods)

Chemicals
2,6-Dichloroindophenol (DPIP) 1g 4B
3,4-dihydroxyphenylalanine (DL-DOPA) 5g 15, 16
5,5 -Dithiobis(2-nitrobenzoic acid)

′
1g 15, 16
(DTNB), (Ellman’s Reagent)
acetone 200 mL 4A, 13
acetylthiocholine iodide (ACTHi) 1g 16
buffer solution, pH 4 500 mL 2 (O2), 2 (Gas Pressure)
cupric sulphate 1g 17
DNA stain
Fast Blast DNA stain, 50X* 100 mL
or
SYBR Safe DNA stain, 0.5X 1L 6B (both options)
or
SYBR Safe DNA stain, 10,000X 400 μL
gel sample loading dye, 5X, 10 mL* 10 mL 6B (both options)
hydrochloric acid, 1 M 150 mL 3, 7, 11
hydrogen peroxide, 3% 1300 mL 2 (all methods)
isopropanol 1 bottle 7, 13, 14
petroleum ether 45 mL 4A, 13
potassium hydroxide 75 g 5 (Gas Pressure)
potassium hydroxide solution, 2% 30 mL 11
potassium iodide 1g 17
potassium phosphate, dibasic 200 g 4B
potassium phosphate, monobasic 150 g 4B
potassium sodium 1g 17
sodium chloride (NaCl, table salt) 350 g 1A, 10A (EHR only)
sodium hydroxide 5g 17
sodium phosphate dibasic 100 g 15, 16

heptahydrate (Na2HPO4)

Appendix F
sodium phosphate monobasic 100 g 15, 16

monohydrate (NaH2PO4)
sucrose 600 g 1A, 1B, 4B
tacrine 1g 16
TAE buffer, 50X 1L 6B (both options)
Toluidine Blue, 0.5% 15 mL 3
tryptophan 5g 17
tyrosine 5g 15, 16
Bio-Rad Kits*
TM
pGLO Bacterial Transformation Kit 1 6A
Analysis of Precut Lambda DNA Kit 1 6B (opt. 1)
Forensic DNA Fingerprinting Kit 1 6B (opt. 2)
Got Protein? Kit 1 17
Suppliers
Bio-Rad Laboratories, Inc. Flinn Scientific Inc.
1-800-424-6723 1-800-452-1261
explorer.bio-rad.com www.flinnsci.com
WARD’S Natural Science

1-800-962-2660
www.wardsci.com

Appendix
G
Bio-Rad Products for Advanced Biology
1-800-424-6723 – explorer.bio-rad.com
Bio-Rad’s goal is to excite and inspire educators about the cutting edge science of biotechnology,
and enable them to take that excitement back to their classrooms. Bio-Rad Explorer kit
applications align with current life science education standards and performance indicators and
meet the most rigorous college preparatory requirements.
Each of the kits includes sufficient material for 32 students (eight student workstations;
four students per workstation). Kits are available to educators at a discount. To place an order,
please call customer service at 1-800-424-6723 to set up an educational account to receive
discounts.
Lab 6A: pGLOTM Bacterial Transformation Kit

