For the assignments in this course, you will be developing a Disas.docx

For the assignments in this course, you will be developing a
Disaster Recovery and Business Continuity (DR/BC) Plan that
defines the objectives, planning process, team creation, risk
analysis, business issues, implementation, testing, and
maintenance required for safeguarding the organization. Your
first task in this process will be to consider situation of an
imaginary company of your choice and create the framework for
a DR/BC Plan.
Project Concept and Executive Sponsorship:
- Provide a brief description a company of your choice and your
assignment to create the DR/BC Plan.
- Provide comments regarding the instructions that you might
receive from corporate executives.
- Provide support through research regarding the need for
executive sponsorship.

DR/BC Introduction and Risk Assessment:
- Provide an introduction to the new DR/BC Plan that the
organization plans to implement.
- Prepare a risk assessment that explains the various types of
threats that could disrupt the business of your chosen
company.
- This should include consideration of both manmade and
natural threats, as well as any threats that may be more likely
given the geographic location of company facilities.
- Support your positions with references obtained from the
university library, Web, text, or other reputable sources.

1
Rutgers University – Newark
College of Arts & Sciences
Department of Biological Sciences
General Biology II (21:120:102)
Lab Learning Objectives & Lab Learning Activities
CONTENT
General Information
...............................................................................................
..... 3 1
Laboratory Safety Rules
...............................................................................................
4 2
Laboratory Syllabus
...............................................................................................
...... 5 3
Microscope Use
...............................................................................................
............ 7 4

4.1 The Compound Microscope
................................................................................. 7
4.2 The Dissecting Microscope
................................................................................... 9
Lab Report Format
...............................................................................................
...... 11 5
Lab 1 – Viewing and Measuring Cells
........................................................................ 12 6
6.1 Exercise 1 - Features of the compound microscope
.......................................... 12
6.2 Exercise 2 – Procedure for viewing specimens
.................................................. 14
6.3 Exercise 3 – The image under a compound
microscope.................................... 15
6.4 Exercise 4 – Mirror images
................................................................................. 16
6.5 Exercise 5 – Features of the dissecting microscope
.......................................... 17
6.6 Exercise 6 – Use of the dissecting
microscope................................................... 18
6.7 Exercise 7 – Calculating total magnification of objects
..................................... 19
6.8 Exercise 8 – Determining the diameter of the field of view.

............................. 20
6.9 Exercise 9 – Using the diameter of the field of view to
measure cells .............. 21
6.10 Exercise 10 – Preparing a wet mount of a naturally
pigmented (red onion) cell.23
2
6.11 Exercise 11 – Staining a wet mount of an unpigmented
(human cheek) cell. ... 24
6.12 Exercise 12 – Estimating the size of a unicellular
organism............................... 26
Lab 2 – Enzymes and Cell Function
............................................................................ 28 7
7.1 Exercise 1 – Browning of fruit
............................................................................ 31
7.2 Exercise 2 – Effect of temperature on enzyme-catalyzed
reactions ................. 36
7.3 Exercise 3 – Effects of pH on enzyme-catalyzed reactions
................................ 39
Lab 3 – Cell Membranes and Water Balance
............................................................ 44 8
8.1 Exercise 1 – Movement of water into a "model cell"

........................................ 47
8.2 Exercise 2 – Using the plasmolysis threshold to estimate the
concentration of
solutes and water inside living cells
.............................................................................. 52
8.3 Exercise 3 – Comparing solute concentrations (isotonic
points) inside plant cells
from different environments
........................................................................................ 56
Lab 4 – Photosynthesis
..............................................................................................
61 9
9.1 Exercise 1 – Separation of leaf pigments by paper
chromatography ................ 62
9.2 Exercise 2 – Internal features of a leaf
............................................................... 64
9.3 Exercise 3 – Stomata
.......................................................................................... 66
9.4 Exercise 4 – The effect of carbon dioxide and pH on the
rate of photosynthesis68
9.5 Exercise 5 – The effect of the color of light on the rate of
photosynthesis ....... 71
Lab 5 – DNA Fingerprinting
........................................................................................ 74
10
10.1 Exercise 1 – extraction DNA from cells

.............................................................. 75
10.2 Exercise 2 – Practice using pipettes to load wells
.............................................. 77
10.3 Exercise 3 – DNA fingerprints of unknown DNA samples
.................................. 78
Copyright: Dr. Douglas Morrison
Modified by Dr. Rola Bekdash
Updated - January 2019
3
General Information 1
protected]);
Boyden Hall 313;
Extension 1267)
protected]);
Biology
Learning Center, Boyden Hall 217; Extension 5108)

([email protected]); Boyden
Hall 111; Extension 1220)
Teaching Assistants
Location of the General Biology Laboratory: Boyden Hall 223A
& 223B
Weekly Laboratory Sessions
Please attend the Lab session that you did register for. The
duration of each lab session is 3 hours.
Section 1 Monday 1 – 3:50 pm
Section 2 Monday 1 – 3:50 pm
Section 3 Tuesday 8:30 – 11:20 am
Section 4 Tuesday 8:30 – 11:20 am
Section 5 Tuesday 2:30 – 5:20 pm
Section 6 Tuesday 2:30 – 5:20 pm
Section 7 Wednesday 8:30 – 11:20 am
Section 8 Wednesday 8:30 – 11:20 am

Section 9 Wednesday 11:30 am – 2:20 pm
Section 10 Wednesday 11:30 am – 2:20 pm
Section 11 Thursday 8:30 – 11:20 am
Section 12 Thursday 8:30 – 11:20 am
Section 13 Thursday 1 – 3:50 pm
Section 14 Thursday 1 – 3:50 pm
mailto:[email protected]
4
Laboratory Safety Rules 2
1. Wear your white lab coat before starting your lab session and
bring your dissection kit with
you.
2. Do not start any procedure until your Teaching Assistant
(TA) has described and discussed
the exercise and the potential hazards associated with it.

3. Wear protective eye wear for all dissection exercises as well
as exercises using hazardous
materials. All students are required to get their own safety
goggles to lab EACH day
whether or not they are needed for the procedure.
4. Wear disposable gloves when you are doing dissection,
working with preserved specimens,
handling blood smaples or working with chemicals.
5. Keep long hair tied back if you are working with dissection
specimens or other hazardous
material.
6. Do not eat or drink or bring any food or beverages into the
lab.
7. Notify your TA and the lab supervisor of any accidents,
including minor cuts, punctures, or
spill of chemicals.
8. If you have any medical condition (e.g. allergies, medication,
and pregnancy) that may
increase your sensitivity to certain chemicals or procedures, you
should notify your TA and
consult your physician.

9. Dispose all sharps such as needles, razor blades, syringes,
slides, Pasteur pipettes and
capillary tubes in the “sharp containers”.
10. Dispose all live specimens and used gloves in the the
container that has an autoclave bag.
11. Clean your work area with disinfectant at the end of each
lab session and return all chairs to
their designated places.
5
Laboratory Syllabus 3
WEEK *LABORATORY TOPIC/QUIZZES *REVIEW & LAB
PREPS
Week 1
1/22 – 1/24
No Labs this week
Week 2
1/28 – 1/31

Lab # 1 – Cells
Week 3
2/4 - 2/7
Review Lab # 1
Quiz 1 on Lab #1
Prep Lab # 2
Week 4
2/11 - 2/14
Lab # 2 – Enzymes
Week 5
2/18 - 2/21
Review Lab # 2
Quiz 2 on Lab #2
Finish Lab#2 Report
Prep Lab # 3
Week 6
2/25 - 2/28
Lab # 3 – Membranes
Week 7
3/4 - 3/7
Review Lab # 3
Quiz 3 on Lab #3
Finish Lab#3 Report

Prep Lab # 4 & Review Lab Exam I
Week 8
3/11 - 3/14
Lab Exam I (Labs # 1, 2 & 3)
Week 9
3/18 - 3/21
Spring Recess – No Labs this week
Week 10
3/25 - 3/28
Lab # 4 – Photosynthesis
Week 11
4/1 – 4/4
Review Lab # 4
Quiz 4 on Lab #4
Prep Lab # 5
Week 12
4/8 - 4/11
Lab # 5 – DNA Technology
Week 13
4/15 – 4/18
Review Lab # 5

Quiz 5 on Lab #5
Review Lab Exam II
Week 14
4/22 – 4/25
Lab Exam II (Labs # 4 & 5)
(*) PLEASE READ THE LABORATORY TOPIC BEFORE
COMING TO CLASS
YOU ARE REQUIRED TO BRING A DISSECTION KIT
YOU ARE REQUIRED TO ATTEND AND PARTICIPATE IN
ALL THESE LAB SESSIONS. IF YOU MISS MORE
THAN TWO LAB SESSIONS, YOU WILL BE ASKED TO
WITHDRAW FROM THE COURSE. YOU CANNOT
MAKE-UP FOR ANY MISSED LAB SESSION. IT IS YOUR
RESPONSIBILITY TO KNOW WHAT YOU MISSED
AND COVER IT. MAKE-UP FOR LAB QUIZZES MAY BE
DONE WITH THE APPROVAL OF YOUR TA/PTL
WITH THE SUBMISSION OF VALID DOCUMENTATIONS.
*NO MAKE-UPS FOR LAB EXAMS. A MISSED LAB EXAM
WILL GET A ZERO GRADE.
6
Grading distribution of the Laboratory
Lab grade represents 25% of the total course grade.

Lab Exam I 10 %
Lab Exam II 10 %
Lab Review/pre Sessions/Quizzes (5 sessions)
Quiz 1 (1%)
Quiz 2 (0.5%)
Lab2 Report (0.5%)
Quiz 3 (0.5%)
Lab3 Report (0.5%)
Quiz 4 (1%)
Quiz 5 (1%)
Total 5 %
In addition to the five lab sessions, you are all required to
attend and participate in the five Lab
Review/Prep sessions. These sessions are worth 5% of the total
course grade. These sessions
are designed to help you prepare for your next week lab and to
review the results/findings of
the lab that you finished the week before. You will have
quizzes. You are also required to
submit with your partner two lab reports based on data obtained
from Labs 2 & 3.
So, come fully prepared to do your experimental work, record
your data and finish the 3 hr lab
session in a timely manner.
Problems

Any health-related problems that may arise from unexpected
accidents (chemical spills, minor cuts ...)
during lab time, please inform your TA, the lab supervisor
and/or the lab supervisor specialist.
7
Microscope Use 4
4.1 The Compound Microscope
Figure 1: Major features of a compound microscope: occular
lens, 2-3 objective lenses, coarse
and fine adjusatment knobs, and iris diaphragm.
8
Using a compound microscope
1. Place the prepared slide on the microscope stage so that
mounted specimen is centered
over the hole in the stage. Use the stage clips to secure the
slide in place.

