The
University of
Lethbridge
BIOLOGY 1010
LABORATORY MANUAL
Fall 2006
BIOLOGY 1010
The Cellular Basis of Life
Laboratory Manual
Fall 2006
Written by:
Helena Danyk
Department of Biological Sciences
University of Lethbridge
Student Name:
Lab Day:
I.D. Number:
Lab Section:
Lab Time:
Instructor’s Name:
Office:
Email address:
Telephone:
Office Hours:
Lab Room:
TABLE OF CONTENTS
Exercise
Page #
Fall, 2006 Laboratory Schedule…………….…………….………………………….…….i
Evaluation and Grading Policies ………………………………………………….……...ii
Safety Procedures ………………………………...………………………………......….iii
Cell Structure and Function, Part 1 …………………………………………………….....1
Cell Structure and Function, Part 2 …………………………………………………...…..8
Cell Membranes: Effects of Stress ……………………………………………...……….16
Enzymes …………………………………………………………………………………26
Bacteriology, Part 1 ……………………………………………………………..………35
Bacteriology, Part 2 ……………………………………………………………….…….45
Fermentation and the Scientific Method ……………………………………………..….53
DNA Structure and Function……………………………………………………….……61
Appendix A: How to Use the Microscope ………………………………………………70
Appendix B: How to Make a Scientific Drawing ……………………………………….73
Appendix C: How to Determine the Size of a Specimen ………………………………..75
Appendix D: Aseptic Technique ……………………………………………………..….76
Appendix E: How to Use a Spectrophotometer ……………………………………....…77
Appendix F: Scientific Inquiry ………………………………………………………….80
Appendix G: Conversions and Taxonomy …………………………………………..…..89
REVISED: Summer, 2006
Written By
Helena C. Danyk
University of Lethbridge, Lethbridge, AB 2006-07
i
BIOLOGY 1010 LABORATORY SCHEDULE: Fall, 2006
Sept. 11-15
Cell Structure and Function, Part 1
Sept. 18-22
Cell Structure and Function, Part 2
Sept. 25-29
Cell Membranes: Effects of Stress
Oct. 2-6
Cell Membranes – Complete (Scientific Method)
Oct. 9-13
Thanksgiving – No labs this week
Oct. 16-20
Enzymes
Oct. 23-27
Bacteriology, Part 1
Oct. 30-Nov. 3
Bacteriology, Part 2
Nov. 6-10
Bacteriology, Part 2 (complete); Fermentation Part 1
Nov. 13-17
No labs this week
Nov. 20-24
Fermentation Part 2
Nov. 27-Dec. 1
DNA Structure and Function
Dec. 4
Final Lab Exam; 4:00-6:00 pm or 6:30-8:30 pm; Room B650 or B660.
For help or advice on any aspect of the Biology 1010 laboratory, please consult your lab
instructor or the lab coordinator (Helena Danyk: D884, 329-2664,
[email protected]).
ii
Evaluation:
The laboratory portion of Biology 1010 is worth 35% of the overall course grade, and is
calculated based on the following allocations:
In-class Quizzes and Assignments:
Laboratory Report:
Final Lab Exam:
12%
8%
15%
Attendance:
You may only attend the lab section in which you are registered due to safety regulations,
equipment limitations, and room capacities. However, if you are aware of a planned
absence, consult your lab instructor who will give you permission to attend an alternate
section if space is available. If you miss a lab due to illness or personal emergency,
inform your instructor as soon as possible so that alternate lab arrangements can be made.
Your lab instructor may request documentation from you (doctor’s note, coach’s letter,
etc) at his/her discretion.
It is your responsibility to ensure that you are properly prepared for all lab quizzes,
assignments and exams even if you are not in the lab. Labs are given one week at a time
and are not repeated in subsequent weeks, so if you miss an entire week of classes you
will not be able to make up the lab exercise.
Missed Assignment and Exam Policy:
In-class quizzes and assignments must be written as scheduled, except in the case of a
medical excuse or personal emergency (validity of excuse to be determined on a case-bycase basis by your instructor, in the event that documentation cannot be provided). If you
must be absent for an approved reason, you will be excused from the quiz or assignment
and the weight of the missed work will be adjusted for in your final lab mark. There are
NO make-up quizzes or assignments. Students will be required to write and submit a lab
report regardless of whether they attended the relevant lab or not. If you miss the final
lab exam for any reason other than medical as previously outlined, you will be required to
apply for an incomplete in the course and write the final lab exam in the following
semester.
iii
GUIDELINES FOR SAFETY PROCEDURES
Students enrolled in laboratories in the Biological Sciences should be aware that there are
risks of personal injury through accidents (fire, explosion, exposure to biohazardous
materials, corrosive chemicals, fumes, cuts, etc). The guidelines outlined below are
designed to:
a) minimize the risk of injury by emphasizing safety precautions and
b) clarify emergency procedures should an accident occur.
EMERGENCY NUMBERS:
City Emergency
Campus Emergency
Campus Security
Student Health Centre
911
2345
2603
2484 (Emergency - 2483)
THE LABORATORY INSTRUCTOR MUST BE NOTIFIED AS SOON AS POSSIBLE AFTER
THE INCIDENT OCCURS.
EMERGENCY EQUIPMENT:
Your lab instructor will indicate the location of the following items to you at the
beginning of the first lab period.
1.
2.
3.
4.
5.
6.
7.
Closest emergency exit
Closest emergency telephone and emergency phone numbers
Closest fire alarm
Fire extinguisher and explanation of use
Safety showers and explanation of operation
Eyewash facilities and explanation of operation
First aid kit
GENERAL SAFETY REGULATIONS:
•
•
•
•
•
•
•
Eating and drinking is prohibited in the laboratory. Keep pencils, fingers and
other objects away from your mouth. These measures are to ensure your safety
and prevent accidental ingestion of chemicals or microorganisms.
Coats, knapsacks, briefcases, etc. are to be hung on the hooks provided, stowed
in the cupboards beneath the countertops, or placed along a side designated by
your instructor. Take only the absolute essentials needed to complete the
exercise* with you to your laboratory bench. (* e.g. manual, pen or pencil)
Mouth pipetting is NOT permitted; pipet pumps are provided and must be used.
Always wash your hands prior to leaving the laboratory.
Students are not allowed access to the central Biology Stores area for any reason.
Consult your instructor if you require additional supplies.
Report any equipment problems to instructor immediately. Do NOT attempt to
fix any of the equipment that malfunctions during the course of the lab.
Use caution when handling chemical solutions. Consult the lab instructor for
instruction regarding the clean-up of corrosive or toxic chemicals.
iv
Contain and wipe up any spills immediately and notify your lab instructor (see
SPILLS below). Heed any special instructions outlined in the lab manual, those
given by the instructor or those written on reagent bottles.
Long hair must be restrained to prevent it from being caught in equipment,
Bunsen burners, chemicals, etc.
Dispose of broken glass, microscope slides, coverslips and pipets in the
specially marked white and blue boxes. There will be NO disposal of glassware
in the wastepaper baskets.
You are responsible for leaving your lab bench clean and tidy. Glassware must
be thoroughly rinsed and placed on paper toweling to dry.
•
•
•
•
SPILLS:
•
Spill of SOLUTION/CHEMICAL: While wearing gloves, wipe up the spill
using paper towels and a sponge as indicated by the lab instructor.
•
Spill of ACID/BASE/TOXIN: Contact instructor immediately. DO NOT
TOUCH.
•
BACTERIA SPILLS: If necessary, remove any contaminated clothing. Prevent
anyone from going near the spill. Cover the spill with 10% bleach and leave for
10 minutes before wiping up. Discard paper towels in biohazard bag. Discard
contaminated broken glass in designated biohazard sharps container.
DISPOSAL:
•
Broken glass, Pasteur pipets, pipets are placed in the upright white ‘broken
glass’ cardboard boxes. NO PAPER, CHEMICAL, BIOLOGICAL OR
BACTERIAL WASTE MATERIALS should be placed in this container
•
Petri plates, microfuge tubes, pipet tips should be placed in the orange biohazard
bags. The material in this bag will be autoclaved prior to disposal.
•
Bacterial cultures in tubes or flasks should be placed in marked trays for
autoclaving.
•
Liquid chemicals should be disposed of as indicated by the instructor. DO NOT
dispose of residual solution in the regent bottles. In case of any uncertainty in
disposal please consult the lab instructor.
•
Slides of bacteria should be placed in the trays filled with 10% bleach that are
located at the ends of the laboratory benches.
HEALTH CONCERNS:
Students who have allergies, are pregnant, or who may have other health concerns should
inform their lab instructor so that appropriate precautions may be taken where necessary.
v
THE UNIVERSITY OF LETHBRIDGE
Policies and Procedures
Occupational Health and Safety Manual
SUBJECT:
CHEMICAL SPILLS PROCEDURE
Precaution should be taken when approaching any chemical spill.
1.
UNKNOWN SPILL
a. Clear the area
b. Call Security at 329-2345
c. Secure the area and do not let anyone enter
d. Call Utilities at 329-2600 and request air be turned on at the spill site
e. Security will respond and determine the severity of the spill
f. Security will immediately notify the spill team as follows:
• Peter Dibble
331-5201
• Michael Gerken
332-2173
• DBS Environmental
only if above not available 328-4483 (24 hrs)
• U of L Occupational Health and Safety 332-2350 (Carolin)
394-8716 (Bill)
2.
KNOWN SPILL
a. Clear the area
b. Call Security at 329-2345
c. Secure the area
d. Call Utilities at 329-2600 and request air be turned on at the spill site
e. Security will respond and determine the severity of the spill
f. Security will immediately notify the spill team as follows:
• Peter Dibble
331-5201
• Michael Gerken
332-2173
• DBS Environmental
only if above not available 328-4483 (24 hrs)
• U of L Occupational Health and Safety 332-2350 (Carolin)
394-8716 (Bill)
3.
NOTIFICATION
a. Occupational Health and Safety will notify the appropriate departments,
including notification of appropriate government agency.
h:\shared\security\Chemical Spills Procedure.doc
1
CELL STRUCTURE AND FUNCTION: PART I
Introduction:
Cells are considered the basic unit of all living organisms because they perform all of the
processes we call ‘life’. All organisms are composed of cells. Although most individual cells
are visible only with the aid of a microscope, some may be up to a meter long (e.g. nerve
cells) or as large as a small orange (e.g. the yolk of an ostrich egg). Despite such differences,
all cells have a similar fundamental design and share many features.
The objectives of today’s lab are to:
• examine some of the features of cells as a means of understanding the life processes of
organisms
• become familiar with the structures and organelles possessed by cells, and to
determine the relative sizes and functions of each
• provide an introduction to microscopy, including the use and care of the microscope
and micrometry to measure cell and organelle size
• learn how to present information in scientific drawings
As background for this lab and the following exercises, you will find it useful to read
pages 6-8 and 94-122 in Campbell and Reece (2005).
A. Elodea Cells
Elodea is a widely distributed pond weed that is often used in studies of photosynthesis.
Elodea is a eukaryote, meaning its cells contain membrane-bound nuclei and other
organelles. For example, chloroplasts are elliptical green structures (organelles) in the cells.
Chloroplasts are the site of photosynthesis and are green because of the presence of the
photosynthetic pigment chlorophyll. Chloroplasts are a feature of plant cells.
Remove a young leaf from the tip of a sprig of Elodea. Place this leaf in a drop of water on a
microscope slide and cover it with a coverslip. Do not let the leaf desiccate (dry up).
Examine the leaf with the 10x power and then the 40x power objective lens. Each of the
regularly shaped units you see is a cell, and each is delimited by a cell wall made of cellulose.
Cellulose is a complex carbohydrate formed of glucose molecules attached end to end. Cell
walls are another characteristic of plant cells.
Most plant cells contain a large central vacuole surrounded by a membrane called the
tonoplast. As a consequence, the liquid cytoplasm containing the chloroplasts and other
organelles is restricted to the periphery of the cell. Look carefully at the periphery of the cells
and chances are you will detect a slow, circular movement of the chloroplasts; this
phenomenon is called cytoplasmic streaming. Because the central vacuole of Elodea does
not contain coloured material, it is sometimes difficult to discern. Chloroplasts that appear to
be in the center of the cell are actually around the edges. To confirm this, focus up and down
through the cell. The vacuole occupies approximately 90% of the volume of each Elodea cell
2
and contains water and a variety of dissolved materials such as sugars, alkaloids and inorganic
salts. In general, vacuoles serve as storage areas for food materials and depots for waste
materials. Vacuoles also help maintain cell turgidity and thus cell shape.