Catalog number: 166-0003EDU, 166-0555EDU (Refill)
Genetic engineering is the process of manipulating the genetic material of an organism – often to
include the DNA from a foreign organism. In this activity, students transform bacteria by
introducing a gene from a bioluminescent jellyfish, Aequorea victoria. Bio-Rad’s exclusive
pGLO plasmid is constructed with the jellyfish gene that encodes Green Fluorescent Protein
(GFP), an antibiotic-resistance gene that encodes β-lactamase protein and the AraC gene
encoding a regulator protein that turns the GFP gene on and off. Bacteria transformed with pGLO
are selected by ampicillin resistance, and, when induced to express GFP, the bugs glow
fluorescent green under UV light!
Key Kit Features
• Transform bacteria with jellyfish gene
• Study gene regulation
• Use controls to select for transformed bacteria
• Complete in two 45-minute lab sessions
pGLO Bacterial Transformation Kit (6A) (catalog number: 166-0003EDU)
Contents Amount Contents Amount
E.coli strain HB101 K-12, lyophilized 1 vial Pipets, sterile, individually wrapped 50
pGLO Plasmid, 20 µg 1 vial Inoculation loops, 10 µL, sterile, 80 8 pkgs
Ampicillin, 30 mg, lyophilized 1 vial Petri dishes, 60mm, sterile, 40 2 pkgs
L (+) Arabinose, 600 mg, lyophilized 1 vial EZ Micro Test Tubes, colored, 2.0 mL 60
Transformation Solution, 15 mL 1 bottle Foam micro test tube holders 8
LB Broth, sterile, 10 mL 1 bottle Instructional manual 1
LB Nutrient Agar Powder, 20 g 1 pouch
Advanced Biology with Vernier G-1

Appendix G
Transformation Kit Refill Package (catalog number: 166-0555EDU)
Contents Amount
pGLO Plasmid, 20 µg 1 vial

E.coli strain HB101 K-12, lyophilized 1 vial
LB Broth, sterile, 10 mL 1 bottle
Transformation Solution, 15 mL 1 bottle
Ampicillin, 30 mg, lyophilized 1 vial
L (+) Arabinose, 600 mg, lyophilized 1 vial
Lab 6B (Option 1): Analysis of Precut Lambda DNA Kit

Catalog number: 166-0001EDU, 166-0011EDU (Refill)
The analysis of precut lambda DNA kit demonstrates basic procedures and principles of DNA gel
electrophoresis, including agarose gel casting, sample loading, size-based separation of DNA
fragments, DNA staining, and graphic analysis. This activity provides in-depth explanations
about how restriction enzymes cut DNA and how electrophoresis can be used to separate and
visualize DNA fragments. By visualizing the effect of three restriction enzymes on four identical
samples of double-stranded lambda virus DNA, students learn that different restriction enzymes
recognize and cut different DNA sequences. DNA size standards are also included for use as
positive controls for the electrophoresis and as a reference to determine the sizes of unknown
DNA fragments. The optional extension guides students through the procedure of DNA fragment
size determination by constructing a standard curve from their gel data and using that to
determine the sizes of the various DNA fragments in their samples.
Key Kit Features
• Study DNA restriction enzyme function
• Use electrophoresis to separate DNA fragments
• Construct standard curves from student data
• Make precise determinations of DNA fragments sizes
Analysis of Precut Lambda DNA Kit (catalog number: 166-0001EDU)
HindIII lambda digest (0.2 μg/μL), 100 μL 1 vial Multicolor micro test tubes 60 tubes
PstI lambda digest (0.2 μg/μL), 100 μL 1 vial Foam micro test tube holders 8
EcoRI lambda digest (0.2 μg/μL), 100 μL 1 vial Agarose, 5 g 1
Lambda DNA uncut (0.2 μg/μL), 100 μL 1 vial Electrophoresis buffer, 50X, TAE 100 mL
Sample loading dye, 5X, 1 mL 1 vial Staining trays 4
™
Fast Blast DNA stain, 500X 100 mL
G-2 Advanced Biology with Vernier

Precut Lambda DNA Kit Refill (catalog number: 166-00011EDU)
Contents Amount
DNA size standard 1 vial

PstI lambda digest 1 vial
EcoRI lambda digest 1 vial
Uncut lambda DNA 1 vial
Sample loading dye, 5X, 1 mL 1 vial
™
Fast Blast DNA stain, 500X, 100 mL 1 bottle
Lab 6B (Option 2): Forensic DNA Fingerprinting Kit