2. Always begin with the lowest power objective. Rotate the
nosepiece to bring the shortest
objective (usually 4x) into position.
3. Turn the coarse adjustment knob to move the objective
downward toward the slide until the
point of the objective lens is just above (but not touching) the
slide.
4. Turn on the microscope light and look into the eyepiece,
holding your eye about 1/2 inch
from the eyepiece. While looking into the eyepiece, use the
coarse adjustment knob to raise
the body tube slowly until the specimen comes into focus.
Sharpen the image with the fine
adjustment knob. If you see nothing, you may need to recenter
the specimen over the hole in
the statge.
5. Note that the contrast between light and dark can be adjusted
using the substage, iris
diaphragm. If the light is too bright, the image may be“washed
out” (invisible).
6. While watching through the ocular lens, move the slide to
the left or right. Which direction
did the image of the specimen move? Now move the slide away
from you or towards you.
What direction did the image move now?
7. For greater magnification, rotate the nosepiece to bring a
higher power (longer) objective
into position. Whenever a higher power objective is in place,
use only the fine focus
adjustment to refocus!

8. Turn off the sub-stage light whenever you are not looking
through the microscope! The
bulbs are expensive and burn out quickly.
9. When you have finished using the compound microscope:
a. Turn off the light source.
b. Rotate the nosepiece to the low power objective.
c. Unplug the microscope and coil the wire loosely over the
body tube.
d. Cover the microscope.
9
4.2 The Dissecting Microscope
The dissecting microscope differs from the compound scope in
many ways:
a. Total magnification is much lower (maximum either 30 or 45
power).
b. Objective lens is equipped with a "zoom" adjustment.
c. Two ocular lenses give a stereoscopic (3-D) view of the
specimen.
d. Only one focus adjustment knob.
e. Longer "working distance" (between specimen and objective
lens).

Figure 2. Major features of the dissecting scope: stage (for
specimen), ocular lenses, objective
(zoom) lens, coarse focusing adjustment, light sources (both
above and below specimen).
10
Using a dissecting microscope
1. Choose the proper illumination (lighting). Some specimens
are better viewed using
reflected light (with the light source above the specimen),
others with transmitted light (with
the light source under the specimen).
2. The total magnification of the microscope can be calculated
by multiplying the power of the
ocular lens (engraved on the side of the eye piece) by the power
of the objective (zoom) lens
(read from the dial on the zoom control knob).
3. The orientation of the image is intuitive – when you move
the specimen, the image moves
in the same direction – unlike the compound microscope.

11
Lab Report Format 5
You will write two lab reports based on data from labs 2 & 3. A
scientific report usually consists
of the following:
1. Title
2. Introduction
3. Materials and Methods
4. Results
5. Discussion & Conclusion
Title
The title should be less than ten words, is straightforward and
reflects the factual content of
the experiment.
Introduction
The introduction defines the subject of the report. It must
outlines the scientific objective(s) for
the experiments performed and give the reader sufficient
background to understand the rest of
the report. A good introduction will answer several questions,
including the following:

s the specific purpose of the study?
The specific hypotheses and experimental design pertinent to
investigating the topic should be
described.
Materials and Methods
Materials and methods used in the experiments should be
reported in this section. Provide
enough detail for the reader to understand the experiment
without overwhelming him or her.
Generally, this section attempts to answer the following
questions:
Results
The results section should summarize the data from the
experiments without discussing their
implications. The data should be organized into tables, figures,
graphs, photographs, and so on.
All figures and tables should have descriptive titles. Figures
and tables should be self-
explanatory; that is, the reader should be able to understand
them without referring to the
text. All columns and rows in tables and axes in figures should
be labeled.
Discussion & Conclusion
This section should not just be a restatement of the results but
should emphasize interpretation
of the data, relating them to existing theory and knowledge.
Suggestions for the improvement
of techniques or experimental design may also be included here.
In writing this section, you

should explain the logic that allows you to accept or reject your
original hypotheses. Provide a
conclusion based on the results that you got.
By Warren D. Dolphin, Iowa State University (Modified by Dr.
Bekdash)
12
Lab 1 – Viewing and Measuring Cells 6
All living things on earth are basically similar: all are
composed of one or more cells. The chemical
reactions that support life occur inside cells. Cells don't arise
spontaneously; they arise only from other
cells. Each cell contains all the organism's hereditary
information. This information is passed from
parent to offspring through cells.
How large is a cell? Are all cells about the same size? Why are
cells so small? What limits cell size?
You’ll soon know answers to all these questions!
Cells are usually way too small to be seen with the naked eye.
The human body is made up of trillions of
cells. One of the largest human cells, the human ovum (egg
cell) is only about the size of the period at
the end of this sentence. Since most cells are much smaller than
that, most of what we know about cells
has been gained with the aid of microscopes.

Lab learning objectives
You will have mastered the content of this minicourse when you
are able to:
1. Given a compound light microscope, locate, name and
describe the functions of the light source,
ocular lens, objective lenses, iris diaphragm, specimen stage,
and coarse and fine focus adjustment
knobs.
2. Demonstrate proper use of the compound light microscope.
Given a prepared slide, properly mount
the slide, adjust the light source, and focus on the specimen
first at low power and then at high
power.
3. Compare and contrast the compound microscope and
dissecting microscope with regard to: range
of magnification, how the specimen is illuminated (transmitted
vs. reflected light), focusing
mechanism, depth of focus, appearance of image (2- vs. 3-
dimensional), and spatial relationships
(up, down, right, left) between specimen and image.
4. Demonstrate the proper techniques for preparing wet mounts
of living cells.

5. Use the diameter of the field of view of your compound light
microscope to measure the overall size
of various single-celled protozoa that use different modes of
locomotion
PART I: THE COMPOUND MICROSCOPE
6.1 Exercise 1 - Features of the compound microscope
The microscope you will use first is called a "compound"
microscope because it uses a combination of
two lenses to magnify the image. The lens closest to your eye
is called the "ocular" lens (oculus is Latin
for eye). The lens closest to the object is the "objective" lens.
13
l. Use Figure 1 to identify the parts of your compound
microscope referenced in bold in Exercise 2: the
eyepiece (ocular lens), nosepiece with 3 objective lenses,
substage iris diaphragm (or disc aperture
diaphragm), coarse and fine focus knobs, and light source.
Figure 1: A Compound Microscope

14
Turn off the microscope light whenever you are not looking
through the microscope!
The light bulbs for the compound scope are expensive ($20
each) and burn out quickly.
6.2 Exercise 2 – Procedure for viewing specimens
1. Remove the dust cover and place the microscope in a
convenient position away from the edge of
the table.
2. Wipe the tips of the objective with lens paper. Do not use
facial tissue or a handkerchief, because
these contain fibers that can scratch the lenses.
3. Obtain a prepared slide of the letter "e," clean the slide with
lens paper, and place the slide on the
microscope stage so that the letter is centered over the hole in
the stage. Use the stage clips to
secure the slide in place.
4. Always begin with the lowest power objective. Rotate the
nosepiece to bring the shortest

objective (labeled 4x) into position.
5. While watching from the side, turn the coarse adjustment
knob to move the objective downward
toward the slide until the point of the objective lens is just
above (but not touching) the cover slip of
the slide. Watch carefully so that the objective does not touch
or crack the cover slip. This is
important when viewing commercially prepared slides and even
more important when viewing the
thick “wet mounts” you’ll be making yourself.
6. Turn on the microscope light and look into the eyepiece.
Your eye should not be too close to the
ocular lens. Instead, the eye should be about 1/2 inch from the
eyepiece (see Figure 1). Keep both
eyes open. (Although awkward at first, this practice minimizes
eye strain and lets you view and
sketch the subject at the same time.)
7. While looking into the eyepiece, use the coarse adjustment
knob to raise the body tube slowly until
the specimen comes into focus. If you see nothing, make sure
the specimen is centered over the
hole in the stage, then repeat steps 5 & 6.
8. Sharpen the focus with the fine adjustment knob. Note that
the contrast between light and dark
can be adjusted using the substage iris diaphragm.

9. For greater magnification, rotate the nosepiece to bring a
higher power (longer, 10x power)
objective into position.
10. Whenever a higher power objective is in place, use only the
fine focus adjustment to refocus!
Otherwise you risk cracking the cover slip with the objective
lens, especially when viewing the slides
you have wet mounted yourself. You should not need to turn
the fine adjustment knob very far,
because the objective lenses are designed to be "parfocal," to
minimize the amount of refocusing
needed after changing magnifications.
15
PART II: RELATIONSHIP OF SPECIMEN AND IMAGE
It is frequently necessary to move the specimen around on the
stage to locate the feature you are
interested in. The relationship between the specimen and its
image may not be what you expect!
6.3 Exercise 3 – The image under a compound microscope

1. Hold the letter "e" slide right side up (that is, the way it
normally appears on a printed page) and
place it on the stage of your microscope. Following the
procedures outlined in Exercise 2 (steps 4-
8), bring the "e" into focus under the low power ("scanning")
objective.
2. You put the specimen (the "e") right-side-up on the
microscope stage. Is the image you see of the
"e" also right side up? Is the opening to the left or right. Is the
base of the e up or down? Draw the
image of the e as it appears under the microscope:
e as seen with the naked eye e as seen through compound
microscope
1. What happens to the image you see as you move the slide of
the specimen to the left or right?
Does the image move in the same or the opposite direction?
What happens when you slide the
specimen toward you and away from you? Does the image
move in the same or the opposite
direction? Record your observations in Table 1.
Table 1: Relationship between the actual orientation of the
specimen and the apparent orientation of
the image. (Enter "same" or "opposite" for each category.)

Specimen (as it is on the
microscope slide when seen
with the naked eye)
Image in
compound
microscope
Image in mirror
(Place letter e on
the table in front of
vertical mirror)
Image in dissecting
microscope
When bottom of "e" is toward
you
opposite opposite same
When open side of "e" is on
right
same
If you move "e" sideways (left
or right)
If you move "e" toward and
away from you (up & down)

16
6.4 Exercise 4 – Mirror images
1. Is the microscope image the same as a "mirror image" of the
specimen? To find out, get a small
mirror from the Central Study Area and use it to study the
relationship between the specimen ("e")
and its mirror image. Hold the mirror vertically on the desk and
facing you. Place the "e" flat on the
table top, between you and the mirror so that when you look at
the "e" directly, its orientation is
the way it should be when you are reading. Now look at the
image in the mirror. In what way is the
image in the mirror similar or different from the “e” printed on
the slide? Specifically: (a) If the
bottom of the printed is toward from you, is the bottom side of
the reflected “e” also away from
you? (b) If the open side of the printed “e” is to the right, is the
open side of the reflected image
also to the right? (c) When you move the “e” to the right, does
its image move right or left? (d)
When you move the “e” toward you, does its image move away
from or toward you? Enter your
observations (same or opposite?) for the left/right and
toward/away categories in Table 1.