Draw representative Elodea cells in the space below and label them. Refer to Appendix B for
rules regarding scientific drawings. Use an ocular micrometer (Appendix C) to measure the
lengths and widths of five cells. Convert ocular units to millimeters, and calculate means
(Appendix F) for length and width. Use the mean length value to calculate your drawing
magnification. As well, measure and report the average diameter of a chloroplast.
B. Structure of Plastids
Plastids are organelles of plants that are the sites of such activities as food manufacture and
storage. You have already examined chloroplasts, a type of plastid in which photosynthesis
occurs. However, other plastids have different functions. You will examine two other kinds
of plastids – amyloplasts and chromoplasts.
Use a razor blade to make a thin section of potato tuber (make this section as thin as possible).
Stain the section for a few seconds with iodine, a stain specific for starch. Add a coverslip,
and examine under the microscope. The intensely stained structures in the cells are
amyloplasts, a type of plastid that stores starch.
Draw a potato cell in the space below. Calculate drawing magnification and include this
information in your figure caption.
3
•
Measure the smallest and largest amyloplasts you can find and report the size range.
Use a razor blade to prepare a thin section of red pepper. Place the section in a drop of water
on a glass slide, add a coverslip, and examine with your microscope. The tiny pinpoint orange
organelles are chromoplasts, a type of plastid containing pigments other than chlorophyll (in
this case, probably carotenes and/or xanthophylls).
Draw red pepper cells in the space below.
•
Compare the size of the chloroplasts, amyloplasts and chromoplasts you have observed
thus far.
C. Paramecium
Add a drop of Paramecia culture to a tiny drop of “Protoslo” (methyl cellulose) in order to
slow down the movement of the Paramecia. Mix gently with a toothpick and add a coverslip.
Observe your slide with the 4x, then the 10-x objective lens to locate a Paramecium. If it is
not moving too quickly, you should be able to switch over to the 40x objective lens to observe
fine details of its structure. Note the beating of the cilia. This serves two purposes; it propels
the organism and it sweeps food into an oral groove. As the food moves down to the bottom
of the groove it is taken into a food vacuole and digested as the vacuole moves around the
cell. You should be able to see the contractile vacuole, a perfectly round and clear vesicle. It
slowly builds in size, then fuses with the plasma membrane and contracts to expel its contents
(water) from the cell. Use the space below to sketch your Paramecium and label the parts you
observed. What is the size of a Paramecium?
4
D. Human Epithelial Cells
Gently scrape the inside of your cheek with the broad end of a toothpick. Stir the scrapings
into a drop of methylene blue on a glass slide, add a coverslip, and observe under the 10x
power lens, then the 40x lens of the microscope. Use the space below to prepare a scientific
drawing.
•
You may have noticed that some of the cells may have had their edges folded over. What
does this indicate about the thickness of the cells?
E. Pond Water
If available, prepare a wet mount of water from the aquarium provided. View your slide
under the low power objective lens initially. You should see examples of autotrophic
(photosynthetic) organisms as well as heterotrophic (ingestive, animal-like organisms).
Consult reference materials available to see if you can identify any of the organisms, and
make some representative sketches in the space below.
5
F. Unusual Cell Types
View the demonstration of unusual cell types present at the back of the lab. Each of these
represents a single cell. Can you think of any other unusual cell types not shown here? Do
you have any thoughts as to why there is such variation in size and shape of particular cells?
6
Thought Questions:
1. Think of the cells you viewed today in lab. Try to correlate cell structure to function. If a
cell has an abundance of certain organelles, can you infer the major function of the cell?
2. Why are most cells microscopic (refer to Figure 6.7 of Campbell and Reece, p. 99)? In
this context, is it better for a plant to have several large chloroplasts or many small
chloroplasts? Explain your answer.
3. What is the usefulness of cytoplasmic streaming?
4. Students often confuse the cell wall with the cell membrane. Compare the structure and
function of the cell wall and cell membrane. Do all cells have both?
5. An inherited disorder in humans results in the absence of a certain critical protein in
flagella and cilia. The disease causes respiratory problems and in males, sterility. What is
the ultrastructural connection between these two symptoms?
6. Why are contractile vacuoles necessary in Paramecium?
7
7. Complete the following Table:
Structure
Function
Cell Wall
Nucleus
Vacuole
Chloroplast
Mitochondrion
Contractile Vacuole
8. Human epithelial cells are highly specialized, for protection. As a consequence of
specialization, they have lost the ability to carry out certain cellular functions. Describe
some of the functions that epithelial cells may have lost.
Literature Cited
Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings,
San Francisco, CA.
8
CELL STRUCTURE AND FUNCTION: PART II
Introduction:
In your laboratory last week, you examined a diversity of eukaryotic cell types, including
animal, plant and protist cells, single-celled organisms capable of performing all of the
functions associated with life, and cells from multicellular organisms that were specialized to
perform a limited number of tasks. You were also introduced to some of the structures and
organelles associated with eukaryotic cells. You will continue your study of cell structure and
function in this exercise.
Today’s laboratory will focus more specifically on processes used by cell biologists to study
particular cell constituents, namely plasma membranes, chloroplasts and mitochondria. In the
first part of the exercise, we will use artificial membranes to study the selective permeability
of membranes, and then will look at the same process in red onion cells. Part two of today’s
lab will make use of centrifugation to isolate cell constituents. A suspension of cell parts and
organelles (termed a homogenate) may be separated using differential centrifugation.
Differential centrifugation relies on using different amounts of centrifugal force to sediment
cell parts of different densities and sizes, and is a technique commonly used by cell biologists
to study cells.
The objectives of today’s lab are to:
• learn about the semi-permeable nature of the plasma membrane
• investigate the roles played by osmosis and diffusion in the proper functioning of a cell
• make use of differential centrifugation techniques with the purpose of separating and
identifying cell components
Please read pages 97, 109-111, and 130-133 in Campbell and Reece (2005) in preparation for
this exercise.
A. Diffusion Through a Selectively Permeable Membrane
One of the most important properties of a cell membrane is its ability to regulate the passage
across of ions and molecules. Typically carbon dioxide and oxygen can pass through
membranes easily, while larger, hydrophilic molecules (those possessing a net charge) like
sugars are impeded. The movement across membranes of molecules like these is facilitated
by the presence of transport proteins imbedded in the membrane. We will examine the
structure of cell membranes in more detail in a future lab. Today we will concentrate on some
of the mechanisms that drive substances across membranes, namely diffusion and osmosis.
Diffusion, the movement of a substance from an area where it is more concentrated to an area
where it is less concentrated, is a spontaneous process that requires no input of energy
(passive transport). This process explains much of the movement of molecules across cell
membranes. The diffusion of water across a selectively permeable membrane is a special
kind of passive transport termed osmosis. The direction that water moves depends on the
total amount of solute present on each side of the membrane. A solution with a higher
9
concentration of solutes compared to another is referred to as a hypertonic solution, while the
solution with the lower concentration of solutes is termed a hypotonic solution. If the net
concentrations of solutes are equal in both solutions, they are said to be isotonic. Water
always moves from a hypotonic to a hypertonic solution.
We can make use of a simple model system to illustrate both of these concepts. Cellophane
dialysis tubing acts as a synthetic semi-permeable membrane that permits the diffusion of
water and other small molecules while prohibiting the movement of larger molecules. Work
in pairs to create and manipulate artificial cells using the procedures outlined below.
Pair 1:
• Cut a 10 cm strip of tubing, and immerse in the container of water provided for about a
minute, until the tubing can be opened easily by rolling between your thumb and
fingers.
• Tie a knot near one end of the tubing using the string provided. Use a plastic
disposable pipette to fill the tube to within 5 cm of the top with an 80% glucose
solution.
• Insert a plastic 1 mL pipette into the tubing, and securely tie the open end of the tubing
shut around the pipette.
• Use the stands provided to support the bag in a beaker of water.
• Observe the position of the column of fluid in the pipette periodically for about 30
minutes. If there is no change after the first five minutes, check your set-up for leaks
(which will appear as wiggly lines in the beaker of water). Record your observations
below.
Pair 2:
• Cut a 10 cm strip of tubing, and immerse in the container of water provided for about a
minute, until the tubing can be opened easily by rolling between your thumb and
fingers.
• Tie a knot near one end of the tubing using the string provided; then use a Pasteur
pipette to fill the tubing with starch solution. Tie the other end of the tubing.
• Immerse the tubing in a beaker of water to which you have added enough iodine to
make the solution light brown in colour.
• When starch molecules come into contact with iodine, a blue or purplish colour
appears (what evidence do we have for this from last week’s lab?). Observe the bag
and the solution for about 15 minutes and record any colour changes that you observe.
Can iodine pass through the membrane? How do you know?
10
Can starch pass through the membrane? How do you know?
Which experiment demonstrates osmosis?
Why is the other experiment not directly a demonstration of osmosis?
Osmosis can also be easily observed in living cells. Work individually to complete the next
part of the exercise:
•
Take one of the red onion leaves provided for you on the side bench and snap the leaf
backwards. Peel back the thin piece of red epidermis formed at the break point (your
instructor will demonstrate this). Place this epidermal tissue in a drop of water on a
microscope slide, add a coverslip, and examine. The entire cell appears red due to
pigment granules (anthocyanin) dissolved within the central vacuole. Sketch what you
see in the space below.
•
Prepare another wet mount as you did above, but replace the water on the microscope
slide with 2-3 drops of 20%NaCl. Add a coverslip, examine, and sketch your
observations in the space below. How do the cells differ in appearance from those
viewed above?
The shrinkage of the cytoplasm because of osmotic water loss is called plasmolysis (Figure
1).
11
Figure 1: Plasmolysis of a Plant Cell
In the first preparation, which solution is hypertonic, the water the cell is immersed in, or the
water inside the cell?
In the second preparation, which solution is hypotonic, the NaCl solution the cell is immersed
in, or the water inside the cell?
B. Cell Fractionation
Cell fractionation is a process in which cells are gently broken apart and their components
separated. Cellular organelles remain intact and biochemically active so that their functions
may be studied. Cells can be disrupted by a variety of means, including electric shock,
sonication (vibration) or grinding. The resulting suspension of cell parts and organelles,
called a homogenate, can then be separated using differential centrifugation. At low speeds,
large nuclei and intact cells sediment to form a pellet at the bottom of the centrifuge tube,
while the remaining organelles are found in the supernatant above the pellet. Centrifugation
of the supernatant at successively higher speeds isolates successively smaller components of
the cell in pellets.
We will make use of differential centrifugation to isolate chloroplasts and mitochondria from
a combined pea and spinach homogenate. We will then examine the different fractions
resulting from our centrifugation procedure for the presence or absence of these two types of
organelles. Please work in groups of four to complete the following experiment.
Each group of four does the following:
1. Fill two 15 mL centrifuge tubes to the 12 mL mark with the pea-spinach homogenate
located at the side bench (the homogenate was prepared by your instructor just prior to
the lab period by placing pea seeds soaked overnight together with two or three
spinach leaves in a blender with phosphate buffer, blending briefly, and then straining
through cheesecloth). Label the tubes with your name and bench number.
12
2. Place your tubes in the centrifuge (the rotor must always be balanced with equal
weights or volumes opposite each other). Centrifuge at 200x gravity for three minutes.
3. While your material is in the centrifuge, remove a small sample of the cellular debris
from the cheesecloth and prepare a wet mount. Place a coverslip on the slide, and
observe the material using the low power objective.
•
Do you see cell wall fragments?
•
Do you see intact cells?
•
Prepare another slide, but use iodine solution in place of water. What do you
see? Sketch your observations in the space below.
•
Use the ocular micrometers provided to measure the length of 10 of the stained
structures. Use these values to calculate their mean size, referring back to
Appendix C if necessary.
4. After centrifugation is complete, remove your test tubes from the centrifuge.
Carefully decant the supernatants into two new centrifuge tubes. Store the original
tubes (each containing a white pellet) on ice for the time being. Return the new tubes
to the centrifuge and centrifuge at 1300xg for 10 minutes.