Catalog number: 166-0007EDU, 166-0027EDU (Refill pack)
This activity provides in-depth explanations about how restriction enzymes cut DNA and how
electrophoresis is used to separate and visualize DNA fragments. The kit guides students through
the procedure of constructing a standard curve using their own gel data, which may then be used
to estimate the molecular weights of the unknown DNA fragments generated by restriction
enzymes.
Electrophoretic technique that distinguish DNA fragments by size are essential in forensics and
in the mapping of restriction sites within genes. With the curriculum in this kit, students also
have the opportunity to read plasmid maps and predict the sizes of DNA fragments from
restriction enzyme digests prior to performing the laboratory activity. They can go one step
further and use restriction digest maps of lambda bacteriophage genomes to design novel
plasmids. By doing these extension activities, students learn how restriction enzymes function
and their use in genetic engineering. Use this kit to open the door to rich discussions about the
scientific, ethical, and legal implications of forensics, DNA profiling, and genetic engineering.
Key Kit Features
• Written in the context of a classic human forensic application and adaptable to other
scenarios
• Study DNA and restriction enzyme functions
• Use electrophoresis to visualize DNA fragments and construct a standard curve
• Make precise determinations of DNA fragments sizes
• Optional extension activities include:
- Reading plasmid maps
- Predicting the sizes of DNA fragments
- Designing novel plasmids based on restriction digest maps of lambda bacteriophage
genomes

Appendix G
Forensic DNA Fingerprinting Kit (catalog number: 166-0007EDU)
Crime Scene (CS) DNA with buffer, Suspect 1 (S1) DNA with buffer,
1 vial 1 vial
lyophilized, 60 μg lyophilized, 60 μg
EcoRI/PstI, restriction enzyme mix, Suspect 2 (S2) DNA with buffer,
1 vial 1 vial
lyophilized, 3000 units lyophilized, 60 μg
HindIII lambda digest (DNA size Suspect 3 (S3) DNA with buffer,
1 vial 1 vial
marker), 0.2 μg/μL, 100 μL lyophilized, 60 μg
™
Fast Blast DNA stain, 500X, Suspect 4 (S4) DNA with buffer,
1 bottle 1 vial
100 mL lyophilized, 60 μg
Suspect 5 (S5) DNA with buffer,
Electrophoresis buffer, 50X, TAE, 100 mL 1 vial
lyophilized, 60 μg
Multicolor micro test tubes, 2 mL 60 DNA sample loading dye 1 vial
Clear micro test tubes, 1.5 mL 30 Sterile water, 2.5 mL 1 vial
Foam micro test tube holders 16 Agarose, 5 g 1
Staining trays 4
DNA Fingerprinting Kit Refill Package (catalog number: 166-0027EDU)
Contents Amount
Crime Scene (CS) DNA with buffer, lyophilized, 60 μg 1 vial

Suspect 1 (S1) DNA with buffer, lyophilized, 60 μg 1 vial
EcoRI/PstI, restriction enzyme mix, lyophilized, 3000 units 1 vial
Sterile water, 2.5 mL 1 vial
HindIII lambda digest (DNA size marker), 0.2 μg/μL, 100 μL 1 vial
DNA sample loading dye 1 vial
™
Fast Blast DNA stain, 500X, 100 mL 1 bottle

Lab 17: Got Protein? Kit

Catalog number: 166-2900EDU, 500-0204EDU and 166-2403EDU (Refill)
Based on the Bio-Rad Quick Start Bradford protein assay, this inquiry-based kit is designed to
introduce students to proteomics. Students use absorbance values from a set of protein samples
with known concentrations to create a standard curve. They learn to use a spectrophotometer,
micropipet, and computer, which are all invaluable tools in modern bioscience research.
This biophotonics laboratory activity allows students to analyze and compare the protein content
in milk, sports drinks, egg, muscle tissue, saliva, tears, or any source of soluble biologically
derived material. Protein quantitation is often necessary before isolation, separation, and analysis
by chromatography, electrophoresis, or western blotting. This lab integrates biology, chemistry,
and physics, allowing students to develop understanding about how the chemical, physical, and
biological properties of proteins determine their structure and function.
Key Kit Features
• Explore biophotonics
• Study protein structure/function
• Apply Beer's law
• Measure protein concentrations
• Learn spectrophotometry
• Complete in one 45 minute lab session
™
Got Protein? Kit (catalog number: 166-2900EDU)
Contents Amount
Quick Start Bradford protein assay kit 4,