2. Referring to Table 1, compare the compound microscope’s
image with the mirror image. Is the
image of the "e" in the microscope the same as a mirror image?
Compound microscope image vs. Mirror image of the “e”
a. In what ways are the microscope image and mirror image
similar?
b. In what ways are they different?
17

PART III: THE DISSECTING MICROSCOPE
6.5 Exercise 5 – Features of the dissecting microscope
1. Locate the dissecting microscope in your carrel or in the
Central Study Area. Identify the major
features of the dissecting scope: stage (for specimen),
eyepieces, objective lens, zoom knob,
focusing knob, and light sources above and below specimen
(Figure 2).
2. The dissecting microscope differs from the compound scope
in many ways:
a. Total magnification is much lower (maximum either 30 or
45 power).
b. Objective lens is equipped with a "zoom" adjustment.
c. Two ocular lenses give a stereoscopic (3-D) view of the
specimen.
d. Only one focus adjustment knob.
e. Longer "working distance" (between specimen and objective
lens).
Figure 2: The Dissecting Microscope

18
3. Why do some microscopes have two eyepieces? Dissecting
microscopes always have two eyepieces,
which are connected to two separate objective lenses. The two
separate optical systems work in
parallel to give a stereo, 3-D image. Although some compound
microscopes also have two eyepieces,
both eyepieces are connected to the same objective lense and so
never can produce a 3-D image. In a
compound microscope the second eyepiece simply serves to
reduce eyestrain during prolonged use.
6.6 Exercise 6 – Use of the dissecting microscope.
1. Illumination (lighting) of the specimen can be from above or
below. Since the e is printed on
translucent paper, it should be clearly visible under both
lighting conditions. So examine an opaque
object instead -- like the leg of an insect. Is the specimen more
easily seen using reflected light
(with the light source above the specimen) or transmitted light
(with the light source under the
specimen)?
2. Total magnification of the dissecting microscope can be
calculated by multiplying the power of the
eyepiece engraved on the side of the eyepiece by the power of
the objective (zoom) lens read from

the dial on the zoom control knob. First set the zoom lens so
that the "e" appears as small as
possible and calculate total magnification. Then set the zoom
so the “e” appears as large as possible
and calculate the highest magnification.
3. How does the orientation of the image you see compare with
the actual letter "e" as you move the
slide around. Enter your observations in the right column of the
Table 1.
Power setting Power of eyepiece
(ocular lens) x
Power of zoom
objective lens =
Total
magnification
Minimum zoom
Maximum zoom
4. Summarize the appearance of the letter e in the four cases
you observed:

Naked eye Compound scope Mirror Dissecting scope
19
PART IV: ESTIMATING THE SIZES OF CELLS
When looking at objects through a compound microscope, it is
difficult to get a feel for just how big (or
small) they really are. We need some kind of "ruler".
The actual size of an object seen under the microscope can be
estimated by first measuring the
diameter ("width") of the viewing field (the circle of light seen
through the eye piece). You can then
estimate the length of the specimen as a fraction of diameter of
the field of view. For example, if you
estimate the diameter of the field of view to be, say, 6
millimeters (mm) and you see that the specimen
is about half as long as the field is wide, then the specimen is
about 3 mm long. To estimate the
diameter of the field of view, complete exercises 8 and 9 below.

When performing these calculations, it helps to keep in mind
that the higher the power, the smaller
the diameter of the field of view. Always!
6.7 Exercise 7 – Calculating total magnification of objects
1. In the table below, record the magnification of the ocular lens
of your compound microscope. It is
engraved on the side of the eyepiece barrel and is usually l0X.
When nothing is indicated, the power is 10X.
a. Power of ocular lens ______
2. Similarly, record below the magnification of each of your
objective lenses.
a. Power of objective lenses ______ ______ ______
3. To calculate total magnification, simply multiply the
magnification of the ocular lens by the
magnification of the objective lens. Compute the total
magnification for each objective lens on your
microscope:
Power setting Magnification of
objective lens x

Magnification of
ocular lens =
Total magnification
Low
Medium
High
20
6.8 Exercise 8 – Determining the diameter of the field of view.
1. First determine the diameter of the field of view (circle of
light) for the microscope under low
power. Place a clear plastic, metric ruler on the microscope
stage across the center of the field of
view and focus on the ruler with the lowest power objective.
2. Move the ruler so that one of the millimeter lines falls
exactly at the edge of the circle of light. Then

count the number of millimeter-long spaces needed to get to the
opposite side. The diameter is
approximately _____ millimeters. (For our purposes you can
round off to the nearest whole number
of millimeters).
3. The unit commonly used for measuring microscopic
specimens is the micrometer (µm), a unit equal
to 1/1000 of a millimeter. There are 1000 micrometers (µm) in
one millimeter (mm). To convert
diameter in mm to diameter in µm, multiply by 1000.
The diameter of your field of view is _____ mm, which is
________ µm.
4. At higher magnifications, the field of view (lighted circle)
covers a much smaller portion of the

specimen, and the image of the plastic ruler becomes so large
that it can no longer be used to measure
the field of view. (Try it!).
However, the diameter of the field of view under higher
magnifications can be calculated from the
diameter of the visual field that you measured under low power.
This is because as magnification
increases, the diameter of the field of view decreases
proportionally.
Example: The easiest way to think about this is with an
example. Suppose you used the ruler and
measured the diameter under low power (40x) to be 6 mm. The
diameter under medium power (100
power) would be simply 40/100ths of 6 mm. The diameter
under high power (450 x) would be simply
40/450ths of 6 mm.
21
Now calculate the actual diameters for your microscope at
medium and high power and enter them in
Table 2. You can just use the example above and plug in the
values for diameter and power you
determined for your own microscope. Or you may find it useful
to use the boxed equation below… but
then again, maybe not!
Unknown diameter = Diameter measured x The power of the

low magnification
under higher power under low power The power of
the higher magnification
Table 2: Relationship between magnification and the diameter
of field of view.
Power Total
magnification
Diameter of field of
view (millimeters) 1
Diameter of field of
view (micrometers) 1
Low
mm
µm
Medium

mm
µm
High
mm
µm
1 For our purposes, you can round off to nearest 0.1

millimeters (mm) or 100 micrometers (µm).
6.9 Exercise 9 – Using the diameter of the field of view to
measure cells
1. The actual size of any microscopic object can now be
estimated by comparing the length of the
specimen to the known diameter of the field of view. To
estimate the length of a cell as it appears
under low, medium, or high power:
a. Determine the diameter of the field of view at this power
(from Table 2).
b. Estimate the number of cells that would fit, end-to-end,
along the diameter of the field of
view. (See diagram below.)
c. Divide the diameter of the field by this number.
2. Estimating cell diameters: In the circles below are two
imaginary cells. The cell on the left is shown
under medium (100x) power. Its length is about 1/8th the
diameter of the field.
From Table 2, what is the calculated diameter of the field at
100X? _____ µm.
22

So how long is this cell? _____ µm.
100 x 430 x
3. A different species of cell is shown on the right, as it
appears under high (430x) power. As you can
see, its length is about one half the diameter of the field.
From Table 2, what is the diameter of the field of view at high
power? _____ µm.
So how long is this cell? _____ µm.
4. According to your calculations, the cell on the right is
smaller than the cell on the left. But looking at
the diagrams, it is the cell on the right that looks larger. How
can this be?

23
PART V: MEASURING LIVING CELLS
6.10 Exercise 10 – Preparing a wet mount of a naturally
pigmented (red
onion) cell.
To view living cells under a microscope, you first have to "wet"
mount them on a glass slide. Prepare
wet mounts of onion cells as follows:
1. Place a clean microscope slide on a paper towel. Put a drop
of water (less than 1 cm diameter) in the
center of the slide.
2. Obtain a wedge of red onion from the Central Study Area.
With your fingers or forceps, remove a
portion of the thin (cellophane-like) tissue that lines the outer
surface of each scale-like leaf. With
scissors or a razor blade, cut off a small (0.5 cm square) piece
of epidermis and place it in the drop of
water on the slide.

3. Place a cover glass over the specimen by holding the cover
glass at a 45-degree angle against one
edge of the water drop. Let the water spread along the width of
the cover glass before slowly lowering
the glass down over the material. Try to avoid trapping air
bubbles under the cover glass. With your
compound microscope, view your wet mount of onion
epidermis. Use the circles below to represent the
field of view (circle of light) as seen through your microscope.
In the left-hand circle, sketch a few of the
onion epidermal cells under high power. Record the
magnification you are using (100x, 430x).
For the onion cells, draw the highest magnification that allows
you see at least one whole onion cell in
the field of view. Use the circle on the left for this drawing.
Onion epidermal cells ( x) Human epidermal cells
(430 x)

24
6.11 Exercise 11 – Staining a wet mount of an unpigmented
(human
cheek) cell.
1. Place a second clean microscope slide on a paper towel. Put
a drop of water (less than 1 cm
diameter) in the center of the slide.
2. Obtain human epidermal cells from the inside of your own
mouth. Shake a clean toothpick out of
the dispenser. Gently scrape the inside surface of your cheek
with the toothpick. Put the cheek cell
scrapings into the water droplet. Spread out the scrapings by
gently tapping the toothpick in the
water droplet.
3. Place a cover glass over the specimen by holding the cover
glass at a 45-degree angle against one
edge of the water drop. Let the water spread along the width of
the cover glass before slowly
lowering the glass down over the material. Try to avoid

trapping air bubbles under the cover glass.
4. Unlike red onion cells, human cheek cells are not naturally
pigmented. Cheek cells are almost
completely transparent and need to be stained. Place a small
drop of methylene blue dye at one
edge of the cover slip and touch a paper towel to the opposite
side of the cover slip. The dye will be
drawn under the cover slip and across the cheek cells, staining
them.
5. After the dye has been in place for one minute, put a drop of
clean water near the edge of the cover
slip and again touch a paper towel to the opposite side. This
will remove excess dye, but leave the
cells stained.
6. View the slide under the microscope. In the right-hand circle
above, sketch a few of the cheek cells
under high power (over 400 x)

25
Table 3: Relative sizes of cells: List the lengths of several
kinds of cells, including your three protozoans
and any other specimens measured by other students in the lab.
Specimen Cell length (µm) Key features
Onion epidermis
Human epidermis
(cheek cells)