5. Meanwhile, examine the white pellet collected in step 4. Mix a drop of iodine with a
small amount of the pellet on a microscope slide, add a coverslip, and observe (pea
seedlings store their food reserves in the form of starch grains).
• Describe the appearance of the starch grains you observe.
• Use the micrometer to measure the size of 10 of these starch grains. Has their
mean size changed?
13
6. After the second centrifugation is complete, carefully remove your test tubes from the
centrifuge. You should see a green layer above another small white pellet. This green
layer will contain the nuclei and chloroplasts.
7. Use a clean Pasteur pipette to carefully remove about 2 pipettes full of the yellowishgreen supernatant. Place it into a new test tube, and store the tube on ice until needed
(the supernatant contains the mitochondrial fraction). The remainder of the
supernatant from your two tubes can be carefully pipeted into a waste beaker. Once
the supernatant has been removed, carefully remove a drop of the green layer from one
of your tubes and place it onto a microscope slide. Add a drop of water and a
coverslip, and examine it with your microscope using the low power, and
subsequently, the high power objective lenses.
•
Do you see aggregations of green-coloured bodies? What are these?
•
Calculate the mean length of 10 of the green structures observed.
•
In some cases nuclei may also be visible. They will appear as large, round, grayish
structures.
8. Make another wet mount of the green residue, but this time use iodine in place of
water.
•
Are amyloplasts still visible? If so, compare the size of these amyloplasts with
those isolated in steps 3 and 5. Why might the sizes be different?
9. Prepare a wet mount of the yellowish-green supernatant from step 7. Add a drop of
Janus Green stain to this wet mount. You should see clumps of small, blue-stained
objects; these are the mitochondria.
•
Use the micrometer to measure and record the size of 10 of the mitochondria you
observe.
•
How does mitochondrial size differ compared to chloroplast size?
14
Thought Questions
1a). What would happen if the Paramecium cells you viewed last week were placed in a
solution of pure water? Use the terms osmosis, hypertonic and hypotonic in your
answer.
1b). What role does the contractile vacuole play in maintaining osmotic equilibrium in a
Paramecium’s natural environment?
1c). Would the same be true of Elodea cells placed in pure water? Why or why not?
2.
What physical properties of cell organelles determine their behaviour during differential
centrifugation? Why are different speeds of centrifugation used? Rank the cell parts
that you have studied over the last two periods based on their size (smallest to largest).
15
3. Organelles are often studied by separating them on a sucrose gradient. A centrifuge tube
is filled with a sucrose solution of increasing concentration from the top of the tube to the
bottom. What would you expect to observe if you put a layer of cell homogenate on top of
the sucrose and then subjected the sample to high speed centrifugation?
4. Janus Green is blue when in an oxidized state, and as it is reduced, it becomes colourless.
Why was Janus Green a useful stain to use to identify mitochondria in this exercise?
What would you expect to happen if you had examined your slide again after several
hours? Why?
Literature Cited:
Helms, D.R., Helms, C.W., Kosinski, R.J. and Cummings, J.R. 1998. Biology in the
Laboratory 3rd Ed. W.H. Freeman and Company, New York.
Wachtmeister, H.F.E., Scott, L.J. and Perry, M.A. 1986. Encounters With Life: General
Biology Laboratory Manual, 2nd Ed. Morton Publishing Company, Englewood, CO.
16
CELL MEMBRANES: EFFECTS OF STRESS
Introduction:
Cellular membranes separate and organize chemicals and reactions within cells by allowing
selective passage of materials across their boundaries. They are composed of a bilayer of
phospholipid molecules interspersed with protein molecules. In addition, most membranes also
contain very small amounts of carbohydrates that are usually associated with the phospholipids
or proteins.
Phospholipids are composed of a phosphate group, glycerol backbone, and 2 fatty acid chains
(Figure 1). They are amphipathic; that is, each molecule has a hydrophilic (water-associating)
region and a hydrophobic (water-avoiding) region. The charged (polar) phosphate group and
glycerol group of each molecule are hydrophilic and the nonpolar lipid tails (fatty acids) are
hydrophobic.
Figure 1. The structural formula (left) and symbolic representation (right) of a phospholipid
molecule.
When phospholipids spontaneously assemble in an aqueous solution, the most stable
conformation is to have the molecules aligned so that the hydrophobic lipid regions turn
inward and face each other, thereby avoiding contact with water (Figure 2). The polar
phosphate head regions are arranged outwardly where they are in contact with water.
Although the hydrophobic forces holding phospholipids in a membranous structure are
individually weak and allow substantial movement of individual molecules, collectively they
confer considerable stability to the overall structure.
17
Figure 2. Artificial membrane cross section depicting the orientation of phospholipids when
exposed to an aqueous solution.
Interspersed within the membrane are protein molecules. Each protein molecule is folded so
that charged hydrophilic amino acid groups project into the aqueous phase inside or outside
the cell and uncharged hydrophobic groups contact the inner lipid phase of the bilayer (Figure
3). As with any protein, relatively weak hydrogen bonds hold the membrane proteins in
specific folded conformations. Within the membrane, proteins are not fixed in position but
rather are free to move about. The proteins within the membrane perform a variety of
functions including transport, enzymatic activity, signal transduction, and intercellular joining.
Figure 3. Lipid bilayer found in all biological membranes with embedded proteins.
The physical and chemical integrity of a membrane is crucial for proper functioning of the cell
or organelle of which it is a part. In lab two, we examined the selective permeability of
artificial cell membranes and membranes from red onion cells. The permeability of
membranes is directly related to its phospholipids and transport proteins. In this exercise, we
will continue our study of membranes by looking at the effects of various stresses
(temperature and detergent concentration) on beet cell membrane integrity, and study how
loss of membrane integrity leads to loss of membrane function.
The roots of beets (Beta vulgaris) contain an abundant red pigment called betacyanin, which
is localized almost entirely in the large central vacuole of beet cells. These vacuoles are
surrounded by a vacuolar membrane and the entire beet cell is further surround by a plasma
membrane. As long as the cells and their membranes are intact, the betacyanin will remain
inside the vacuoles. However, if the membranes are stressed or damaged, betacyanin will leak
18
through the membranes and produce a red color in the water surrounding the beet tissue. The
intensity of this red color will allow you to assess the damage produced by experimental
treatments.
The objectives of today’s exercise are thus to:
• Continue to learn more about membrane structure and function by determining the
effects of temperature and detergent treatments on membrane integrity and function
• Become familiar with using a spectrophotometer to collect scientific data
• Learn about scientific methodology, in particular, how to collect, present and analyze
experimental results.
Please read pages 99-101 and 124-130 in Campbell and Reece (2005) in preparation for this
exercise. As well, review Appendix F and read chapter 9 in Pechenik (2007) prior to attending
the next lab.
Procedure:
Based on information provided to you in the lab manual and text readings, prepare hypotheses
for the experiments investigating [a] the effect of SDS concentration and [b] the effect of
temperature on beet membranes.
[a] SDS:
[b] Temperature:
1. Working with your partner, use a cork borer with a 5-mm inside diameter to cut five
uniform beet cylinders, and use a razor blade to trim each to 15 mm in length. Place all
cylinders in a beaker and gently run cool tap water over them for two minutes to wash
betacyanin from the injured cells on the surface.
2. One group at a bench will test the effect of various temperatures (see below), while the
other group will test the effect of the detergent sodium dodecylsulfate (SDS).
3. For those testing SDS:
• Add 6 mL of the appropriate concentration (see Table 1) to labeled tubes and place a
cylinder of beet in each.
• Let stand at room temperature for 20 minutes, shaking (gently) occasionally.
• Use a dissecting needle to spear the cylinders and remove them quickly from the tubes,
and then arrange the tubes in order from palest to darkest in colour.
• Quantify the relative colour of the solutions, beginning with the palest and ending with
the darkest (refer to Appendix D for information regarding use of a
spectrophotometer). You will be reading the Absorbance at 460 nm. In order to set
“100” on the percent transmittance scale, use the test tube containing the SDS solution
(the reagent blank) which should be located next to the spectrophotometer. The light
absorbance is a direct measure of the concentration of betacyanin and an indirect
measure of membrane damage.
• Record absorbance readings in Table 1.
19
• Rinse all test tubes and place in the baskets provided.
4. For those testing the effect of temperature:
• Label five test tubes 1-5.
• Gently, using forceps, remove two beet cylinders from the wash beaker, and touch
each briefly to a paper towel to remove most of the adhering water. Place one in Tube
1 and one in Tube 2. Your lab instructor will collect Tubes 1 and 2 and place them in
a freezer (-5oC) and refrigerator (5oC) respectively, for 15 minutes.
• Obtain hot water from the bath at the side bench (caution: water is very hot) and mix
in cold tap water (stirring with the stir stick, not the thermometer) until the
temperature is 70oC. Select a third beet cylinder from the wash beaker and place it in
the 70oC water for exactly 3 minutes. Then transfer it to Tube 5 and cover with 6 mL
of distilled water at room temperature for 15 minutes.
• Cool the beaker of water to 45oC by adding more tap water. Immerse a fourth beet
cylinder for 3 minutes, place it in Tube 4 and add 6 mL of distilled water; let stand for
15 minutes.
• Cool the water to 25oC and repeat the procedure for tube 3 with the final beet cylinder.
• After 15 minutes of cold treatment, add 6 mL of distilled water to Tubes 1 and 2; let
stand for 15 minutes.
• After all beet cylinders have soaked for 15 minutes (no more – each cylinder will have
to be timed on its own), remove them from the tubes. Use a dissecting needle to spear
the cylinders and remove them quickly.
• Arrange the tubes from palest to darkest in colour. Then quantify the relative colour
of the solutions, beginning with the palest and ending with the darkest (refer to
Appendix D for information regarding use of a spectrophotometer). You will be
readingthe Absorbance at 460 nm. In order to set “100” on the percent transmittance
scale, use the test tube containing the distilled water (the reagent blank) which should
be located next to the spectrophotometer. The light absorbance is a direct measure of
the concentration of betacyanin and an indirect measure of membrane damage.
• Record absorbance readings in Table 1.
• Rinse all test tubes and place in the baskets provided.
A standard curve is used to determine the concentration of a substance. It is prepared by
assaying various known concentrations of the substance you are trying to measure. In our
case, we have measured the absorbance of known concentrations of betacyanin to make our
standard curve (Figure 5).
5. Use the standard curve provided to determine the concentration of betacyanin that leaked
from the beet cells for each SDS and temperature treatment (your instructor will
demonstrate how to use the standard curve). Record these values in the appropriate
column in Table 1 and on the black board.
6. Record your group’s betacyanin concentrations for the various SDS or temperature
treatments in the table on the black board. Copy all of the class data into the appropriate
spaces in Tables 2 and 3. Use this data to calculate the means and standard deviations of
betacyanin concentration for each SDS and temperature treatment (your instructor will
provide you with formulae).
Betacyanin Absorbance (460 nm)
20
Betacyanin Concentration (µM)
Figure 4: Standard Curve for known betacyanin concentrations.
Table 1. The absorbance (A460) and concentration of betacyanin leaked from beet (Beta vulgaris) cells
following different SDS and temperature treatments.
Tube #
Temperature
Treatment
(°C)
0
1
-5
2
0.025
2
5
3
0.05
3
25
4
0.25
4
45
5
0.50
5
70
Tube #
%SDS
1
A460
Concentration (µM)
A460
Concentration (µM)
21
Table 2. Class data for the concentrations of betacyanin leaked from beet (Beta vulgaris) cells
following different SDS treatments as determined using spectrophotometry and a betacyanin standard
curve.
Tube #
%SDS
1
0
2
0.025
3
0.05
4
0.25
5
0.50
Class Betacyanin Concentrations (µM)
Mean
+
s.d.
Table 3. Class data for the concentrations of betacyanin leaked from beet (Beta vulgaris) cells
following different temperature treatments as determined using spectrophotometry and a betacyanin
standard curve.
Tube #
Temp
Trmt
(°C)
1
-5
2
5
3
25
4
45
5
70
Class Betacyanin Concentrations (µM)
Mean
+
s.d.
22
Use the means and standard deviations from Table 2 to complete a scientific graph that
demonstrates the relationship between the concentration of betacyanin released and the %SDS
treatment.
•
Compare your results to your hypothesis for the experiment. Are your results as
expected? Explain.