includes 1X dye reagent (1 L), bovine γ-
1 kit
globulin standard set (2 sets of 7
standards,0.125-2.0 mg/mL)
10X phosphate buffered saline (PBS), 100 mL 1 bottle
1.5 mL semimicro cuvettes, 100 1 pack
Got Protein? Instruction manual 1
™
Got Protein? Refill Items
Catalog number Item Amount
Quick Start Bradford Assay Kit 4
500-020EDU 1X dye reagent (1 L) 1L

bovine γ-globulin standard set (2 sets
2 sets
of 7 standards,0.125-2.0 mg/mL), 2 mL
166-2403EDU 10X PBS 100 mL
223-9955EDU 1.5 mL semimicro cuvettes, 100 1 pack

Appendix G
TEACHER RESOURCES
Website: explorer.bio-rad.com
Discover the Bio-Rad online classroom biotechnology teaching resources at explorer.bio-
rad.com. Whether you’re just getting started or looking for something new, take a look at the
large portfolio of educational resources that Bio-Rad offers:
• Educator discounts & price quotes • Kit curricula & instruction manuals
• Online ordering • Special offers, free posters
• Grants and grant writing • Research equipment and supplies
• Tutorials and animations • Technical support
• Complete classroom biotech lab • PowerPoint presentations, images, and
equipment packages lectures
• Professional development workshops and • Quality classroom kits and kit refill
conference schedules packages
Explorer Catalog
The Explorer catalog is informative and easy-to-read with its vivid photographs and colorful
diagrams and flow charts. Biotechnology Explorer kits help you teach more efficiently by
integrating multiple core content subjects into a single lab, connecting concepts with techniques,
and putting them into context with real-world scenarios. The majority of the kits are designed to
be completed in 45-minute lab sessions. Request your free Explorer catalog for detailed
descriptions on all kits, consumables, equipment, and resources.
Partners
Collaborations with teachers, schools, and educational organizations around the world have
provided invaluable insight into developing kits, curricula, and professional development
programs that align with state and national education standards.
Bio-Rad Laboratories has partnered with Vernier Software & Technology to bring a complement
of quality kits and data analysis tools to your classroom.
NON-CONSUMABLES
Bio-Rad Catalog Lab 6B Lab 6B
Item Lab 6A Lab 17
Number (Option 1) (Option 2)
Adjustable micropipet, 100–1000 µL 166-0508EDU
BLUE-VIEW
BlueView Transilluminator
(Vernier)
or
WHT-TRANS
*
White Light Transilluminator *BlueView
(Vernier)
or or UV only
166-0500EDU
Long wave UV lamp
(Bio-Rad)
Fixed volume micropipet, 10 µL 166-0512EDU
Horizontal electrophoresis chamber 166-4000EDU

Microcentrifuge 166-0602EDU
Microcentrifuge tube racks 166-0481EDU
Micropipet rack 166-0554EDU
Mini centrifuge 166-0603 EDU
Mini incubation oven 166-0501EDU
Power supply 165-5050EDU
Rocking platform 166-0709EDU
Temperature controlled water bath 166-0504EDU
CONSUMABLES
Bio-Rad Catalog Lab 6B Lab 6B
Item Lab 6A Lab 17
Number (Option 1) (Option 2)
1.5 mL, EZ micro test tubes, 500/box 223-9480EDU
1
2.0 mL, EZ micro test tubes 223-9430EDU
Certified Molecular Biology Agarose, 25 g 161-3100EDU
Disposable plastic transfer pipets, sterile, 500 166-0474EDU
DNA stain 166-0420EDU
Fast Blast DNA stain, 50X, 100 mL (Bio-Rad)
or SYBR-05X
SYBR Safe DNA stain, 0.5X, 1L (Vernier)
or SYBR-10KX
SYBR Safe DNA stain, 10,000X, 400 μL (Vernier)
Gel support film 170-0554EDU
LB nutrient agar powder 166-0600EDU
Petri dishes, 60mm, sterile, 500 166-0470EDU
Pipet tips, 2–200 µL, 1000/box 223-9347EDU
Pipet tips, 100–1000 µL, 1000/box 223-9350EDU
Sample loading dye, 5X, 10 mL 161-0760EDU
TAE buffer, 50X, 1 L 161-0743EDU
1
Refill micro test tubes are not colored.