A protozoan with cilia
(Paramecium)
A protozoan with a flagellum
Name:
A protozoan with pseudopodia
Name:
Virus (influenza)
Limit for light microscopes
Bacteria (anthrax)
Red blood cell (human)
Airborne pollen
Limit for naked eye
Human ovum
Grain of sand
0.02 µm
0.5 µm
1.0 µm

7.0 µm
25 µm
100 µm (= 0.1 mm)
100 µm (= 0.1 mm)
500 µm (= 0.5 mm)
26
6.12 Exercise 12 – Estimating the size of a unicellular organism
1. Water from ponds contains many interesting unicellular
organism. One of the most common is the
single-celled Paramecium – a fast moving organism covered
with beating, hair-like cilia. Place a drop of
Paramecium culture onto a clean microscope slide. For best
results, take a sample of the "sludge" from
the bottom of the container.
2. To see the fast-moving Paramecium better, add a drop of
"Proto-slo" to the sample on you slide and
stir using a dissecting needle or toothpick. Proto-slo slows
down swimming organisms by "thickening"
(increasing the viscosity of) the water.
3. Add a cover slip by holding a cover glass at a 45-degree
angle against one edge of the water drop.
Permit the culture solution to spread along the width of the
cover glass. Then slowly lower the cover

glass. Try to avoid trapping air bubbles under the cover glass.
4. Use the low power objective to find a Paramecium. Center
the Paramecium in the field of view
before switching to higher power.
5. Sketch the Paramecium under medium and high power in the
circles below. Estimate its length,
using the diameter of the field of view. Enter your estimate in
Table 3.
Paramecium (medium x) Paramecium (high x)

27
Sketch two additional protozoans (other than Paramecium). Use
your drawings to estimate the size of
these protozoans, and enter results in Table 3.
Name _______________________ ( x) Name
____________________ ( x)
Length ________ µm Length ________ µm
Locomotion type ___________________
Locomotion type __________________
Based on the cells you have measured, are all protozoans about

the same size? Are any of the species
significantly larger -- say, 10-times larger -- than the others?
CHECK OUT PASS:
1. Return your carrel to its original condition. An instructor
will check your carrel and give you a "check
out pass" to turn in at the front desk as you leave.
2. When you have finished using the compound microscope:
a. Turn off the light source.
b. Rotate the nose piece to the low power objective.
c. Unplug the microscope and coil the wire loosely over the
body tube.
d. Cover the microscope.
3. Rinse off and dry the slides and cover slips you used for wet
mounts. Any broken cover slips should
be placed in the “broken glass” container near the fish tanks.
28
Lab 2 – Enzymes and Cell Function 7
YOU NEED TO WORK WITH A PARTNER FOR THIS LAB
AND ALL REMAINING LABS THIS SEMESTER.

Please plan ahead. Partners share the data they collect, but the
reports you write for labs #2 and #3
must be written individually.
Growing, reproducing, digesting, and the many other processes
of "life" involve thousands of different
biochemical reactions. Without enzymes, almost none of these
biochemical reactions would proceed
quickly enough to sustain life. Enzymes are catalysts that can
help break larger molecules into smaller
molecules, or help join two molecules together, all while
remaining unchanged themselves. Enzymes are
involved in everything from photosynthesis to the fertilization
of eggs by sperm, from the digestion of
food to the clotting of blood.
Enzymes are proteins that catalyze (speed up) vital biochemical
reactions by reducing the "activation
energy" needed to get the reaction going.
Without enzymes, the temperature inside living cells is too low,
and the concentration of reacting
molecules is too dilute, to sustain the biochemistry of life.
Enzymes are extremely efficient. Minute quantities of an
enzyme can accomplish at low temperatures
what otherwise would require much higher temperatures and/or
harsh chemical reagents. For example,
one ounce of the stomach enzyme pepsin can digest two tons of
egg white in a few hours. Without
pepsin, digesting this much egg white would require 10-20 tons

of strong acid working for 24-48 hours
at high temperature.
Enzymes are so extraordinarily efficient for four reasons:
(1) Enzymes can be used over and over again because they are
not themselves changed by the
reactions they catalyze. In the process of converting one
molecule (the substrate) to another (the
product), the enzyme binds temporarily with the substrate to
form an enzyme-substrate complex. The
enzyme returns to its original form as soon as the
transformation of the substrate into the product is
complete.
29
Figure of “Lock-and-key model” is from Wikipedia.com
(2) Enzymes are extremely specific. They are very choosy
about what substrates they will bind with
and what reactions they will catalyze. Most enzymes bind with
only one particular kind of molecule
(like a "lock and key") and cause only one particular kind of
change in that molecule. Some enzymes
specialize in synthesis (joining two substrates) while others
specialize in splitting the substrate into
products.

(3) Enzymes are extremely reactive, much more reactive than
ordinary chemical catalysts. For
example, hydrogen peroxide (H2O2) by itself slowly
decomposes into water and oxygen. A small
amount of powdered iron will act as a catalyst and speed up this
decomposition several fold. But a
single molecule of the enzyme catalase (found in human blood)
can, in one minute, split more than five
million peroxide molecules! Catalase is one of the fastest
acting enzymes known. Other enzymes
operate on their substrates at rates ranging from 1000 to
500,000 molecules per minute.
(4) Most enzymes function best within a narrow range of
temperature and pH (acidity). For example,
as temperature rises, the rate of an enzyme-catalyzed reaction
will at first increase because the enzyme
and substrate molecules move around more quickly and so
encounter each other more often. But above
a certain temperature most enzymes become denatured (lose
their shape) and so lose their catalytic
activity.
In this lab, you will learn about some of the qualitative
characteristics of enzyme catalyzed reactions
(Part I) and then quantify the effects of environmental factors
(temperature, pH) on the rate of enzyme-
catalyzed reactions (Part II).
30

are able to:
1. Explain why life on earth could not exist without enzymes.
Using specific examples, explain how the
metabolic efficiency of living organisms is improved by the
extreme reactivity and specificity of
enzyme catalysts (See Introduction.)
2. Use a spectrophotometer and other qualitative and
quantitative techniques to measure the activity
of an enzyme under different environmental conditions.
3. Graph and analyze the effects of temperature and acidity (pH)
on the rate of an enzyme catalyzed
reaction.
4. Explain, at the molecular level, why enzymes work better at
certain temperatures and pH’s. Include
the effects of pH and temperature on molecular motion and
shape.
PART I: A SIMPLE ENZYME-CATALYZED REACTION
When you cut open an apple, banana or pear, you cut through

many cell walls and release the complex
contents of many cells. Left exposed to the air, the white
surface of the fruit will turn brown in a short
time. This familiar color change is a good example of an
enzyme catalyzed reaction. The brown color
comes from the reaction of oxygen in the air with catechol in
the fruit to form benzoquinone. Catechol
(the substrate) is clear and benzoquinone (the product) is
brown. This browning reaction is catalyzed by
catecholase, an enzyme that occurs naturally in the fruit.
catecholase
Catechol + 1/2 O2 ------------> benzoquinone + H2O
(clear) (brown)
This simple browning reaction can be used to illustrate several
important properties of enzymes in
general. In Part I, you will make some preliminary qualitative
observations on the nature of enzyme
catalyzed reactions by recording how environmental factors
affect the browning reaction. For example,
must the fruit be exposed to air to turn brown? Does
temperature affect the rate of browning? Is the
browning rate changed by the presence of an acid, like lemon
juice? You will be asked to use the
observations you make in Part I to formulate qualitative
hypotheses about how enzymes work. In Part II
you will test your hypotheses quantitatively.
31

7.1 Exercise 1 – Browning of fruit
In this exercise you will compare the relative amount of
browning that occurs in banana slices placed
under six different conditions.
Methods
1. Work with a partner.
2. Set up the six experimental treatments (described below)
before you obtain a piece of banana. The
comparison of browning rates will be easier if all the slices
begin browning at the same time.
Slice 1: leave open to the air at room temperature.
observe the effect of acidity (pH):

3. Acidity is measured in terms of a unit called pH. On the pH
scale, any value below 7 is considered
acidic, anything above 7 is basic, and 7 is neutral. Acids
typically have pH values between 2 and 6. The
stronger the acid, the lower the pH value.
Use litmus paper to determine the acidity (pH) of lemon juice
and water.
Lemon juice pH = ______
Distilled water pH = ______
4. From a fresh banana, cut 6 equal, unpeeled slices. Working
quickly, place one slice in each of the
conditions described in step 2.
5. Check the sections every 3-5 minutes for the first half hour.
In Table 1 record (a) the time you first
detect browning, and (b) the extent of subsequent browning (if
any).
Between observations, begin Part II.
32
Results
Table 1: Extent of browning under different environmental

conditions.
Minutes elapsed to
first browning
Extent of further browning
Slice #1
(open to air)
Slice #2
(wrapped)
Slice #3
(cold)
Slice #4
(warm)
Slice #5
(distilled
water)
Slice #6
(lemon juice)

Analysis and Discussion
l. Look at the browning rates of slice #1 (exposed to air) and
slice #2 (wrapped in plastic).
a. Which one turned brown faster? Why? (With what is the
catechol reacting?)
b. If your observations are not what you expected, consider
this. Ripening fruit releases
ethylene, a gas that speeds the ripening process. Saran Wrap
Classic is made of polyvinyl and
Saran Wrap with Cling Plus (formerly HandiWrap) is
polyethylene. How might this affect
browning rate?
c. How could you test whether the wrapping material is
increasing browning rate?
33
2. If you use the browning rate of slice #1 (left out at room

temperature) as your standard of reference
(called your experimental "control") :
a. What effect (if any) did cooling have on the browning rate?
b. What effect (if any) did warming have on browning rate?
c. What do these observations suggest about the effect of
temperature on enzyme-catalyzed
reactions?
-- Based on these observations, what should happen to the rate
of browning as temperature
increases? State this in the form of a hypothesis.
HYPOTHESIS A: "As temperature increases, the rate of an
enzyme catalyzed reaction
(browning) should ___________________________."
(Choose one: increase, decrease, or remain the same)
3. Comparing the browning rates of slice # 5 (treated with
distilled water) and slice #6 (treated with
lemon juice).
a. What effect did lemon juice have on the rate of browning?
b. What do these observations suggest about the effects of
acidity on enzyme activity?
-- Based on these observations, what should happen to
browning rate as pH increases?
(Remember, the stronger the acid, the lower the pH.) State this

in the form of a hypothesis.
HYPOTHESIS B: "As pH increases (meaning the environment
becomes less acidic), the rate of an
enzyme catalyzed reaction (browning) should
_________________________________."
(increase, decrease, or remain the same)
These last two hypotheses are based on the qualitative
observations you made in Part I. They may or
may not be correct. In Part II, you will test these last two
hypotheses quantitatively.
34
PART II: EFFECTS OF ENVIRONMENTAL FACTORS ON
RATE OF ENZYME-CATALYZED REACTIONS
In this section you and a partner will design and carry out
experiments to test the two hypotheses you
made at the end of Part I concerning the effects of temperature
and pH on enzymes. The quantitative
techniques you will be using will allow you to measure rates of
the browning reaction far more
accurately than before.
The experiments are intended to give you first hand experience
with the methods of scientific inquiry.
They also reveal some interesting features of enzyme-catalyzed

reactions.
The Spectrophotometer
The Spec 20 is a device that measures how "dark" (opaque) a
liquid is. The Spec 20 shines a beam of
light through the liquid in a test tube and measures how much of
the light is absorbed as it passes
through the test tube. Measurements are expressed in terms of
"absorbance" on a scale from 0 to 2.
You will be using the Spec 20 to measure the rate of the
browning (benzoquinone formation) under
different conditions. As more and more (clear) catechol is
converted to (brown) benzoquinone, less and
less light will be able to pass through the test tube, and the
percentage of light absorbed will increase.
We will be using absorbance to measure the amount of
benzoquinone produced under different
temperatures and acidities (pH's).
35
Follow this overall, 3-step procedure whenever you need to use
the Spec 20.