•
Based on the class results, what SDS concentration(s) were the most damaging to the beet
cell membranes? Explain .
•
Based on the class results, what SDS concentration(s) had little or no effect on the beet
cell membranes? Explain.
23
Use the means and standard deviations from Table 3 to complete a scientific graph that
demonstrates the relationship between the concentration of betacyanin released and the
temperature treatment.
•
Compare your results to your hypothesis for the experiment. Are your results as
expected? Explain.
•
Based on the class results, what temperature(s) were the most damaging to the beet cell
membranes? Explain.
•
Based on the class results, what temperature(s) had little or no effect on the beet cell
membranes? Explain.
24
Thought Questions:
1. You probably noticed that there was some variability in the absorbance values that were
obtained by different groups of students for the same experiment. What factors would
affect the readings obtained from the spectrophotometers? (All machines are calibrated in
the same manner and in good working order, so instrument error will not be an important
factor.)
2. What is the advantage of using a spectrophotometer rather than your eyesight to measure
color intensity?
3. In 1925, Gorter and Grendel obtained lipid from cell membranes by bursting and
removing the contents of red blood cells. They spread the lipid in a layer one molecule
thick on the surface of a tray of water. The area covered by this lipid layer was twice as
large as the surface area they had calculated for the original cells. Can you explain the
discrepancy?
4. You probably noted damage to membranes at both low and high temperatures. Is the
mechanism of damage the same? If not, explain each.
25
5. After looking at Figure 5 below, explain in words how you think a detergent act on the
molecules in the membrane.
6. In the experiment on the effect of temperature on beet membranes, name:
a) the independent variable
b) the dependent variable
7. In the experiment on the effect of SDS concentration on beet membranes, name:
a) the dependent variable
b) a controlled variable
Literature Cited
Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San
Francisco, CA.
26
ENZYMES
Introduction:
Without enzymes, most biochemical reactions would take place at a rate far too slow to
keep pace with the metabolic needs and other life functions of organisms. Enzymes are
catalysts that speed up chemical reactions but are not themselves consumed or changed
by the reaction.
The cell’s catalysts are proteins. As with all proteins, enzymes have a primary structure (a
unique sequence of amino acids), a secondary structure (a folding or coiling of the chain), and
a tertiary structure (a folding of the molecule into a complex three-dimensional structure). A
combination of weak hydrogen bonds and stronger covalent bonds maintain protein structure.
Enzymes speed up chemical reactions by lowering the activation energy required to complete
the reaction; they are not consumed or changed by the reaction. An enzyme reacts with a
substance known as a substrate to form a transient stage called the enzyme-substrate
complex (Figure 1). The enzyme has a specific three-dimensional site, the active site, into
which only one kind of substrate fits. Thus the enzyme and substrate relationship is very
specific. The substrate remains in the active site for only a very short time before the product
or products are released, whereupon another substrate molecule takes its place.
Figure 1. An enzyme and substrate interaction resulting in the formation of a product.
Changes in temperature, alterations in pH, the addition of ions or molecules, and the presence
of inhibitors all may affect the structure of an enzyme’s active site and thus the activity of the
enzyme and the amount of product which results. Enzymatic activity can also be affected by
the relative concentrations of enzyme and substrate in the reaction mixture.
During the lab period today, you will investigate how changes in substrate concentration and
temperature affect the enzymatic activity of catechol oxidase. You are familiar with the
activity of this enzyme if you have ever seen brown spots or “bruises” on fruits and
vegetables. The browning reaction is the result of the oxidation of phenolic compounds such
as catechol to quinones, which subsequently polymerize into complex branched chains.
Quinones are toxic to microorganisms and thus aid in the prevention of infection at a wound
site. In normal undamaged cells, the catechol substrate molecules are maintained apart from
the oxidizing enzyme molecules (catechol oxidase) by being sequestered in different parts or
components of the cell. Following damage, the reaction proceeds as shown in Figure 2.
27
Figure 2. The reaction between the substrate (catechol) and enzyme (catechol oxidase), to
form the product (benzoquinone).
The objectives of today’s lab are to:
• provide an introduction to enzyme structure and function
• determine the effect of temperature and substrate concentration on enzyme activity
• provide practice collecting, representing and interpreting data.
As background for this lab, you will find it useful to read pages 77 - 85 and 150-157 in
Campbell and Reece (2005).
A. Effect of Substrate Availability on Enzyme Activity
In the presence of a large quantity of enzyme, the amount of product formed in a given period
of time depends on the availability of substrate molecules. However, as more and more
substrate is made available, at some point the initial amount of enzyme becomes limiting; that
is, all the enzyme molecules are participating in reactions, and no matter how much more
substrate is available, the amount of product formed in a given time period is the same.
•
Based on the information provided to you in the lab manual and in text readings, prepare a
hypothesis for the experiment investigating the effect of catechol availability on catechol
oxidase.
Procedure:
Work in pairs to complete this part of the exercise.
1.
2.
3.
4.
Label six test tubes in this sequence: 1, 2, 4, 8, 16 and 24.
Using the pipet and pipet pump provided, add 5 ml of pH 7 buffer to each of the tubes.
Add 1, 2, 4, 8, 16 and 24 drops of catechol to each of the appropriately labeled tubes.
Add 23, 22, 20, 16, 8 and 0 drops of pH 7 buffer to the six tubes respectively to make all
volumes of solution in the test tubes equal.
5. Add 30 drops of potato juice extract to each of the six tubes; shake to mix. The “juice”
contains the enzyme catechol oxidase and is obtained by homogenizing potatoes in a highspeed blender.
6. Maintain the tubes at room temperature for 5 minutes, shaking each tube briefly at 1minute intervals. Don’t shake so hard that froth builds up at the top!
28
7. The yield of the enzyme-catalyzed reaction (i.e., the amount of product formed) will be
proportional to the intensity of colour developed in each reaction mixture. After zeroing
the spectrophotometer (Appendix D), record the intensity of colour of each solution after 5
minutes. Line up your six test tubes from palest to darkest in colour and pour each one in
turn into the special spectrophotometer tube to read absorbance. Shake out residual liquid
from the special test tube after each solution is measured; however it isn’t necessary to
rinse the tube. In the instrument, light is absorbed by the benzoquinone molecules.
Record your results and class results on Table 1.
•
Why is it important to measure the absorbance of the palest liquid first, then the
second palest, and so on?
Table 1. Class absorbance (A460) values for the substrate availability experiment.
Drops of
Substrate
Benzoquinone Absorbance (A460)
Bench 1
Bench 2
Bench 3
Bench 4
Bench 5
Mean +
s.d.
1
2
4
8
16
24
8. Work as a group to calculate the means and standard deviations for the different substrate
treatments and place these in the appropriate spaces in Table 1.
9. Prepare a completely labeled graph (on the graph grid provided) that demonstrates the
effect of substrate availability on the production of benzoquinone by the enzyme catechol
oxidase.
29
•
Describe the trend (pattern) shown by the data.
•
Which treatment(s) displayed maximal product formation?
•
Do the results support your original hypothesis regarding substrate concentration and
enzyme activity? Explain.
30
B. The Effect of Temperature on Catechol Oxidase
As temperature increases, so does the movement of molecules. The rate of collisions between
reactants (substrate and enzyme molecules) increases, and hence, the amount of product formed
increases. Eventually however, the temperature reaches a point at which the weakest bonds
maintaining the secondary and tertiary structure of the enzyme protein molecule are ruptured.
The enzyme loses its catalytic function and is said to be denatured.
Procedure:
Work as a pair of students to complete the following exercise.
1. Label six test tubes with the temperature treatments to be used (3oC, 12oC, 20oC, 35oC,
50oC and 70oC) and add 3 mL of pH 7 buffer to each.
2. Place the test tubes in their respective water baths; set the 20oC tube on your bench. Allow
15 minutes for the buffer in each tube to temperature-equilibrate.
3. Add 10 drops of potato juice (containing the catechol oxidase enzyme) to each tube,
without removing them from the baths, followed by 10 drops of catechol to each. Don’t
forget to add potato juice and catechol to the tube at room temperature as well. Shake
each tube, and allow them to incubate at their respective temperatures for 5 minutes,
briefly shaking each at 1-minute intervals.
4. Move all of the test tubes (including the room temperature treatment) to the 3°C water
bath and organize them from palest to darkest. Immediately remove the palest solution
from the 3oC bath, pour it into the special spectrophotmeter test tube and read absorbance
(see Appendix D for a review of spectrophotometer use). Do likewise with the other
tubes, removing them one at a time from the cold bath, and ending with the darkest
solution. The amount of product produced during a given period of time (the “yield) will
be proportional to the intensity of colour developed in each reaction mixture. Record your
results and class results on Table 2.
•
Why did you move all tubes to the 3oC bath after their respective incubations?
5. Calculate the means and standard deviation for the different substrate treatments and place
these in the appropriate spaces in Table 2.
6. Prepare a completely labeled graph on the graph grid provided to demonstrate the effect
of temperature on the production of benzoquinone by the enzyme catechol oxidase.
31
Table 2. Class absorbance (A460) values for the temperature experiment.
Temperature
(oC)
3
12
20
35
50
70
Benzoquinone Absorbance (A460)
Bench 1
Bench 2
Bench 3
Bench 4
Bench 5
Mean
+ s.d.
32
•
Describe the trend (pattern) shown by the data.
•
Do the temperature experiment results support your hypothesis? Explain.
33
Thought Questions:
1. At low levels of substrate availability, what limits the yield of an enzymatic reaction? At
high levels of substrate availability, what limits the yield? At what point does the change
take place?
2. Enzymes that lose their three-dimensional structure cannot interact with substrate
molecules and thus lose their catalytic function; they are said to be denatured. The
activity of catechol oxidase decreases with both high and low temperature. Does this
mean that denaturation is occurring at both instances? Explain.
3. If you wish to preserve vegetables in the freezer, you must first boil them for a few
minutes before packaging them. Why is this boiling step necessary? Wouldn’t the low
temperature alone be sufficient to preserve the taste and appearance of the vegetables?
4. Is there an optimum temperature for catechol oxidase? Would all enzymes have the same
optimal temperature? Explain why or why not.
34
5. How would your results differ if you had not shaken the tubes periodically during
incubation?
Literature Cited:
Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San
Francisco, CA.
35
BACTERIOLOGY PART I:
GENERAL LABORATORY PROCEDURES, BIOSAFETY and
MORPHOLOGY
Introduction:
Up to this point, we have limited our examination of cell structure and function strictly to
eukaryotic cells. In the next two labs we will focus our attention on prokaryotic cells.
Prokaryotic cells differ from eukaryotic cells in several important features. Firstly, prokaryotic
DNA is not packaged in a membrane bound nucleus but rather found in a region inside the cell
called the nucleoid. The DNA in bacterial cells is primarily a single, circular chromosome
whereas eukaryotic nuclear DNA is organized into discrete, linear chromosomes. The internal
organization of prokaryotic cells lacks the elaborate membrane systems (e.g., endomembrane)
and structures (e.g.: mitochondria and chloroplasts) that are typical of eukaryotic cells. Lastly,
like plant cells, bacterial cells also have a cell wall; however, the composition of the bacterial
cell wall is unique and contains the molecule peptidoglycan (a polymer of amino sugars).
A primary feature of the microbiology laboratory is that living organisms are employed
as part of the experiment. Most of the microorganisms are harmless; however, whether
they are non-pathogenic or pathogenic (capable of causing disease), the microorganisms
are treated with the same respect to assure that personal safety in the laboratory is
maintained. Careful attention to technique is essential at all times. Care must always be
taken to prevent the contamination of the environment from the cultures used in the
exercises and to prevent the possibility of the people working in the laboratory from
becoming contaminated. This care is referred to as aseptic technique.
You will learn the appropriate procedures for manipulating bacterial cultures and will use
these procedures to perform some basic microbiological techniques in the first part of this
exercise. The second half of the exercise makes use of stains as well as well as immersion
oil in order to view and make observations of bacterial morphology.
The objectives of today’s exercise are thus to:
• become familiar with using aseptic techniques to handle microorganisms.