Appendix
AP ∗ Correlations H
Correlation of AP Recommended Experiments
The College Board AP Biology Course Description booklet (Acorn book) describes
12 recommended laboratory experiments. Data show that student scores on the AP Biology
Exam improves with increased time spent doing these experiments. As shown in the table below,
we have correlated the 12 experiments in Biology with Vernier to the 12 lab experiments
recommended by AP in the Acorn booklet.

AP Lab and Objectives Met
Experiment
Lab 1: Diffusion and Osmosis

• Relate osmotic potential to solute concentration and
water potential.
Experiment 1A: Diffusion
• Measure the water potential of a solution in a
Experiment 1B: Osmosis controlled experiment.
• Determine the osmotic concentration of living tissue
or an unknown solution from experimental data.
• Describe the effects of water gain or loss in animal
and plant cells
Lab 2: Enzyme Catalysis

• Measure the effects of changes in temperature, pH,
Experiment 2: Enzyme Action Testing and enzyme concentration on reaction rates of an
Catalase Activity enzyme catalyzed reaction in a controlled
experiment.
• Explain how environmental factors affect the rate of
enzyme-catalyzed reactions.
Lab 3: Mitosis and Meiosis

• Recognize the stages of mitosis in a plant or animal
cell.
• Calculate the relative duration of the cell cycle
Experiment 3: Mitosis and Meiosis
stages.
• Compare and contrast the results of meiosis and
mitosis in plant cells.
• Compare and contrast the results of meiosis and
mitosis in animal cells.
∗
AP and Advanced Placement Program are registered trademarks of the College Entrance Examination Board, which was not involved in the
production of and does not endorse this product.
Advanced Biology with Vernier H-1

Appendix H

Experiment
Lab 4: Plant Pigments and Photosynthesis

• Compare photosynthetic rates at different light
Experiment 4A: Plant Pigment
intensities.
Chromatography
• Separate pigments and calculate their Rf values.
Experiment 4B: Photosynthesis • Describe a technique to determine photosynthetic
rates.
• Explain why the rate of photosynthesis varies under
different environmental conditions.
Lab 5: Cell Respiration

• Calculate the rate of cell respiration from
experimental data.
Experiment 5: Cell Respiration
• Relate gas production to respiration rate.
• Test the effect of temperature on the rate of cell
respiration in non-germinated versus germinated
seeds in a controlled experiment.
Experiment 6A: Transformations Lab 6: Molecular Biology

• Use plasmids as vectors to transform bacteria with a
Experiment 6B: Analysis of Precut gene for antibiotic resistance in a controlled
Lambda DNA experiment.
• Describe the biological process of transformation in
Experiment 6B: Forensic DNA bacteria.
Fingerprinting
• Calculate transformation efficiency.
Lab 7: Genetics of Organisms

• Investigate the independent assortment of two genes
Experiment 7: Genetics and determine weather the two genes are autosomal
or sex-linked using multigeneration experiment.
• Analyze the data from your genetic crosses using chi-
squared analysis techniques.
Lab 8: Population Genetics and Evolution

• Calculate the frequencies of alleles and genotypes in
the gene pool of a population using the Hardy-
Experiment 8: Population Genetics
Weinberg formula.
• Discuss natural selection and other causes of
microevolution as deviations from the conditions
required to maintain Hardy-Weinberg equilibrium.
H-2 Advanced Biology with Vernier