For exercises 2 and 3, you will need to use the Spec 20 to
measure rates of reaction. Use these three
steps as a reference when you do exercise 2 (page 9) and
exercise 3 (page 12).
1. Prepare a reference solution (or "blank")
The starting solution of enzyme and substrate is not perfectly
clear and so absorbs some light even
before the browning reaction begins. To correct for this fact,
you will be using a "blank," a test-tube
filled with the starting solution as a reference. You use the
amount of light absorbed by the blank as
your reference point. The Spec 20 can be adjusted to treat the
amount of light absorbed by the
blank as "zero". Any additional light absorbed can then be
attributed to newly formed
benzoquinone. To make a blank:
a. Label a small test tube with the letter "B" for "blank". This
test tube will hold the reference
solution for setting the "0% absorbance" level on the Spec 20.
b. Use a pipette and pipette pump to measure 1 ml of potato
extract and 6 ml of distilled
water into the tube.
c. Cover the tube with a small square of parafilm and invert to
mix.
2. Calibrate the Spec 20 (i.e., standardize the internal light
level):

a. Set the wavelength knob on the Spec 20 to 540 nanometers,
a wavelength in the “green”
portion of the light spectrum. We use 540 nanometers in this
experiment because benzoquinone (the
product) absorbs this wave length better than any other.
b. With the sample compartment empty and the cover closed,
set the transmittance to zero by
using the Zero Control (left hand) knob or by following the
directions posted next to the Spec 20.
c. Wipe off the "blank" tube with a Kimwipe (to remove light-
absorbing fingerprints) and insert
it into the sample compartment and close the cover.
d. Set the absorbance reading to zero by using the Absorbance
(right hand) knob, or by
following the posted instructions.
Calibrated in this way, the Spec 20 will measure only increases
in absorbance above the reference level
established by the “blank”.
3. To measure the absorbance of your samples in Exercises 2 &
3:
a. Remove the blank. (Blanks get old fast. When setting up
the testube series you
will be using for each experiment, make a fresh
blank at the same time.)
b. Insert the sample tube into the sample compartment.
c. Close the cover of the sample compartment.
d. Read "absorbance " off the lower dial.
e. Remove sample tube and repeat steps 3b-d with each
sample.

In Part II, you and your partner should work together on both
the
temperature experiment (Exercise 2) and the pH experiment
(Exercise 3).
36
7.2 Exercise 2 – Effect of temperature on enzyme-catalyzed
reactions
For Exercise 2, you will be setting up test tubes containing
potato extract (a good source of the enzyme
catecholase), water, and catechol (the substrate). Even though
potato contains catechol, you will be
adding extra substrate (catechol from a commercial supplier) to
make the reaction go faster.
There are several different ways to speed up an enzyme
catalyzed reaction. One way is to add more
enzyme. Another way is to increase the concentration of the
substrate on which the enzyme is working.
In this experiment, we will be making the reaction go faster by
adding extra substrate (namely catechol).
We are not adding extra enzyme. There is plenty of the
catecholase enzyme in the potato extract
already.
Set up all your tubes with everything in them (see below) except
the catechol. When everything is ready
add the catechol last, so all the reactions start at the same time.

1. Obtain 6 test tubes and a test tube rack. With a wax pencil,
mark the tubes near the top with your
initials and numbers 1 through 5, plus B (for "blank", the tube
that will hold the reference solution used
to calibrate the Spec 20.)
2. With a pipette, measure into each of the 5 tubes: 1 ml of
potato extract (a rich source of the enzyme
catecholase) and 4 ml of water. Avoid picking up any of the
particulate matter (cloudy with particles)
that has settled to the bottom of the potato extract flask. Any
cloudy material will throw off the
measurement of browning rate.
3. To make a "blank," put 1 ml potato extract and 6 ml of water
into the sixth tube. The extra 2 ml of
water makes up for the 2 ml of catechol that you will not be
adding to the control blank. Cover all 6
tubes with parafilm, invert to mix, and stand the tubes in rack.
4. Do not add catechol yet. Put one sample tube into each of
the 5 different water baths in the Central
Study Area. In Table 2 record the actual temperatures of each
of the baths.
5. BEFORE ADDING THE CATECHOL to your samples, use a
thermometer to make sure the temperature
of the solution inside your test tube has actually reached the
temperature of its water bath. This should
take 3-7 minutes.
6. Plan ahead! Read steps #7-9 completely before proceeding.
Try to run the 5 tubes simultaneously,
or closely together in the same sequence, so that reaction times
in the 5 samples will be comparable.

7. Add 2 ml of catechol solution to each of the 5 sample tubes.
You will need to remove the tube from
the water bath, remove the parafilm, add the catechol, put the
parafilm back on, and invert tube to mix
the contents. Return each tube to its bath for 5 minutes.
8. Use the blank (test tube “B") you made earlier to recalibrate
the Spec 20.
9. Exactly 5 minutes after adding the catechol, remove each
sample tube from its water bath, dry it with
a Kimwipe, insert the tube into the sample holder of the Spec
20, and measure absorbance. Quickly
repeat for the other 4 tubes, one at a time, in numerical order.
Record these values in Table 2.
37
Table 2: Effect of temperature on extent of browning
Sample Temp (oC) Absorbance after (
) minutes
Any color changes?
(see #11 below)
Blank
1

2
3
4
5
10. If you experience measuring delays, you will need to
control for the fact that the reaction product
continues to form while you are waiting to measure it. After
measuring the absorbance in tubes 1, 2, 3,
4, and 5, measure them again in reverse order (5, 4, 3, 2, 1) and
use the average absorbance for each
temperature.
11. If the reaction is proceeding correctly, you should see (with
your naked eye} a darkening of the
solution. This rusty brown precipitate is benzoquinone, the
desired product of the reaction. If you see
“cloudiness,” it means you mistakenly picked up particulate
matter (sludge) from the bottom of the
flask.
12. Plot your values for absorbance as a function of
temperature using the graph paper below. If
necessary, you may change the absorbance scale on the y-axis to
0.5 and 1.0.
Graph 1: Effect of temperature on browning rate
Temperature of water bath (oC)

38
1. At what temperature(s) was the rate of reaction greatest?
____ At what temperature(s) was the
rate lowest? ____ Are these observations consistent with the
hypothesis about thermal effects you
formulated at the end of Part I? ______
What do these observations suggest about the "optimal" range
of temperatures for enzyme catalyzed
reactions? State your conclusion in the form of a revised
hypothesis.
HYPOTHESIS A (Revised): The rate of an enzyme catalyzed
reactions is greatest at temperatures
that are...
2. At the molecular level, what might explain the rate of the
enzyme-catalyzed reaction at low
temperatures? (Hint: For a reaction to occur, the two reactant
molecules must “bump into’ each other.
How does temperature effect the motion of molecules?)

3. At the molecular level, what might explain the rate of the
enzyme-catalyzed reaction at high
temperatures? (Hint: How does temperature effect the
molecular structure of enzymes?)
39
7.3 Exercise 3 – Effects of pH on enzyme-catalyzed reactions
The pH (acidity) of the environment can affect the molecular
bonds that maintain the shape of an
enzyme’s “active site,” the site on the enzyme molecule
responsible for binding to the substrate or
substrates. A pH value of 2 is highly acidic, 7 is neutral, and
11 is highly basic.
For Exercise 3, set up test tubes containing potato extract (a
good source of the enzyme catecholase),
catechol (the substrate), and 5 different pH buffer solutions.

Set up all 6 test tubes with everything in
them (see below) except the catechol. Add the catechol last, so
all the reactions will start at about the
same time.
1. Obtain 6 test tubes and use a wax pencil to mark all the
tubes near the top with your initial and either
numbers 1 through 5 or "B" for the calibration blank.
2. Fill the 5 tubes as follows:
Sample 1: 1 ml potato extract, 4 ml buffer for pH 3 .
Sample 2: 1 ml potato extract, 4 ml buffer for pH 5.
Blank: 1 ml potato extract, 4 ml buffer for pH 7, plus 2 ml
distilled water.
The 2 ml of water is a substitute for the 2 ml of catechol that
will be added to the other test tubes but
not to the blank tube. You will need only one blank, because all
5 of the pH buffer solutions are clear
and have identical absorbency properties.
3. Cover each tube with parafilm and invert to mix. Stand all 6
tubes in the test tube rack.
4. Now add 2 ml of catechol to the 5 sample tubes, put the
parafilm back on, and again invert the tube
to mix the contents. You do not need to uncover the tube for
the reaction to proceed; the solution has
plenty of dissolved oxygen. Keep the test tubes at room
temperature. DO NOT PUT THE TEST TUBES IN
THE WATER BATH! In this experiment we are measuring the
effects of pH, not temperature.