• gain experience in handling microorganisms by using fluorescein dye-labeled
E. coli cultures to perform a series of exercises
• illustrate the potential for contamination that is always present when working
with microorganisms
• learn basic microbiological techniques such as streaking for single colonies
and inoculation of liquid cultures
• provide an introduction to the Gram stain and allow you to practice using this
differential stain
• use light microscopy and oil immersion to assess bacterial characteristics such as size,
shape, and association
• encourage proper representation of bacteria using scientific drawings
36
As background for this lab, please read pages 534-535 in Campbell and Reece (2005). Please
ensure that you have read over the guidelines on Safety (pages v-vi), and those on aseptic
technique (Appendix D) prior to attending your lab. As well, you should become familiar
with the contents of the University of Lethbridge Biosafety web site:
http://www.uleth.ca/fas/bio/safety/biosafety.html
A. Introduction to Handling Microbes and Aseptic Techniques
Procedure:
Wear gloves and a lab coat for the entire exercise.
1. Tape bench coat onto the bench to cover your working surface.
2. Work individually over the bench coat and prepare a streak plate for single
colonies using the following techniques:
• Transfer (aseptically) a loop of the fluorescein dye-labeled E. coli culture
provided to a sterile plate in the area shown by Figure 1a.
• Once the first set of streaks has been made, flame the inoculating loop until red
hot. Do not reintroduce the loop into the original culture.
• Cool the loop by holding it in the region around the Bunsen burner flame for a
few moments, and then make a second set of streaks as shown in Figure 1b, only
crossing over the initial set of streaks once.
• Flame the loop again, cool, and make a third set of streaks as shown in Figure 1c.
Note, try not to gouge the agar while streaking the plate.
Figure 1: Procedure for inoculating a streak plate.
Figure 1a
Figure 1b
Figure 1c
37
Label your plate and place it in the tray provided with the agar side up.
Labeling plates of media:
• Label on the agar side on the Petri dish itself (not on a piece of tape)
• Label close to the edge of the dish following the edge
• Do NOT write across the plate in big letters!
3. From the same fluorescein dye-labeled E. coli culture, inoculate one tube of
nutrient broth using the following procedure. Again, the important thing to
remember is that exposure of sterile liquids or bacterial cultures to air must be
minimized.
• Ensure that you have the tube of inoculum (microbe used for setting up a culture),
inoculating loop and a sterile tube of medium available within easy reach.
• Flame the inoculating loop until red-hot. Remove the cap from the tube of
inoculum by grasping the cap between the last finger and the hand that is also
holding the inoculating needle (Figure 2). Do not place the cap on the bench!!
Figure 2: Technique for manipulating test tubes aseptically.
• Flame the mouth of the tube by passing it rapidly through the Bunsen burner 2-3
times. This sterilizes the air in and immediately around the mouth of the tube.
• Cool the loop on the inside of the tube, and then remove a loopful of the
•
•
•
•
inoculum..
Reflame the mouth of the tube and replace the cap.
Remove the cap for the tube of sterile medium in the same way, and gently place
the inoculating loop into the medium.
Flame the mouth of the tube, and replace the cap, and then flame the inoculating
loop before replacing it on the bench.
Note, when removing inoculum from a plate, cool the loop before picking up the
bacteria.
38
Work in pairs to complete steps 4 – 11.
4. Place a watch glass in the centre of the bench coat.
5. Obtain and label one Nutrient Agar (NA) plate with your names, date, organism
tested and distance from the watch glass).
6. Place the agar plate on one side of the glass plate, either 5 cm or 10 cm from the
watch glass (consult with the other pair at your bench to ensure that both distances
are tested). Remove the lid from the plate and set aside (off the bench coat).
7. Using a pipette pump, and aseptic technique, draw up 1 mL of bacteria/fluorescein
suspension.
8. Hold pipette tip 30 cm from glass plate and allow 10 drops to fall (one drop at a
time) onto the glass plate. Put any remaining bacterial culture and the pipette into
the disinfectant tray provided (be careful as this contains 10% bleach).
9. Put the glass plate into the disinfectant tray provided and place the cover back on
the agar plate. Place on a tray on the side bench.
10. Use the hand-held UV lamps to inspect your bench coat, gloves, and lab coat.
Caution: UV rays are harmful to skin and eyes. Ensure you wear proper eye
protection.
• What do you observe?
• What can you conclude about your techniques for handling microorganisms?
How can you improve?
11. Your plates will be incubated for 16-20 hours at 37oC, and then refrigerated at
4oC. During the next laboratory period, count and record the number of colonies
present (use Table 1).
Table 1. Results from falling drop exercise
Distance of plate from watch
glass (cm)
5
10
Number of Colonies
• How far can droplets containing bacteria spray when a liquid culture is
dropped?
39
B. Microscopic Characteristics of Individual Cells
The small size of bacteria leads to a series of challenges involved with their study. Because
living bacteria are almost colorless, they do not show enough contrast with the medium in
which they live to be seen clearly under the microscope. Bacteria have a marked affinity for
certain dyes, and when treated with these dyes they become readily visible. Their affinity for
dyes is based on the fact that the cell wall has a net negative charge and therefore will attract
positively-charged stain molecules.
Observation of bacteria also requires a careful adjustment of the amount of light striking the
specimen to increase the contrast between the cell and its background. Today, you will learn
a special technique with the light microscope called oil immersion, which enables you to
maximize both the magnifying power and the resolution of the light microscope in order to
view very small objects.
Work as a group of four students to complete the following exercise. You should review
Appendix A (microscopy) prior to attending the lab.
1. Obtain a prepared slide of bacteria from one of the trays. Place the slide on the stage of
the microscope, and focus with the 4X objective lens in place.
2. Once objects come into focus, switch to the 10X and then 40X objective. Remember that
only the fine focus adjustment knob is required to bring the image into sharp focus when
the higher power lenses are used.
3. Rotate the 40X objective away from the slide in preparation for using the 100X lens.
Before clicking the 100X lens into position, add a single, small drop of immersion oil
directly onto the slide surface at the spot where the light from the condenser is shining
through.
4. Now rotate the 100X objective lens into place. Do not lower the stage; although it may
appear that there is insufficient room for the 100X lens. Watch carefully from the side to
ensure the lens slides into position. This lens will be sitting in the immersion oil.
5. Open the iris diaphragm as wide as possible to let more light into the field. Using only
slight adjustments of the fine focus adjustment knob, bring the image into clear focus. Do
not return the 40X lens back into position once you have immersion oil on your slide.
6. Note the shape of the bacteria. Typically bacteria come in three shapes, coccus
(spherical), bacillus (rod-shaped), and spirillum (spiral-shaped) (Figure 3).
40
Figure 3. Examples of different bacterial shapes and associations commonly observed in
culture.
7. Use the ocular micrometer (see Appendix C) to determine the size of the bacteria in your
field of view. Typically it is the length of the bacteria that is used to indicate size.
8. Note the association of cells on your slide. The bacterial association refers to the
relationship of different cells of the same species. Bacteria may occur singly or in pairs,
chains or clusters (Figure 1).
9. Note whether your bacterial specimen has flagella. Some bacteria, but not all, have
flagella that aid in locomotion.
10. Use the space below to make a proper scientific drawing of one of the four types of
bacterial cells (coccus, bacillus-type, Spirillum volutans, and Proteus vulgaris) that are
available for examination. Your drawing should depict the shape and association of the
bacteria, while the drawing magnification will provide some insight on their actual size.
41
C. Gram Staining
The Gram stain is a routine differential staining technique that aids in bacterial identification.
A differential stain can be used to chemically distinguish between bacterial species that may
be morphologically indistinguishable. Bacteria are generally divided into two groups, Gram
positive and Gram negative, depending on their reaction to the staining procedure. The
response of the cells to the stain is due to differences in the complexity and chemistry of the
bacterial cell wall (Table 1).
In the following lab exercise, you will prepare and stain two bacterial species to determine
their size, shape, association, and Gram reaction.
Table 1. Characteristics of Gram negative and Gram positive bacteria.
Characteristic
Bacteria Type
Gram negative
Gram positive
Cell wall complexity
More complex
Relatively simple
Peptidoglycan content
Thin layer
Thick layer
Outer lipopolysaccharide wall layer Present
Absent
Stain displayed by cell
Crystal violet
Safranin
Cell color following Gram staining Red / pink
Dark purple
Work as a pair of students to complete the following exercise.
1. Wash your hands and disinfect the bench top using bleach.
2. Obtain broth cultures of Escherichia coli and Bacillus subtilis and label them using the
tape provided.
3. Write the name of the bacterium to be stained at one end of the slide.
4. Flame an inoculating loop and keep it in your hand while it cools.
5. While the loop is cooling, open the tube of inoculum as you did in Part A.
6. Remove a loop full of culture; reflame the lip of the tube and replace the cap.
7. Spread the culture toward one side of your slide – the size of the smear should be at least
the size of a dime.
42
8. Reflame the loop and set it aside.
9. Let the smear air-dry. You may use a warming plate or bench lamp if available.
10. The cells, although dry, will wash away when you apply liquid stain unless you first heat
fix the cells. Pass your slide five times through the flame to attach cells to the slide.
11. Place your slide on a paper towel, add one drop of crystal violet to the smear, and let the
preparation stand for 30 - 60 seconds.
12. Hold your slide at the edges and gently rinse the slide with tap water for about 5 seconds.
Be careful – crystal violet stains your fingers as well as the bacteria! (At this stage of the
Gram stain, all bacteria will be colored purple regardless of their cell wall nature.)
13. Cover your smear with two drops of Gram’s iodine solution and let the preparations stand
for 30 seconds.
14. Gently rinse the slide with water.
15. Holding the slide at an angle over the sink, gently trickle 95% ethanol (EtOH) onto the
smear until very little dye washes off. Immediately gently rinse the preparations with
water to prevent further destaining.
16. Apply one drop of safranin to your slide and let stand for 30-60 seconds.
17. Gently rinse the slide with water and remove any excess by gently tapping the side of the
slide against a paper towel. Let both preparations air dry.
18. Observe your slide starting with the scanning objective (4X) and ending with the oil
immersion lens (100X) of your light microscope.
19. Note the size (using ocular micrometry), shape, association, and Gram reaction of each
type of bacteria in Table 2.
20. Tidy the bench and properly store the microscope.
•
Are the species Gram positive or Gram negative? How do you know?
Table 2. Gram characteristic, size, shape and association of two bacterial species, B. subtilis,
and E. coli, as determined following Gram staining and observation using a light
microscope.
Bacterium
Bacillus subtilis
Escherichia coli
Gram
Reaction
Cell Size (µm)
Cell Shape
Association
43
Thought Questions:
(Use the University of Lethbridge Biosafety web site:
http://www.uleth.ca/fas/bio/safety/biosafety.html to help with answering questions 15)
1. What is an MSDS and where can you find one?
2. In Canada, the Laboratory Centre for Disease Control has classified infectious
agents into 4 Risk Groups using pathogenicity, virulence and mode of
transmission (among others) as criteria. What do these terms mean?
3. Lab facilities themselves are classified according to containment level. What
does this mean? Provide 2 characteristics for containment levels 1, 2, 3, and 4.
What containment level is your classroom?
4. What criteria would characterize an organism classified in Risk Group 1, 2 3 or 4?
Provide an example of an organism found within each group.
44
5. There are many “Golden Rules” for Biosafety. Identify 4 common sense practices
that will protect you in your microbiology labs.
6. a) What ultrastructural information about bacterial cells is revealed by the Gram stain?
b) Based on the Gram stain results, which bacteria were Gram positive?
c) Based on the Gram stain results, which bacteria were Gram negative?
7. List the four reagents used in the Gram stain and their respective purposes.
Gram Reagent
Purpose
Literature Cited:
Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San
Francisco, CA.
45
BACTERIOLOGY PART II:
ANTIBIOTIC-RESISTANT BACTERIA
Introduction:
In your lab last week you were introduced to general morphological characteristics of bacteria,
and you also learned ways to manipulate them safely. In this exercise we are going to
continue our studies of prokaryotes, but instead of concentrating on morphology, we are going
to delve into aspects of their physiology and metabolism.
Bacteria are adapted to survive in very diverse environments in which few other organisms
could survive, including those with extremely high or low temperatures, or high amounts of
acids or salts. Because of this tremendous adaptability, bacteria dominate the biosphere,
making up at least 90% of the biomass on earth. They play a variety of roles in the natural
world, but also have clear and direct impacts on human society as well. We have taken
advantage of their genetic diversity (and hence, metabolic diversity) in a number of different
ways, including bioremediation (the detoxification of polluted ecosystems), food production,
mining, and more recently, have gained the ability to genetically modify them to overproduce
compounds beneficial to human health. However, they also have negative impacts on our
society. It is thought that bacteria are responsible for about 50% of all human diseases,
including tuberculosis, cholera, anthrax, botulism and hamburger disease (caused by E. coli
strain O157:H7).