AP Correlations

Experiment
Lab 9: Transpiration
• Test the effects of environmental variables on rates of
Experiment 9: Transpiration transpiration using a controlled experiment.
• Make thin sections of stem, identify xylem and
phloem cells, and relate the function of these vascular
tissues to the structures of their cells.
Lab 10: Physiology of the Circulatory System

• Measure the heart rate and blood pressure in a human
Experiment 10A: Blood Pressure as a volunteer.
Vital Sign • Describe the effect of changing body position on
heart rate.
Experiment 10B: Heart Rate and • Explain how exercise changes heart rate.
Physical Fitness
• Determine a human's fitness index.
• Analyze cardiovascular data collected by the entire
class.
Lab 11: Animal Behavior

• Describe some aspects of animal behavior, such as
Experiment 11: Animal Behavior orientation behavior, agonistic behavior, dominance
display, or mating behavior.
• Understand the adaptiveness of the behaviors you
studied.
Lab 12: Dissolved Oxygen and Aquatic Primary

Experiment 12A: Dissolved Oxygen in Production
Water • Measure primary productivity based on changes in
dissolved oxygen in a controlled experiment.
Experiment 12B: Primary Productivity • Investigate the effects of changing light intensity on
primary productivity in a controlled experiment.
Advanced Biology with Vernier H-3

Index
Index
(by Experiment Number)
A E
Absorbance, 4B, 13, 14 E. coli, 6A
Acetylcholine, 16 Electrophoresis, 6B
ACh, 16 Ellman method, 16
AChE, 16 Environmental changes, responses to, 11
Acid, 10A Enzymes, 2, 15, 16
Active transport, 1B Exercise, 10A
Allele frequencies, 8
Amino acids, 17 F
Aortic valve, 10A Field study, 1B
ATP, 4 Fight or flight response, 10A
B G
Beer’s Law, 17 Gel electrophoresis, 6B
Binding affinity, 15 Genes, 3, 6A
Biuret Reagent, 17 Genetic drift, 8
Blood pressure, 10A Germination, 5
Bradford assay, 17 Green Fluorescent Protein (GFP), 6A
Gross productivity, 12B
C
Casein, 17 H
Catalase, 2 Hardy-Weinberg Principle, 8
Catalysts, 2 Heart rate, 10A
Cellular membrane, 1A, 1B Heat shock method, 6A
Chi-square statistical analysis, 7 Heterozygous advantage, 8
Chlorophyll, 4A, 4B Hydrogen peroxide, 2
Chloroplast, 4B Hypertension, 10A
Chromatography, 4A
Chromosome, 3 I
Circulatory system, 10A IC50, 16
Cold, response to, 10A Initial rate, 2
Concentration gradient, 1A Ions, 1A
Conductivity, 1A
K
D Km, 15
Denature, 2
Detergents, 9
Diastolic pressure, 10A
L
Life cycle of Sordaria, 3
Diffusion, 1A, 1B
Dihybrid crosses, 7
Dissolved oxygen, 12A, 12B M
DOPA, 15 Macromolecules, 17
Dopachrome, 15 Mass, 9
Dose-response curve, 16 Mean arterial pressure (MAP), 10A
Drosophila, 7, 11 Membrane, 1A, 1B
Mendelian inheritance patterns, 7
Advanced Biology with Vernier Index - 1