5. If you see "cloudiness,” it means either than you mistakenly
picked up particulate matter from the
bottom of the flask, or that an unwanted precipitate is forming.
A cloudy white precipitate often forms
if the pH gets too low (pH = 3). A grayish black precipitate
sometimes forms if the pH gets too high (pH =
11). When precipitates are formed by reactions that have
nothing to do with “browning,” they can
distort your data, causing you to overestimate the rate of
browning at very low and very high pHs. So
record any color changes and include them in your lab report.
6. About a minute before step 6, use your blank to calibrate the
Spec 20.
7. Allow the browning reaction to proceed for exactly 5
minutes. Then insert the sample tubes, one at a
time in numerical order, into the Spec 20 and record the
absorbances in Table 3. If the reaction ran
longer than 5 minutes before you took your measurement,
record the exact number of minutes and use
40
this same time interval when you measure the other sample
tubes. Record the time in Table 3. Note
any color changes in the test tubes.
8. If you experience measuring delays, you will need to control
for the fact that the reaction product
continues to form while you are waiting to measure it. After
measuring the absorbance in tubes 1, 2, 3,

4 and 5, measure them again in reverse order (5, 4, 3, 2, 1) and
use the average absorbance for each pH.
Results
Table 3: Effect of pH on extent of browning
Sample pH Absorbance after (
) minutes
Any color changes?
(See #5 above)
Blank * zero
1 3
2 5
3 7
4 9
5 11
9. Wash all test tubes and return them to the rack.
10. Plot your values for absorbance as a function of pH on
Graph 2.
Graph 2: Effect of pH on the browning rate

pH
41
1. At what pH values was the rate of reaction greatest? ____
At what pH values was the rate lowest?
____ Are these observations consistent with the hypothesis
about the effects of acidity you formulated
at the end of Part I? ______
What do these observations suggest about the "optimal” range
of pH for enzyme catalyzed reactions?
State your conclusion in the form of a revised hypothesis.
HYPOTHESIS B (Revised): The rate of an enzyme catalyzed
reactions is greatest at pH values that
are...
2. The pH (acidity) of the environment can affect the hydrogen
bonds that maintain the shape of an
enzyme’s “active site,” the site on the enzyme molecule
responsible for binding to its specific
substrate(s). At the molecular level, what might explain the
changes you observed in the rate of the
enzyme-catalyzed reaction at low and high pH values?

3. Consider the effects of pH on these two enzymes. A pH
value of 2 is highly acidic, 7 is neutral, and 11
is highly basic.
From these data, do all enzymes necessarily have the same
optimal pH?
Which one of these two enzymes do you think is more typical of
the enzymes found in the human body?
Where in the body would you find an enzyme whose optimal pH
is 2?
42
Questions For Further Thought
1. Predict what would happen to a living organism exposed to
temperatures that fall outside
the optimal range for its enzymes.
2. List some familiar adaptations animals have to reduce the
effects of temperature extremes

on their many vital, enzyme-catalyzed reactions.
a.
b.
c.
d.
3. In general, the rates of chemical reactions double for every
10 degree (Celsius) increase in
temperature. At the molecular level, what changes account for
slower reaction rates of enzyme-
catalyzed reactions at high temperatures? (Hint: enzymes are
three-dimensional protein molecules.
What happens to the shape of protein molecules when they are
heated? How would this change the
vital “active site” of the enzyme?)
-- Why are enzyme catalyzed reactions also slower at low
temperatures? (Hint: In a liquid or
gas, what happens to the motion of molecules as they cool?
Why would this affect the rate at which
enzyme and substrate molecules collide and react?)
4. The binding of enzymes with their substrates is most
efficient under certain (so called
"optimal") pH conditions. An enzyme molecule’s 3-
dimensional shape is maintained by hydrogen bonds
and other chemical bonds sensitive to pH. How might extremes

in pH change the efficiency of the
enzyme?
5. Human cells and body fluids contain hundreds of natural
buffer systems that keep pH at or
very near optimal levels. Potato cells and human cells are both
living and so have thousands of
biochemical reactions in common. Given your results for
catecholase, what value of pH is most likely to
be found inside human cells?
43
LAB REPORT
At your next Discussion class, hand-in a lab report to your TA.
You and your partner can submit identical
cover pages and data tables, but your introduction and
discussion must be written by you, in your own
words.
(1) Cover page: including the title of the experiment (in this
case, use "Effects of environmental
factors on the rate of enzyme catalyzed reactions"), your name,
the date, your discussion leader's name,
and the number of your discussion section. Also include the

names of all your partners and their
discussion section leaders.
(2) Introduction: State your two hypotheses about the effects
of temperature and acidity (pH)
on the rate of this enzyme catalyzed reaction. Explain why each
hypothesis makes sense to you. State
the prediction you generated from these hypotheses and describe
(in general terms) how you tested
them. You don’t need to detail the methods (because they are
already in the lab guide), but you do
need to say enough to show you understand the experiment;
e.g., which substance is the enzyme, which
is the substrate, and why your Spec 20 data can be used to
compare the rate of the reaction under
different conditions.
(3) Results: On a separate page, summarize your data from
the tables and graphs on pages 10
and 13.
(4) Discussion: In 1- 2 pages, explain why each of your two
curves is shaped the way it is.
Explain why your curves went up or down at low, intermediate,
and/or high temperatures and pH's.
Compare your actual curves to the theoretically-expected shapes
for these curves. You should include
any relevant parts of your answers to the questions raised in the
“Analysis and Discussion" and
“Questions for Further Thought” sections of the lab guide.

44
Lab 3 – Cell Membranes and Water Balance 8
PLAN AHEAD! This lab requires working with a partner and
writing a lab report.
Osmosis is the vitally important process by which water moves
in and out of cells. Overall, the cells of
living organisms are composed of about 75-85% water. The
concentration of water is even higher in the
fluid inside cells; i.e., the fluid containing dissolved substances
that are not attached to membranes. Life
depends on having the proper concentration of water inside the
cell. If there is too little water, the
chemical reactions that support life will not be able to proceed
and the cell will die. If there is too much
water inside, the cell may burst. In this minicourse, we will
examine factors affecting the movement of
water in and out of cells.
The earliest cells are thought to have originated and evolved in
the ocean. Is the internal environment
(i.e., the intracellular fluid) of cells still a lot like sea water
(97% water, 3% dissolved salts)? In this
minicourse, you will also compare the concentration of water
and "solutes" (dissolved substances or
"salts") found inside plant cells from different environments --
fresh water, marine and terrestrial.

are able to:
1. Define diffusion and osmosis in terms of concentration
gradients and the movements of molecules.
Explain (in terms of concentration gradients and internal
pressure) the effects of osmosis on cells placed
in hypertonic, hypotonic and isotonic environments.
2. Use an osmometer to measure changes in internal pressure in
model "cells" (dialysis bags) placed in
different "environments" (solutions) and graph your results.
Explain why internal pressure increases
over time and then stabilizes.
3. Describe how the effects of osmosis in plant cells are
modified by the presence of a cell wall.
Contrast turgor and plasmolysis.
4. Use the plasmolysis threshold of cells to compare the
isotonic points (internal solute concentrations)
of plant cells from fresh water, marine, and terrestrial
environments.
PART I: OSMOSIS
Molecules (including water, oxygen, carbon dioxide) are in
continual motion, colliding and bouncing off
each other in random directions. Because the movement of each
molecule is random, it is impossible to
predict the direction any one molecule will be moving at any
given time. However, if you look at huge
groups of randomly moving molecules (billions of molecules at
a time), predictable patterns emerge.

Namely, the "net” (or overall total) movement of molecules is
from areas where they are in high
concentration to areas where they are in low concentration.
45
One of the principal ways molecules get in and out of cells is
diffusion. Diffusion is defined as the net
movement of molecules from areas of higher concentration to
areas of lower concentration. For
example, if you place a crystal of a purple dye in a glass of
water, the purple color spreads out as the dye
molecules diffuse through the water. Another example of
diffusion is inside the air sacs of your lungs,
where the net movement of oxygen is out of the air and into
your blood. Because oxygen molecules
move in random directions, there will always be some molecules
moving in the "wrong" direction. But
overall, more oxygen molecules move out of the air than out of
the blood, simply because there are
more oxygen molecules in the air than in the blood.
The diffusion of water molecules is so important to living cells
that it is given a special name, osmosis.
Osmosis is the net movement of water molecules across a semi-
permeable membrane.
[Semipermeable membranes allow small molecules to pass
through them, but not larger molecules like
sugar and dye.] When the concentration of water molecules is
higher outside than inside the cell, the
net movement of water will be into the cell and the cell will

swell. When the concentration of water
molecules is lower outside, the net movement of water will be
out of the cell and the cell will shrivel.
The cell membrane of a living cell is similar to a semipermeable
membrane in that it permits some
substances to cross it but not others. For example, the cell
membrane allows small, uncharged
molecules (water, oxygen and carbon dioxide) to diffuse freely,
but blocks the diffusion of molecules
that are large (e.g., sugar, proteins) or are electrically charged
(e.g., sodium and chloride ions).
In the exercise below, you will make a simple "model" cell from
(kidney) dialysis tubing – plastic tubing
manufactured with holes just large enough to be permeable to
water but not permeable to large
molecules like sucrose (table sugar). Now suppose you filled
the dialysis bag with water and a little
sucrose, and then placed the bag in a beaker of water. Water
will begin to move by osmosis into the
bag. But why? Think of it this way: the concentration of water
inside the bag is lowered by the
presence of the sucrose molecules. (In a sense, the dissolved
sugar molecules "dilute" the water,
lowering the water's concentration.) Because the concentration
of water is higher outside than inside,
water diffuses "down" its concentration gradient and into the
bag.
Unlike water, sucrose is unable to flow down its concentration
gradient, because sucrose molecules are
too large to get through the pores and out of the bag. Because
sucrose molecules can't get out, their
presence will keep the concentration of water molecules always
lower inside the bag compared to the

pure water outside the bag. So water will continue to move into
the bag (a) until the bag bursts, or (b)
until the internal fluid pressure gets high enough to prevent
more water from diffusing in. Notice that
because of the presence of sucrose inside the bag, the
concentration of water inside can never become
the same as the concentration of the water outside.
With this background, you can now understand the formal
definition of osmosis: the passive
movement [= diffusion] of water across a semi-permeable
membrane in response to differences in
pressure and solute concentrations on either side of the
membrane. (We define solute as any
dissolved substance. Sucrose is the solute in the above
example.)
46
The Osmometer
Osmotic pressure is the tendency of water to move into a cell.
An osmometer is a device
designed to measure osmotic pressure by measuring the amount
of internal hydrostatic pressure
needed to stop the movement of water into a cell. The osmotic

pressure experienced by a cell differs
under different environmental conditions.
Figure 1. An osmometer.
The height of the standing column of liquid in the glass tube
provides a way to measure the hydrostatic
pressure of the liquid inside the dialysis bag. The higher the
column, the greater the internal pressure.
The osmotic pressure for different experimental setups is
measured as the height of the column after
the fluid level has stopped rising. The fluid stops rising when
the water pressure inside the bag (the
hydrostatic pressure) has increased to the point where it it is
equal to the osmotic pressure and stops
any more water from moving in.
47
8.1 Exercise 1 – Movement of water into a "model cell"
In Exercise 1, as water moves by osmosis into a dialysis bag
(our "model cell"), the pressure in the bag
increases. You will quantify this pressure change by measuring
the height of the water in a glass tube
inserted into the bag (See Figure 1). A taller column contains
more water, so it weighs more and exerts
more downward pressure.