Currently, one of the most important issues society faces is the increase in the populations of
microbes that have evolved resistance to antibiotics. This has led to our increasing inability to
treat bacterial infections. Antibiotic resistance may arise naturally in bacteria, such that an
antibiotic becomes ineffective in reducing numbers of bacteria of a particular species.
Exposure of a population of bacteria to a given antibiotic will kill those that are sensitive to
the antibiotic, but not those which have evolved resistance. Our society’s indiscriminate use
of antibiotics further exacerbates this problem. Furthermore, genes encoding resistance to
antibiotics are often located on plasmids; circular, double-stranded, replicable fragments of
DNA that can be easily transferred to other bacterial cells, hastening the rate at which
bacterial populations become resistant.
Proliferation of antibiotic resistant pathogenic bacteria in our food supply over the past
several years has also occurred. Antibiotic sprays are often applied to crop plants which
encourages the proliferation of resistant microbes associated with crops and soil. The use of
antibiotics in the livestock industry may lead to increased bacterial resistance in these animals,
and these bacteria become integrated into soil via fecal deposition.
The objectives of this exercise are thus to:
• determine typical numbers of bacteria found on a common food source
• determine what proportion of these bacteria are antibiotic resistant
• apply the principles of the scientific method to analyze and interpret data collected
46
Please read pages 351 and 545-547 in Campbell and Reece (2005) in preparation for this
exercise.
Week 1 Procedure:
Please work in pairs to complete this exercise. Practice aseptic techniques when handling all
lab materials.
1. One pair at the bench will test an unwashed vegetable sample; the other will wash their
vegetable material first, and then follow the same procedures as for the unwashed
material. Pairs responsible for washing vegetables should follow the directions provided
by the container of vegetable wash at the side bench. Ensure that the pair working with
unwashed material begins the exercise first!
2. Preparation of dilution series:
• Collect four test tubes and four, 1mL pipettes from the side bench, and use the tape
provided to label them with the name of the vegetable used and the dilution (10-1, 10-2,
10-3 or 10-4).
• Weigh out 1 gram of vegetable tissue using the balance on the side bench. To obtain
your material, use a razor blade to scrape off shavings from the outer surface of the
vegetable material into a plastic weigh boat.
• Use the spatula on your bench to transfer the shavings into the tube labeled 10-1. Add
10 mL of sterile water using the 10 mL pipette and pump provided (your instructor
will show you how to use the pump). This represents a 1:10 dilution; in other words, 1
gram of tissue is now suspended in a total volume of 10 mL (or 0.1 grams of tissue per
mL of water).
• Vortex the contents of the tube vigorously for 30 seconds.
• Use the 1 mL pipette labeled “10-1” to remove 1 mL of the solution from your 10-1
tube. Add it to your tube labeled 10-2. Add 9 mL of sterile water, and mix vigorously.
This represents another 1:10 dilution. The 0.1 g of tissue transferred in the 1 mL from
the 10-1 tube is now suspended in a total volume of 10 mL (or, 0.01 grams of tissue per
mL of water)
• Remove 1 mL of the solution from the second dilution tube (10-2) using the
appropriately labeled pipette, and place it into the tube labeled 10-3. Add 9 mL of
sterile water, and vortex vigorously.
• Remove 1 mL of the solution from the third dilution tube (10-3) using the appropriately
labeled pipette, and place it into the tube labeled 10-4. Add 9 mL of sterile water, and
vortex vigorously.
• What is the final dilution achieved? It may be easier to determine this by
calculating how many mg of tissue are present per mL of water in the 10-4
dilution.
3. Plating:
• Your instructor will demonstrate the plating procedure for you using sterile water to
which vegetable material has not been added. This plate will act as a control.
47
• What is a control? Why was it necessary to incorporate a control into this
procedure?
•
•
•
•
•
•
•
Collect three Nutrient Agar (NA) plates from the side bench and label them with your
name, lab number, vegetable (indicate washed or unwashed), and the date. You will
plate only your 10-2, 10-3 and 10-4 dilutions.
Collect two Nutrient Agar + Kanamycin (NA + KAN) plates from the side bench, and
label them as above. You will plate only your 10-2 and 10-3 dilutions.
Use the 1 mL pipette labeled 10-2 to remove 0.1 mL of solution from your 10-2 tube.
Remove the lid from the agar plate you labeled “10-2”, and add your 0.1 mL aliquot to
the agar side.
Remove a sterile glass spreader from the beaker at your bench, and use it to spread the
drop of solution over the entire surface of your agar plate. Replace the lid on your
Petri dish, and place the used spreader in the appropriately labeled beaker on the side
bench.
Repeat this procedure four more times, using a new sterile spreader each time.
Once your five plates are prepared, cut a thin strip of Parafilm and wrap each of the
plates.
Place all five of your plates in an inverted position (agar side up) on the tray provided.
Plates will be incubated at room temperature for 48 hours, and then will be stored at
4oC until your next lab period.
Week 2 Procedure:
4. Determination of total number of bacteria:
• Collect your three NA plates from the tray at the side bench. Examine them for the
presence of colonies. When examining plates, look for those that have between 30 and
300 colonies present. Any plates with fewer than 30 or more than 300 colonies are not
useful from a statistical perspective, as there is more experimental error associated
with these.
• Count the number of colonies on the plate with between 30 and 300 colonies, and
record the number of colonies and from which dilution they were obtained in Table 1
in your lab manual.
48
Table 1: Class data for the number of colonies growing on Nutrient Agar (NA) plates
following the inoculation of 100 µL of a series of dilutions of unwashed and washed vegetable
tissue and subsequent incubation at room temperature for 48 hours.
Bench Number
Number of colonies on Amount of Dilution Total bacteria
plate (between
extract
plated
per gram of
30-300)
plated (µL)
tissue
1: unwashed
100 µL
2: unwashed
100 µL
3: unwashed
100 µL
4: unwashed
100 µL
5: unwashed
100 µL
1: washed
100 µL
2: washed
100 µL
3: washed
100 µL
4: washed
100 µL
5: washed
100 µL
Control (water)
100 µL
•
Calculate the total number of bacteria per gram of original vegetable material.
Remember that each colony is thought to represent progeny from one original bacterial
cell. Record numbers in Table 1, and in the Table on the board. Use the equation
below as a guide.
Number of colonies
Dilution
*
x 10* =
total number of bacteria per gram of
vegetable tissue
multiplying by 10 corrects for plating only 0.1 mL of your dilution onto your
NA plate, and brings the number of bacteria up to the number of bacteria per
mL of the dilution.
49
•
Prepare a bar graph showing the mean (+/- s.d.) total number of bacteria per gram of
vegetable material versus the condition of the vegetable (washed and unwashed).
•
What conclusion(s) can you draw from these results with respect to numbers of
bacteria found on unwashed and washed vegetable material?
5. Determination of the number of bacteria resistant to kanamycin:
• Collect your two NA+KAN plates from the tray at the side bench. Examine them for
the presence of colonies. Again, look for the plate that has between 30 and 300
colonies present, and count the number of colonies. Record the number of colonies
and from which dilution they were obtained in the appropriate part of Table 2.
• Calculate the total number of antibiotic resistant bacteria per gram of vegetable
material. Record your numbers in Table 2, and in the Table on the board.
50
Table 2: Number of colonies growing on Nutrient Agar plus Kanamycin (NA+KAN) plates
following the inoculation of 100 µL of a series of dilutions of unwashed and washed vegetable
tissue and subsequent incubation at room temperature for 48 hours.
Bench Number
Number of colonies on Amount of Dilution Total bacteria
plate (between
extract
plated
per gram of
30-300)
plated (µL)
tissue
1: unwashed
100 µL
2: unwashed
100 µL
3: unwashed
100 µL
4: unwashed
100 µL
5: unwashed
100 µL
1: washed
100 µL
2: washed
100 µL
3: washed
100 µL
4: washed
100 µL
5: washed
100 µL
Control (water)
100 µL
•
Calculate means for total numbers of bacteria per gram of tissue from Tables 1 and 2.
Record the mean values in Table 3, and use these values to calculate the percentage of
antibiotic resistant bacteria found on your vegetable tissue.
51
Table 3: Percentage of kanamycin-resistant bacteria found on washed and unwashed
vegetables.
Vegetable tissue
Mean total number of Mean total number Percentage of
kanamycin-resistant
of bacteria/gram of kanamycinbacteria/gram of tissue tissue
resistant bacteria
Unwashed:
Washed:
Control (water)
•
Prepare a second bar graph, but this time plot the mean total number of bacteria and
the percentage of resistant bacteria versus the condition of the vegetable tissue
(washed or unwashed).
• What conclusions can you draw with respect to this figure?
52
Literature Cited:
Brock, D., Boeke, C., Josowitz, R. and Loya, K. 2004. Stalking Antibiotic Resistant Bacteria
in Common Vegetables. The American Biology Teacher, 66 (8) pp. 554-559.
Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San
Francisco, CA.
53
FERMENTATION AND THE SCIENTIFIC METHOD
Introduction:
Many metabolic reactions that occur in cells do not happen spontaneously, but instead
require a source of chemical energy in the form of adenosine triphosphate (ATP).
Typically cells manufacture ATP by oxidizing glucose using a series of enzymecatalyzed reactions. The oxidation of glucose takes place in two distinct stages:
[i] glycolysis, where one molecule of glucose is converted to two molecules of
pyruvic acid, a small amount of ATP and NADH. Glycolysis occurs in the
cytoplasm of all living cells, and does not require oxygen to be present.
[ii] aerobic cellular respiration, which consists of the Krebs Cycle and electron
transport. These reactions occur in the mitochondria of cells, and oxygen is
required as the final electron acceptor. Aerobic cellular respiration produces many
more ATP’s per molecule of glucose than does glycolysis alone.
If oxygen is not available or is not able to be utilized, then glycolysis becomes the major
source of ATP production. In some organisms, like the yeast cells you will use in your
experiment today, pyruvic acid is metabolized by another set of reactions termed
fermentation. Fermentation results in the production of alcohol and carbon dioxide, but
more importantly, oxidizes NADH back to NAD, enabling glycolysis, and hence, energy
production, to continue. The fermentation reaction may be summarized as follows:
C6H12O6 (sugar)
C2H5OH (ethanol) + 2 CO2 + energy
Yeasts are single-celled eukaryotic organisms related to molds and mildews. Under
anaerobic conditions, yeasts break down sugars, releasing ethanol and carbon dioxide
gas. Baker’s yeast (Saccharomyces bayanus) is a readily available organism that can be
manipulated easily within a laboratory setting. We will use it in this exercise to explore
what variables affect the process of fermentation. However, unlike other exercises you
have completed thus far, it will be up to you to design and carry out an experiment
illustrating some aspect of fermentation in yeast. You will use today’s period to think
about what variable you want to test, to develop a hypothesis, and to design an
experiment. Next week, you will carry out your experiment and collect and analyze your
data.
Thus, the objectives of this exercise are to:
• Develop an understanding of fermentation and the generation of cellular energy
• Allow students to gain experience in experimental design using a yeast system
• Illustrate the Scientific Method through a hands-on approach
Please read pages 160-162, 165-167 and 174-176 in Campbell and Reece (2005) and
review Appendix F prior to attending your lab.
54
Week 1: Experimental Design
Students will work in groups of four to design and perform an experiment.
Your experimental design will be constrained somewhat by what materials we will be
able to provide for you. The following reagents and equipment will be available for you
to you carry out your experiment – please consult this list as you generate your
hypothesis and design your experiment.