Index
Mesophyll, 9 T
Michaelis-Menten constant, 15 Tacrine, 16
Mitosis, 3 Temperature, 2, 5, 12A
Monohybrid crosses, 7 Transformation efficiency, 6A
Transpiration, 7
N Tyrosinase, 15
NADPH, 4B
Natural selection, 8 V
Net productivity, 12A Vasovagal syncope, 10A
Neurons, 16 Velocity, maximum, 15
Neurotransmitters, 16 Velocity, reaction, 15
Nitrogen, 12A Vital sign, 10A, 10B
O W
Osmosis, 1B Water potential, 1B, 9
Osmotic balance, 1B
Oxidation, 5 X
Xylem, 9
P
Passive transport, 1B Y
Permeability, 1A, 1B Yeast, 2
Peroxidase, 2
pH, 2
Phosphates, 12B
Photosynthesis, 4B, 12B
Physical fitness, 10A
Pressure, 1B, 2 (Gas Pressure), 5 (Gas Pressure), 9
Primary productivity, 12B
Proteins, 17
Pulse, 10A
R
R-group, 17
Rf, 4
Rate, 1B, 2, 5, 9, 12B, 15
Respiration, aerobic (cellular), 5
Respirometer, 5 (Gas Pressure)
Risk factor, 10A
S
Sex-linked crosses, 7
Spectrum, 13, 14
Standard curve, 17
Stomata, 9
Stroke, risk of, 10A
Sugars, 1A
Sympathetic nervous system, 10A
Systolic pressure, 10A
Index - 2 Advanced Biology with Vernier

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SECOND EDITION

Advanced Biology with Vernier

Experiments for AP* and College Biology

Vernier Software & Technology

Experiments for AP and College Biology

Sensors Used in Experiments............................................................................................ vii

White or Blue Digital

* - Included in the Blue or White Digital Bioimaging System

There are three different versions of each experiment in this book:

Vernier Software & Technology Biology Department

Diffusion through Membranes

Advanced Biology with Vernier 1A - 1

Part I Concentration gradients

1A - 2 Advanced Biology with Vernier

6. Place a 1% salt solution into a section of dialysis

10. Determine the rate of diffusion. To do this:

Advanced Biology with Vernier 1A - 3

Part II Effect of other molecules

Test to determine if water or a sugar solution conducts electricity.

20. Obtain 300 mL of a 5% sugar solution in a clean 400 mL beaker.

Test if 5% sugar interferes with the diffusion of a 5% salt solution.

1A - 4 Advanced Biology with Vernier

Salt concentration Rate of diffusion

Table 3 Table 4: Summary of Data

Solution Concentration Solution Rate of diffusion

Distilled water 5% salt

Sugar water 5% salt / 5% sugar

Advanced Biology with Vernier 1A - 5

1A - 6 Advanced Biology with Vernier

Ideas for Inquiry

Advanced Biology with Vernier 1A - 1 T

Salt concentration Rate of diffusion

Table 3 Table 4: Summary of Data

Solution Conductivity Solution Rate of diffusion

Distilled water 1.5 5% salt 1.23

Sugar water 1.9 5% salt / 5% sugar 1.21

Diffusion through dialysis tubing of differing salt concentrations

1A - 2 T Advanced Biology with Vernier

Advanced Biology with Vernier 1A - 3 T

Advanced Biology with Vernier 1B - 1

1B - 2 Advanced Biology with Vernier

9. Click to begin data collection.

11. Rinse out the dialysis tubing and Styrofoam cup.

12. Determine the rate of change in osmotic pressure.

14. Repeat Steps 6–13 for the remaining solutions.

Sucrose solution Rate of pressure

Advanced Biology with Vernier 1B - 3

PROCESSING THE DATA

3. Does sucrose move in or out of the cell? Explain.

1B - 4 Advanced Biology with Vernier

EXTENSION – WATER POTENTIAL

1. Pour 100 mL of the assigned sugar solution into a 250 mL beaker.

3. Put the four cores into the beaker of sugar solution.

7. Repeat Steps 1–6 for any additional assigned sugar solutions.

Advanced Biology with Vernier 1B - 5

Sugar solution Initial mass Final mass Percent change in

Sugar molar concentration

PROCESSING THE DATA

1B - 6 Advanced Biology with Vernier

2. What factors affect water potential?

Advanced Biology with Vernier 1B - 7

4. Soak 15 cm segments of dialysis tubing in distilled water prior to the lab.

1.0 M 250.0 mL 0.0 mL