1. Work with one or two partners. Write your partners’ names
here:
Partner’s name Discussion leader’s name
2. After looking at the osmometer set up in the Central Study
Area, obtain the following materials:
20 cm of dialysis tubing 400 ml beaker
scissors 10% sucrose solution
string (with dye added)
glass tube marking pencil
ruler supporting clamps
4. Cut a piece of dialysis tubing 20 cm long. Soak the tubing
under running water until soft. Tie a tight
knot in one end of the dialysis tubing. Open the other end by
rubbing it between your fingers.
5. While your partner holds the bag, carefully pour in 8-10 cm
worth of 10% sucrose solution containing
dye. (The dye is used as a tracer to let you see if any sucrose
solution is leaking out of the bag.) Insert
the glass tube into the dialysis bag. Tie the upper end of the
bag around the glass tube by wetting a
piece of string and wrapping it a few times around the tubing
before knotting it. Try to minimize the air
pocket left inside the bag.
6. Make sure that:
(a) the filled portion of the bag is less than 10 cm long

(b) the submerged end of glass tube is about 5 cm from the
bottom of the bag
(c) the air space inside the bag is as small as possible,
(d) that top end of the tube is at least 15-18 cm above the fluid
level in the bag,
(e) there are no leaks. (There should be no green dye outside
the tube.)
7. Hold the bag and glass near the upper knot and carefully
rinse off the dialysis bag with water. Avoid
squeezing the bag. If properly tied, no dye should leak out.
8. Fill the beaker with about 360 ml of room temperature water
from the carboy and lower the dialysis
bag into the beaker (See Figure 1). Support the upper end of
the glass tube with clamps in such a way
that (a) the water level in the beaker is about l.5 cm below the
upper end of the fluid inside the dialysis
bag, and (b) the bag is not touching the bottom of the beaker.
48
9. Immediately mark the level of the solution in the glass tube
by wrapping a second piece of wet string
twice around the tube. Move the string to mark the starting
level of the fluid in the glass tube. At 5
minute intervals, measure how far (in millimeters) the top of the
column of solution in the tube has
risen above the starting level. Record your observations in
Table 1, then plot these results on Graph 1.

10. You will need to know: (a) When was the rate of rise the
greatest? and (b) At what level (in mm)
did the fluid in the tube stop rising (if it stopped)?
While monitoring the fluid level rising in the tube, begin Part
II.
Table 1: Changes in internal pressure (height of fluid column)
Elapsed time
(minutes)
Distance above mark
(millimeters)
Change (# of mm)
per 5 min. interval
0
5
10
15
20
25
30

35
40
45
49
From Graph 1, at what height did the column of fluid in the tube
stop rising? _______mm
This height can be used as a measure of the “osmotic pressure”
inside our model cell in this particular

“environment”.
50
1. In terms of water molecules and solute concentration
gradients, why does the fluid level in the
osmometer rise, at least at first? (Hint: Think about the
concentration gradient and the net movement
of water at the molecular level.)
2. Look at the numbers in Table 2, and at the steepness of the
slope of the graph. Why does the rate of
rise (the number of mm of rise per 5 min. interval) change as
the experiment progresses? (Hint: Again

think at the molecular level.) How might the net inward
movement of water molecules be affected by
the water pressure (or “hydrostatic pressure”) that is building up
inside the bag?
3. When the fluid level finally stops rising, what keeps more
water from moving in? Warning: The
reason is not that the concentration of water is equal on both
sides! The concentration of water inside
the bag is always going to be lower, because all the sucrose
molecules are trapped inside and so are still
“diluting” the water.
4. “What If” Questions: What would have happened …
… if all the sucrose had been placed in the beaker rather than
inside the bag?
… if the glass tube had been only 5 cm long?

51
PART II: WATER CONTENT OF CELLS
Although living tissue is about 75-85% water, the effective
concentration of solutes inside cells is not 15-
25%. Most of the non-water components of cells are bound into
cell structures and are not in solution,
so do not influence osmotic pressure.
It is the concentration of solutes (substances actually dissolved
in the fluid portion of the cell) rather
than overall water content that determines whether water will
diffuse into or out of a cell. Just what is
the actual concentration of solutes (or "salts") in intracellular
fluids, the internal fluids of cells? Is it
anything like sea water (97% water, 3% salts) in which the first
cells originated and evolved? Let’s find
out.
In Part II you will learn a technique to estimate the
concentration of solutes and water in living cells
without killing them. You will use this technique to determine
and compare cells from different
environments -- fresh water, marine, and terrestrial.
The direction of osmosis

Whether osmosis will move water into a cell or out of it
depends on the "environment" surrounding the
cell. Unfortunately, environments are not described in terms of
their water content (e.g., 97% water).
Instead environments are described in terms of their "solute
concentration,” meaning the concentration
of substances dissolved in the water (e.g., 3% salt).
What is most important is the relative concentration of solutes
in the environment compared to the
solutes inside the cell. For example, when the environment’s
solute concentration is higher than inside
the cell, its water concentrations will be lower than in inside the
cell, so water will move by osmosis out
of the cell, dehydrating the cell.
There are three possible environmental conditions:
Environment Solute concentration Water concentration Water
movement
Hypertonic Higher than in cell Lower than in cell Out of the
cell
Hypotonic Lower than in cell Higher than in cell Into the cell
Isotonic Same as in cell Same as in cell No net movement
1. In hypertonic environments, solute concentrations are higher
in the environment than they are
inside the cell. (Hyper means more, in this case more solute.)
That means water moves out of the cell.

2. In hypotonic environments, solute concentrations are lower
outside than inside the cell, and water
moves into the cell. (Hypo means less, in this case less solute.)
3. In isotonic environments, solute concentrations are the same
outside as inside, so there is no net
movement of water. Water moves out at the same rate it moves
in. (Iso means equal.)
Turgor and plasmolysis
Everyone knows that cut flowers need to be kept in water. But
why? In plant cells, the cell membrane is
surrounded by a rigid cell wall. When plant cells are placed in
water -- a hypotonic environment -- water
diffuses into the cell, pressure builds inside, and the cell’s
plasma membrane becomes pressed tightly
52
against the cell wall. The cell does not burst, but instead
becomes turgid. Turgor pressure is the
pressure exerted on the cell wall by the fluid contents of the
cell, keeping the flowers erect.
When a plant cell is placed in a hypertonic environment, water
diffuses out of the cell. Having lost its
turgor pressure, the cell’s plasma membrane collapses and pulls
away from the cell wall. This shrinking
of the cell membrane away from the cell wall is called
plasmolysis.
Figure 2. Turgid (left) and plasmolyzed plant cells.

8.2 Exercise 2 – Using the plasmolysis threshold to estimate the
concentration of solutes and water inside living cells
Under your compound microscope, the cell membrane of a
turgid cell is invisible because it is pressed
up against the inner surface of the cell wall. But in a
plasmolyzed cell, the cell membrane is visible
because it has pulled back away from the cell wall. We will use
this fact to estimate the concentration of
water and solutes inside living cells.
In Exercise 2 you will be placing cells into a series of
"environments"; i.e., test solutions with known
solute concentrations. Your objective is to find the “isotonic
points” for each cell type-- the test solution
whose solute concentration is equal to the solute concentration
inside the cell.
At the “isotonic point” point cells are close to the balance point
between turgid and plasmolyzed.
Viewed under a microscope, an isotonic environment is one in
which about half the cells appear turgid
and half are plasmolyzed.

53
Table 2: Using plasmolysis and turgor to determine the solute
concentration inside plant cells.
When the cell
membrane is ...
The environment is
said to be ...
The concentration of solute in
the environment is...
plasmolyzed
(shrunken & visible)
in all cells
hypertonic greater than that inside the cell
turgid (invisible)
against the cell wall
in all cells.
hypotonic less than that inside the cell
turgid in about half
the cells, plasmo-
lyzed in about half

isotonic equal to that inside the cell
Procedure
1. Continue to work with 1-2 partners. View the videotape on
plasmolysis and how plasmolysis is used
to determine the isotonic points of cells. In the videotape, the
solute used is sucrose (sugar). We will be
using sodium chloride (table salt) as the solute.
2. Obtain a wedge of red onion and peel off a few thin sheets of
outer epidermal tissue. To see the
difference between plasmolyzed and unplasmolyzed cells, place
a sample of onion epidermis in small
beakers containing10 ml pure water (0% salt solution) and one
with 10% salt solution. Wait for 10
minutes. The cells in pure water (0% salt) will be
unplasmolyzed, so can be used as a standard for
comparison.
3. To make sure you have actually seen the difference between
plasmolyzed and unplasmolyzed red
onion cells, sketch some actual cells in the circles below.

Unplasmolyzed onion cells ( x) Plasmolyzed onion cells
( x)
Being able to see the difference between plasmolyzed from
unplasmolyzed cells is crucial for your
experiment in Part III. If you are having trouble, be sure to ask
for help from one of the learning center
instructors.
54
4. While waiting for the samples in step 2, put additional
samples of onion tissue into a series of 4-5
beakers containing concentrations of salt between 0 and 10%.
Label a series of microscope slides with
0%, 10%, and your other testing solutions.
5. Allow the samples in step 4 to remain in solution for 10
minutes. Then prepare a wet mount of each
specimen. One by one, examine each specimen under the
microscope. Work quickly so the specimens
do not begin to dry out under the microscope.
6. For each specimen, look only at the cells that contain red
pigment. Count 50 cells and record in Table
3 how many of the 50 are plasmolyzed and unplasmolyzed. The
natural pigmentation in red onion
makes it easier to see whether the cell membrane has pulled
away from the cell wall.

7. Calculate the percentage of cells plasmolyzed =
Number plasmolyzed_ x 100%
Number of cells counted
and enter the data in Table 3 on the next page.
8. Repeat steps 5 and 6 (above) for several salt solutions.
HINT: You are looking for the concentration at which about
half (40-60%) of the cells are plasmolyzed,
half unplasmolyzed. This indicates a concentration close to the
boundary between plasmolyzed and
turgid.
If you plan ahead, you won’t need to test all of the
concentrations! Look at the results of 0%, 5% and
10%, and then decide whethr you need to test 2 - 4%, or 6- 9%.
You may then need to run only one or
two additional concentrations to "zero in" on the solute
concentration isotonic for onion epidermal
tissue.
9. For each of the solutions you test, the percentage of cells
plasmolyzed =
Number plasmolyzed_ x 100%
Number of cells counted
Enter these results in Table 3.
10. From Table 3, the isotonic point for red onion appears to be
about _____ %.