•
•
•
•
•
•
•
•
•
Yeast suspension (7%)
Sugar solutions: 0.5% glucose (all concentrations are in w/v), 0.5% sucrose,
0.5% lactose, 0.5%maltose, 0.1%, 0.2%, 0.3% and 0.4% glucose (all in water)
0.5% aspartame
Waterbath at 40oC
15 mL Falcon tubes with holes punched in the lids (10 per bench)
500 mL beakers
Tube templates showing level corresponding with volume
Wax pencils
Ethanol: 0%, 5%, 10%, 20% and 40% solutions containing 0.5% glucose
1. In order to assess fermentation, you will be measuring CO2 production by yeast over
a 35 minute time period. To start, you should answer the following questions:
a) What components are necessary in order for fermentation in yeast to occur?
b) What factors will affect the process of fermentation? (One place to start may be
to think about the fact that as the reactions of fermentation are controlled by
enzymes, the same factors that affect enzyme activity may also affect
fermentation)
55
2. Choose a variable that you would like to test for its effect on fermentation in yeast
(remember to consult the list of supplies available). Prior to going any further, you
should confirm your choice with your Instructor.
3. Complete the worksheet found at the end of this exercise and hand it in by the end of
the laboratory period. Note that you should be as detailed as possible as this will
form the basis of your experiment next week.
The following tips have been provided for you to help you design a workable experiment.
Please consult these tips and incorporate the information into your experimental protocol
(Page 2 of your worksheet).
Procedural Tips:
•
•
•
•
•
•
•
•
•
Add yeast (8 mL) to tubes first.
Fill with sugar solution of choice (note, if testing a variable other than carbon
source, use 0.5% glucose solution in water and a 40oC waterbath for incubation).
Ensure that the volume of liquid in your tube is right up to the very top.
Screw the cap on. Some liquid may come out of the holes in the cap – this is fine.
Invert the tube gently to mix the contents.
You will be placing your tubes in a 500 mL beaker filled with water that has been
placed into the waterbath so that the beaker water is the appropriate temperature.
You don’t want to place your tubes directly into any of the waterbaths in order to
avoid contaminating the water with yeast cells.
Place inverted tube into the beaker containing water in the waterbath. Start time =
0 immediately when you do this.
At 5 minute intervals, lift the tube out of the waterbath, and mark the level of the
solution in the end of the tube. Invert the tube to mix again and place back into
the waterbath.
Carry out readings for 35 minutes.
At the end of 35 minutes, use the wax pencil markings to determine volume of
CO2 produced at each time point. Use the tube template for small amounts.
Week 2: Experiment
Collect your worksheet from your lab instructor and make note of any suggested changes
to your experimental procedures. Carry out your experiment as designed, and collect
your data. Use the data to prepare two figures, as described on the following pages.
56
1. Plot the change in CO2 production over your sampling times (this figure will
give you information about the rate of the fermentation reactions).
•
Consider the rate of the fermentation reactions illustrated by your figure. You
may notice that there is a lag initially (this is not an unexpected result). What
may cause the lag in the fermentation rate?
•
Regardless of whether you saw a lag or not, how might you redesign your
experiment to ensure that a lag does not occur?
57
2. Plot the independent variable you chose versus the total amount of CO2
produced.
•
Relate your results as illustrated by this figure to your experimental objectives
(and to your null and alternative hypotheses). Do your results support your
alternative hypothesis?
58
BIOLOGY 1010 FERMENTATION WORKSHEET
Lab Number:
Date:
Group Members:
______________________________
______________________________
______________________________
______________________________
Hypothesis:
Independent Variable:
Dependent Variable:
Control(s)/Purpose of control(s):
Controlled Variables:
Supplies Required (only 12 tubes per bench are available)
59
Step by Step Experimental Summary (this will be the ONLY copy of the protocol so
ensure that you can follow it!):
60
Thought Questions
1. Alcohol becomes toxic to yeast around a concentration of 12%. Why were you still
seeing evidence of fermentation when you tested your cells with 20% ethanol?
2. How can the differential rates of fermentation using different sugar substrates be
explained? Why did you not see fermentation when you used aspartame as a
substrate?
3. Recognizing that fermentation reactions are catalyzed by enzymes, explain why we
saw an increase in CO2 production as the concentration of glucose increased.
Literature Cited:
Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San
Francisco, CA.
Black, S., Moore, R. and Haugen, H., eds. (2000). Biology Labs That Work: The Best of
How-To-Do-Its, Volume II. National Association of Biology Teachers, Maryland.
61
DNA STRUCTURE AND FUNCTION
Introduction:
Up to this point in Biology 1010 we have studied, either directly or indirectly, the structure
and function of three of the four major groups of macromolecules. In your exercise last week,
we looked at how carbohydrates contribute to the generation of cellular energy. Earlier, we
examined the structure of phospholipids and the role they play in maintaining membrane
structure and function. We also studied enzymes, particular kinds of proteins whose function
is to catalyze biological reactions. Our exercise this week will focus on the final group of
macromolecules, known as the nucleic acids.
There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). DNA and RNA are polymers consisting of many identical or similar monomers
linked together in a chain. The monomeric units, termed nucleotides, in turn are composed of
a grouping of [i] a pentose sugar, [ii] a phosphate molecule, and [iii] a nitrogenous base. A
strand of DNA resulting from a chain of connected nucleotides is paired with another,
complementary strand by way of bonds between the nitrogenous bases. As a result of the
pairing, the double strand becomes helically twisted, and is referred to as a double helix. The
genetic information in the cell is stored within the nucleotide sequence of DNA. This
information is organized into discrete units called genes.
DNA molecules do not exist in nuclei as linear double helices. In humans, each DNA
molecule is made up of about 2 x 108 nucleotide pairs, which would be equivalent to a total
length of about six cm (remember in humans that there are 46 such molecules in each nucleus,
which represents about 2.8m of DNA!). A typical eukaryotic nucleus is only about 5µm in
diameter, so the DNA must be packaged in order to fit. Proteins called histones have a net
positive charge and thus bind to negatively charged DNA molecules to form a DNA-histone
complex termed chromatin. When DNA is in this form it directs the synthesis of RNA (in a
process called transcription) and in turn, RNA controls the synthesis of cellular proteins
(translation).
DNA is also the molecule responsible for the transmission of genetic information from one
generation to the next. Although DNA is replicated while in the form of chromatin, the actual
transfer of genetic information to subsequent generations occurs after chromatin becomes
even more tightly condensed to form transient structures called chromosomes. Chromosomes
are allocated in an organized and predictable manner to newly formed cells in a process called
cell division. Although there are two different types of cell division that occur, mitosis and
meiosis, we will focus only on mitosis in this exercise. Once mitosis is complete,
chromosomes uncoil and remain as chromatin until the next round of mitosis occurs. The cell
cycle is the term used to describe the series of events that occur in an actively dividing cell
from the beginning of one cell division to the beginning of the next.
In lab today, we are going to isolate chromatin from liver cells in order to get a sense of how
much is present and what it looks like. In the second part of the exercise, you will learn about
the events associated with the different stages of the cell cycle. You should be able to
62
distinguish between chromatin and chromosomes and when each is found within the cell
cycle.
The objectives of today’s lab are thus to:
• Provide an introduction to nucleotides and nucleic acids, and to relate their structure to
the functioning of these molecules
• Provide students with the opportunity to extract and visualize DNA.
• Learn about the cell cycle and where mitosis fits in
• Learn how to prepare onion root tip squashes
• Understand the stages of mitosis and what happens in each
Please read pages 86-89, 293-298 and 359-361 in Campbell and Reece (2005) prior to
attending your lab.
A. Extraction of Chromatin From Liver:
In this portion of the lab exercise you will extract and view chromatin from non-dividing
liver cells. First, cells will be ground and suspended in a saline (salt) solution. The next
step is to release the chromatin from the cells – remember that membranes surround both
the nucleus and the cell. Cell membranes are composed mainly of phospholipids and as
such have an “oily” property. As dishwashers know, detergents “cut” grease. Likewise,
sodium dodecylsulfate (SDS), a laboratory detergent, will cut or solubilize the oil-based
cell membranes enabling extraction of chromatin. Once the membranes are solubilized
(cells are said to be lysed), then chromatin can then be precipitated from the cell solution
and visualized.
Work in groups of four up to and including step six, then work in pairs.
1. Place a small amount of liver (about 1 cm by 2 cm) into a mortar and pestle. Chop
the liver with scissors or a razor blade several times (for about 1 minute).
2.
Add 10 mL of 0.9% saline to the well-diced liver. Grind the tissue thoroughly (for
about 5 minutes) with the pestle using a circular motion.
3.
To remove any unpulverized liver, strain the cell suspension through four layers of
cheesecloth, into a small beaker. Rinse the mortar and pestle at this point to make
clean-up easier later.
4.
Remove a drop of the cell suspension using a Pasteur pipet. Place the drop on a slide
and next to it place a small drop of methylene blue. Use a toothpick to vigorously
mix the two solutions on the slide. Add a cover slip and observe the cells with the
10X and then the 40X objectives. Identify the cells with their distinct, stained nuclei.
5.
Use a Pasteur pipet to add 10 drops of SDS to your cell suspension. Mix the
suspension and remove a drop (with the pipet you used earlier for the cell
suspension) to prepare another wet mount slide (clean and reuse the same slide each
63
time). Add methylene blue, mix together, add a coverslip and observe at 10X and
40X.
6.
Repeat Step 5 until membranes are no longer visible. This may require at least four
to six additions of SDS. When you have determined that the majority of the cells
have lysed, refocus using the 4X objective and remove the slide from the stage
without altering the stage position.
7. Place a drop of cell suspension onto a clean slide. Do not add methylene blue or a
coverslip but do spread out the drop so that it is a thin layer. Place the slide on the
microscope stage and position it to view the drop. Do not alter the coarse focus; it
should be in the correct focal plane from the last slide.
8.
As you view the preparation, your partner will add one drop of 95% ethanol (from
the dropper bottle or with a clean Pasteur pipet) The ethanol will precipitate the
chromatin and you should see masses of strand-like material ranging from thick to
very thin.
9.
Because the preparation is uncovered, the alcohol-saline solution will evaporate
quickly. Clean the slide and repeat Step 8 as you add the alcohol and your partner
views the preparation.
10. Your preparation is not pure DNA. It contains protein and some RNA. If
purification was your goal, you would have to remove these materials. As the first
step, you would want to precipitate all chromatin from the cell suspension. To show
how much is present use a Pasteur pipet to carefully add two or three pipets-full of
alcohol, expelling it down the side of the beaker so that it forms a layer on top of the
aqueous solution. You will see the chromatin appear as a white, mucus-looking
substance at the interface between the solutions. Slow stirring will mix in the
alcohol sufficiently to precipitate all of the chromatin. Centrifugation, re-suspension
and washing, deproteination and chromatographic procedures would then have to be
employed to purify the DNA.
• In hindsight, what should you have done to prove that the nuclear and plasma
membranes have to be ruptured before the chromatin can be extracted?
64
B. The Cell Cycle
The orderly, complex series of events that occur in an actively dividing cell from the
beginning of one cell division to the beginning of the next is termed the cell cycle. The
cell cycle is divided into two major phases, interphase and the mitotic phase (M)
(Figure 1). Interphase can be further divided into three stages: the first gap phase (G1),
the S phase, and the second gap phase (G2). The G1 phase follows mitosis and
cytokinesis and precedes the S phase. During this stage, a variety of growth processes
such as gene expression and metabolism occur. The S phase is sandwiched between the
gap phases and is when DNA synthesis and replication occur. Following replication,
each chromosome is made up of a pair of sister chromatids held together at the
centromere. The G2 phase follows the S phase and is a period of continued growth and
initiation of activities in preparation for mitosis, the following step. Collectively, the
three phases of interphase account for roughly 90% of the time spent in the cell cycle.
Figure 1. Stages of the cell cycle.
65
The other major phase of the cell cycle is the mitotic (M) phase, consisting of mitosis and
cytokinesis. Mitotic cell division is necessary for the growth and repair of multicellular
organisms. It is also a form of asexual reproduction for single-celled, eukaryotic
organisms. Mitosis is nuclear division that results in two daughter nuclei, each with the
same number and kind of chromosomes as in the nucleus of the parent cell. Mitosis is
followed by cytokinesis, the cleavage of the cell cytoplasm into two halves each
containing a nucleus.
For convenience, mitosis is subdivided into five stages: prophase, prometaphase,
metaphase, anaphase, and telophase (Figure 2). During prophase, the long thin strands
of chromatin become condensed into discrete recognizable bodies called chromosomes.