55
Table 3: The number of plasmolyzed and unplasmolyzed onion
cells in different concentrations of salt
solution.
Salt
concentration
Number of cells
plasmolyzed
Number of cells
counted
Percent of cells
plasmolyzed
0%
1%
2%

3%
4%
5%
6%
7%
8%
9%
10%
1. Red blood cells will burst if placed in distilled water (0%
salt). Why didn't the onion cells burst?
(Hint: what do plant cell have that animal cells don’t.)
2. Unfortunately the isotonic point doesn’t always give a good
estimate of the actual concentration of
solutes inside the cell. How does the concentration of solutes
you found for onion cells compare to the
concentration of solutes in sea water? If the solute
concentrations are similar, why might this make
sense? If they are not similar, what cell structures or
mechanisms might help explain the difference?

The Role of Evolution by Natural Selection
Living cells and their membranes are much more complicated
than the “model cell” you made out of
semi-permeable dialysis tubing. In some plant species,
plasmolysis is due primarily to the loss of water
from the vacuole, a large storage chamber for relatively dilute
fluids whose solute concentration may be
lower than that in the cytoplasm proper. In other plant species,
the cells may actually be able to resist
plasmolysis in high-salt (hypertonic) environments due to
properties of the cell membrane that actively
pump solute ions in, or otherwise alter permeability of water
and solute ions across the membrane. In
your lab report, when you interpret your results from Part III,
keep in mind that cells have adaptations
that evolved for survival in hypotonic (freshwater) and
hypertonic (marine/salt water) environments.
56
PART III: DESIGN YOUR OWN EXPERIMENT
Do plants living in a freshwater pond have the same solute
concentrations inside their cells as plants
living in the salty ocean or on dry land? All plants are thought
to have evolved from a common ancestor
– single-celled green algae living in the ocean.
In Part III you will design an experiment to test one of the
following hypotheses:

Hypothesis 1: Since water is so critical for the life’s
biochemical process, all plant cells should maintain
the same internal solute concentrations as found in their marine
ancestors.
Hypothesis 2: Since plant species evolved for survival in
different environments, their internal solute
concentrations should be different from their marine ancestors.
Which hypothesis do you think is more likely to be true? Circle
1 or 2.
8.3 Exercise 3 – Comparing solute concentrations (isotonic
points)
inside plant cells from different environments
Using the plasmolysis threshold technique from Part II, design
and carry out an experiment to determine
whether the isotonic points of terrestrial and freshwater plants
are the same or different than the
isotonic points of marine plants.
Procedure
1. Work in groups of 3-4. Write your partners’ names here:
Partner’s name Discussion leader’s name

2. Select three or four organisms from those available in the
Central Study Area.
In table 4A-D, record the names of the organisms and where
they live.
a. freshwater algae
b. salt water algae
c. terrestrial plants
d. other plants, including any you bring in yourself
57
3. Based on the hypothesis you circled above (that is, that the
cells will have the same or different
internal concentrations), formulate a reasonable prediction
about the outcome you expect. Your
prediction should have the general form:
Prediction: "If my hypothesis (1 or 2) is correct, then the
isotonic point should be (higher/lower/the
same) in cells from (aquatic, marine and/or terrestrial)
environments than in cells from (aquatic, marine
and/or terrestrial) environments.

Write below the prediction you plan to test:
Prediction: If my hypothesis is true, then…
4. Working with your group, test your prediction by following
the procedures used in Exercise 2. Enter
your results in Table 4A-D.
Table 4A: Plasmolysis of the cells of
____________________________________in different
concentrations of salt solution.
This species lives in
____________________________________________________
Salt
concentration
Number of cells
plasmolyzed

Number of cells
counted
Percent of cells
plasmolyzed
58
Table 4B: Plasmolysis of the cells of
____________________________________in different
____________________________________________________
Salt
concentration
Number of cells
plasmolyzed
Number of cells
counted

Percent of cells
plasmolyzed
Table 4C: Plasmolysis of the cells of
____________________________________in different
____________________________________________________
Salt
concentration
Number of cells
plasmolyzed
Number of cells
counted
Percent of cells
plasmolyzed

Table 4D: Plasmolysis of the cells of
____________________________________in different
____________________________________________________
Salt
concentration
Number of cells
plasmolyzed
Number of cells
counted
Percent of cells
plasmolyzed
59

For cells to survive in environments whose salinity is much
lower or much higher than 3%, the cells may
need to pump salt in (or out) to maintain a suitable internal
environment. However, molecular pumps
require a lot of energy (ATP), so we would expect cells to have
evolved various ways to reduce this
energy expenditure.
1. Freshwater plants are surrounded by water in which the salt
concentration is much lower than that of
sea water. Do any of your isotonic point data suggest that
freshwater plant cells might have a lower
internal salt concentration than marine plant cells? What
advantages and/or disadvantages might this
have for freshwater plants?
2. Intertidal organisms that live at the edge of the ocean
experience wide fluctuations in salinity. For
example, when the tide is in, the intertidal area is covered with
sea water (3% salt). As the tide goes
out, tidal pools are left behind to evaporate in the sun, greatly
increasing salinity in the pools. However,
after a rain, freshwater running into the tidal pools can greatly

reduce salinity. Do any of your data
suggest that marine plant cells may have evolved ways to
stablilze their internal environments in the
face of the wide fluctuations in salinity found in intertidal
environments? Explain.
60
LAB REPORT
Hand in to your discussion leader a typed lab report. You and
your group members can submit
identical cover pages and data tables, but your introduction and
discussion must be written by you, in
your own words.
(1) Cover page: including the title of the experiment (in this
case use "Comparing cell contents
of plants from different environments" ), your name, the date,

your discussion leader's name, and the
number of your discussion section. Also include the names of
all your partners and their discussion
section TAs.
(2) Introduction: State your hypothesis about whether the
concentration of solutes inside the
cells of plants from different environments should be the same
or different. Explain briefly why your
hypothesis makes sense to you. State the prediction you
generated from this hypothesis and describe
(in general terms) how you tested it. You don’t need to detail
the methods (because they are already in
the lab guide), but you do need to define an isotonic point
(especially what you consider to be its
relationship to the cell’s internal solute concentration) and
explain how you used isotonic points to test
your prediction.
(3) Results: On a separate page, summarize your data from
tables 3 (onion cells) and 4 (three
other kinds of cells) into one table, clearly labeled.
(4) Discussion: In about 2 pages, explain what an isotonic
point is and compare the isotonic
points of your specimens with each other and with sea water.
Does there appear to be a relationship
between isotonic points and environment in which the plants are
found? If not, then what cellular
mechanisms (salt pumps, impermeable cell membranes, or
others?) might these plant cells be using to
maintain a stable internal environment despite widely differing
external environments? Include some of
the analysis and discussion questions raised on the previous
page.

61
Lab 4 – Photosynthesis 9
Almost all life on earth depends directly or indirectly on
photosynthesis: the ability of certain organisms
(notably green plants) to capture and store the energy of
sunlight. Photosynthesis in green plants
involves a complex series of reactions, but the overall process
uses carbon dioxide and water to make
glucose (a simple sugar) and other carbohydrates, with oxygen
as a by-product.
sunlight
6 CO2 + 12 H20 ----------------> C6H12O6 + 6 O2 + 6
H2O
The sun’s energy is stored in the bonds that hold the glucose
molecule together. Photosynthesis occurs
inside the green, chlorophyll-rich cell organelles called
chloroplasts. Inside the chloroplasts are stacks of
"thylakoids" (flattened sacs), aligned to keep the light-
absorbing chlorophyll exposed to sunlight.
The energy stored in the glucose can later be released by a
process called respiration. Respiration

involves another complex series of steps, but the overall
reaction is roughly the reverse of
photosynthesis:
C6H12O6 + 6 O2 -----------------> 6 CO2 + 6 H2O +
Energy
During respiration, glucose is broken down into carbon dioxide
and water. The energy stored in the
glucose is released gradually, a little at each step, and stored in
small ATP molecules. The ATP's then
carry the energy to wherever it is needed inside the cell.
Respiration occurs inside mitochondria, which
are cell organelles packed with internal membranes in which the
enzymes of respiration are embedded.
This minicourse focuses on photosynthesis the effects of
environmental factors on the rate of this
enzyme-catalyzed reaction.
are able to:
1. Explain how paper chromatography is used to separate the
light-absorbing pigments in a leaf.
Prepare and interpret a chromatogram.
2. Given a leaf cross section, identify the major cell layers and
explain in what way(s) each layer aids

photosynthesis by the chloroplasts.
3. Analyze and discuss the results of experiments concerning
the effects of the environment and
osmosis on guard cells and stomata.
4. From your own experiments and data from others, describe
how the rate of photosynthesis is
affected by the following factors: the availability of carbon
dioxide, light intensity, light quality,
temperature and/or pH.
62
5. Given what you know about the molecular properties of
enzymes, explain why the rate of
photosynthesis should be affected by each of the environmental
factors listed above.
PLAN AHEAD! You will need to work with a partner for lab
#4.
A lab report is NOT required this lab.
PART I: LIGHT-ABSORBING PIGMENTS
Sunlight is "white" light composed of all the wavelengths
(colors) of the visible spectrum: red-orange-

yellow-green-blue-indigo-violet (or "Roy G. Biv.").
Photosynthesis begins with the absorption of sunlight
by pigments in the chloroplasts. These pigments absorb some
colors of light better than others. For
example, a green leaf contains the pigment chlorophyll. The
leaf looks green not because chlorophyll
absorbs green light, but because it’s molecular bonds absorb all
the colors of the spectrum except green.
The green light either reflects back to your eye, or passes
completely through the leaf and out the other
side..
Since chlorophyll can’t absorb any green light, does that mean
all the energy in green light goes to
waste? Might the leaf contain other pigments that absorb green
light? To find out, do Exercise 1.
9.1 Exercise 1 – Separation of leaf pigments by paper
chromatography
Chromatography is a technique used to separate substances in a
mixture. It can be used to determine
what pigments are present in a leaf. Paper chromatography can
separate different pigments only if
those pigments (a) have different solubilities, and/or (b) differ
in the degree to which they adhere to the
paper.
Paper chromatography involves placing a drop of leaf extract
near the bottom of a strip of paper. The
bottom edge of the strip is then dipped into a solvent. As the
leading edge of solvent moves up the
paper and across the spot of leaf extract, the pigments dissolve
and are carried along with the solvent.
The more soluble a pigment, the farther it will travel up the

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