At this stage, the nucleoli temporarily disappear and the mitotic spindle apparatus begins
to form in the cytoplasm. As the nuclear membrane fragments and disappears, the
mitotic spindle becomes fully assembled during prometaphase. At the conclusion of
metaphase, the chromosomes have aligned in an area that is equidistant from the spindle
poles called the metaphase or equatorial plate. Anaphase is characterized by the
splitting of the sister chromatids at the centromere followed by the migration of the
recently separated chromosomes to opposite poles of the spindle apparatus. Telophase
essentially reverses the early events of mitosis to form two, complete daughter nuclei: the
mitotic spindle disassembles, two nuclear membranes reassemble, the chromosomes
become decondensed and return to the long thin strands of chromatin, and the nucleoli
reform. The completion of mitosis results in the formation of two separate daughter
nuclei that have the same genetic composition as the parental cell.
The first indication that cytokinesis is occurring can be viewed during telophase. In
plant cells, a cell plate develops in the center of the cell. The cell plate enlarges until it
joins with the plasma membrane at the perimeter of the cell. In animal cells, the initial
indication of cytokinesis is the development of a shallow groove around the midline of
the cell called a cleavage furrow. A contractile ring composed of microfilaments
associated with the cleavage furrow is used to deepen the furrow and results in the
cleavage of the original cell into two completely separate cells. Regardless of the nature
of the cell, cytokinesis results in the formation of two daughter cells having identical
nuclei and similar amounts of cytoplasm and associated organelles.
Mitosis is easily studied using root tips from actively growing plants. In this lab exercise
you will prepare onion root tip squashes and use prepared longitudinal sections of onion
(Allium cepa) and broad bean (Vicia faba) to observe the various stages associated with
mitosis. Although this part of the exercise emphasizes the events associated with mitosis,
you should be able describe and identify all stages associated with the cell cycle.
66
Figure 2. Mitosis and cytokinesis in a typical plant cell.
Preparation of Onion Root Tip Squashes:
Work individually to complete this part of the exercise.
1. Label a microscope slide with your name. Using a razor blade, carefully cut off one onion
root as close to the bulb as possible. In the plant root, cell division does not occur along
the entire length, but only in one region near the tip. Place the root on your slide and trim
the root so that you have a two mm root tip segment. Save the apical root tip and discard
the remainder of the root.
2. Use an eye dropper to add two drops of 1N HCl to the root tip (be careful, acid will burn
your skin).
•
What is the purpose of adding HCl to your preparation?
67
3. Place your slide on the slide warmer (65°C) for five minutes. Do NOT let your
preparation dry out during the incubation time. If required, add another drop of HCl to
your preparation.
4. Use a paper towel to wipe away excess acid. Add two drops of toluidine blue stain to the
root tip tissue. Continue to warm the slide at 65°C for three to five minutes, adding more
stain if required.
5. Wipe away any excess stain and add one drop of fresh stain to your preparation. Cover
with a cover slip and use a cork to press firmly, straight down (avoid lateral movement of
the cover slip). This will separate and spread the cells.
6. Examine your preparation with the compound light microscope. With the 10X objective,
you should be able to scan around and find cells in stages of mitosis, which can then be
observed more closely with the 40X objective. Most of the cells will not be dividing, and
their nuclei will be blue, often with one or more unstained nucleoli. Be patient and you
will discover dividing cells. The chromosomes will again be coloured blue, and you ought
to be able to pick up cells in all stages of division.
If you are having difficulty identifying any mitotic stages, consult the Allium root tip or
Vicia faba prepared slides available at the side bench. Share your good examples with
your lab partner.
•
Why did you observe so few cells displaying mitosis in your preparation?
•
Which mitotic phase did you see most frequently?
•
What features distinguish a cell in prometaphase from a cell in interphase?
7. Use the space below to sketch cells in each of the stages you observed.
68
Thought Questions:
1. Describe three structural differences between RNA and DNA molecules.
2. In the table below, name the five nitrogenous bases found in nucleic acids and indicate
whether they are found in DNA, RNA, or both.
Nitrogenous Base
Base Present in: DNA, RNA or Both
3. Compare (provide a similarity) and contrast (provide a difference) for the following
pairs of terms:
a) Chromosome and gene
b) Diploid and haploid
c) Centromere and chromosomal arm
4. What is a karyotype? How are chromosomes differentiated from each other?
69
5. Although interphase is not actually part of mitosis, recognition of its events is essential to
understanding mitosis. Describe the events that occur during the G1, S and G2 phases of
interphase.
6. Plot the amount of DNA in a cell as a function of time over the course of a complete cell
cycle.
7. a) What aspects of cell division are common to both plants and animals?
b) What aspects of cell division are different between plants and animals?
8. Explain the advantages of having genetic material (genes) grouped into chromosome
format, instead of all genes existing as separate entities.
Literature Cited:
Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San
Francisco, CA.
70
APPENDIX A: HOW TO USE THE COMPOUND MICROSCOPE
Your instructor will review the use of the compound light microscope with you. You may
find it beneficial to label the parts discussed on the diagram provided (Figure 1).
Figure 1: The Compound Light Microscope
Compound light microscopes have two sets of magnifying lenses, the ocular and the
objective lenses. The ocular lens (eyepiece) is the lens that is closest to your eye. The ocular
lenses on your microscope magnify objects to 10X their size. The set of lenses (typically four,
the smallest being that with the least magnifying power, and the largest being that with the
greatest magnifying power) closest to the specimen are the objective lenses; they are located
on the revolving nosepiece. The body tube connects the two sets of lenses.
The arm and base of the microscope are the heavy cast metal parts of the microscope and are
important support parts of the microscope that can be used to transport it. The arm supports a
horizontal surface called the mechanical stage, upon which slides are placed for viewing. It
is equipped with a spring-loaded clip that holds the slide in place, and two slide adjustment
knobs used to move the slide on the stage.
Place a slide on the stage and center it over the hole in the stage. Adjust the distance between
the oculars to match your interpupillary distance (distance between your pupils). Revolve
the nosepiece so that the lowest power objective lens (generally the 10x power lens) is in
71
position. To focus the microscope, locate the coarse and fine focus adjustment knobs at the
base of the microscope, and use the coarse adjustment focus to move the slide close to, but not
touching, the objective lens. Look at the stage from the side as you do this. On most
microscopes this involves raising the stage, but on some the lenses are lowered. Also, on
most microscopes an automatic stop will prevent you from moving the stage closer than about
one centimeter from the lens. Now, look through the ocular lenses, and move the slide away
from the objective lens until the specimen becomes clear (is in focus). Finish focusing with
the fine adjustment knob. Once you have focused with the low objective power lens, you may
switch over to the next higher power lens with only fine focus adjustments (the microscope is
said to be parfocal).
As you switch from one objective lens to another, you will notice that the working distance,
the clearance between the lens and the stage, decreases with increasing lens power. This is
illustrated in Figure 2 below.
Figure 2: The working distance (above) and the field of view (below) change with
magnification of the objective lens.
It should be obvious to you why, when using the high power objective lenses (40X or 100X),
you must use ONLY the fine focus adjustment knob; otherwise risk of contact and
subsequent damage become high. Also shown in Figure 2 is the diminishing field of view
(the illuminated area and contents that are seen when looking through the ocular lenses) as the
objective lens power increases; this is due to a smaller and smaller aperture at the bottom of
the lens through which light enters. This means that [a] things are harder to find on a slide
when you are using high power since only a small fraction of the slide can be seen, and [b]
72
less light enters your eye and everything in the field appears darker. As a consequence, you
will learn to [a] switch back to a lower power objective lens when you want to ‘scan’ around
the slide, and [b] manipulate the amount of light coming into the lens using the iris diaphragm
so that you can see the objects clearly.
The amount and concentration of light coming through the specimen and hence to your
eyes can be adjusted in several ways. First, of course, is the on/off light switch, generally
located at the base of the microscope, and often associated with a rheostat to control light
intensity. A condenser lens is mounted below the stage, and concentrates light on to the
specimen; it generally needs no adjustment of position. An iris diaphragm is located
below the condenser lens. Find the lever which controls the diaphragm; it can be very
useful in adjusting illumination and contrast.
Finally, some useful hints and cautions:
• Never drag the microscope across the counter-top. Lift it with both hands by its arm,
being careful not to tip it.
• Use lens paper to clean glass slides and lens surfaces before using your microscope.
• Water damages objective lenses. If water does contact a lens, wipe it off immediately.
Also avoid getting water under the slide as it will stick to the stage.
• If you have used immersion oil, use lens paper dipped in 60% ethanol to remove it from
the 100x objective lens when you are finished.
• Always start the focusing procedure with the low (10x) power lens.
• When attempting to locate an object on a slide, remember that the image you see is
reversed; that is, as you move the slide toward you on the stage, the slide is apparently
moving away from you as you view it through the lens.
• Some ocular lenses are equipped with pointers; they appear as a dark black line that will
rotate if the lens is rotated in its tube.
• When finished with the microscope, rotate the 4x or 10x objective into working position,
lower the stage, turn off the light, wrap the cord so that it will not fall away when the
microscope is lifted, and cover with the dust cover.
73
APPENDIX B: HOW TO MAKE A SCIENTIFIC DRAWING
A scientific drawing is a graphical means of presenting results or observations. Guidelines for
constructing a scientific drawing are given below.
•
•
•
•
•
•
•
•
Although the cells are microscopic, your drawing should be larger in size.
Use a sharpened, hard leaded (H or 2H) pencil, never ink or colored pencil crayons.
Place the drawing slightly to the left side of the space, leaving room for labels to the right
of the drawing.
Always draw more than one cell in your drawing. This indicates the cellular association
to an observer. (e.g.: found singly or as part of a tissue) The cellular detail of only one of
the cells needs to be complete for labeling.
Draw with one continuous line and do NOT retrace your lines. Do NOT shade in your
drawing. Do NOT color your drawing.
Place label lines horizontally (use a ruler to make them straight), with no crossed lines.
Structure or organelle labels should be singular unless the label line branches to multiple
structures (see examples below).
• ‘mitochondrion’ is singular and ‘mitochondria’ is plural
• ‘bacterium’ is singular and ‘bacteria’ is plural
• ‘flagellum’ is singular and ‘flagella’ is plural
Only draw and label structures that are visible in the field of view. Do NOT include
structures that you know are present but are not visible or detectable with the light
microscope.
A complete scientific drawing will always have a figure caption that is located below the
drawing. The figure caption will contain three pieces of information: i) a figure number
identifier (e.g.: Figure 1), ii) a brief description of what was observed and drawn, including
the scientific name of the organism from which the specimen was taken and how the
observation was made, and iii) the drawing magnification.
The drawing magnification is a simple calculation that indicates the relationship between the
size of your drawing and the actual size of the specimen. A diagram of a cell would be much
larger than the actual cell, whereas a diagram of an elephant would be much smaller than the
actual elephant.
Magnification is defined as:
Size of drawing
Actual specimen size
Where: - the size of the drawing is determined by measuring it with a ruler
- the actual size of the specimen is determined by ocular micrometry
- the number calculated has as many significant figures as the accuracy of your
measurement (usually 2, if you measure in mm)
An example of a proper scientific drawing is provided on the following page. Notice the
positioning of the labels and figure caption and the type of information that is included in the
figure caption.
74
Figure 1. A dividing Brachydanio rerio (zebra fish) cell in anaphase as observed
using the light microscope. (1300X)
75
APPENDIX C: HOW TO DETERMINE THE SIZE OF A SPECIMEN
METHOD 1: Use of an ocular micrometer.
An ocular micrometer is simply an ocular lens that has a scale etched into the glass of the
lens. A lens that has been so modified will have a red dot pointed on it. To measure the
object you are viewing, simply replace your regular ocular lens with the micrometer lens.
What you see is a scale overlying your object. By moving the stage, one end of the
micrometer scale can be positioned at one end of your object.
The scale is a line divided into tenths and each tenth is again divided into tenths. Each small
division is called an ocular unit (ou). The scale and ocular unit graduations do not have any
imperial or metric units assigned to them and so must be calibrated. The calibration of the
ocular micrometer for the various objective lenses has been done for you (see Table 1).
Table 1. The calibrated values of an ocular unit for various Olympus light microscope
objective lenses as determined by a stage micrometer.
Objective Lens
Value of 1 Ocular Unit (mm)
4X (scanning)
0.026
10X (low power)
0.01
40X (high power)
0.0026
100X (oil immersion)
0.001
To calculate the length of the cell or structure you must always know the power of the
objective lens used (so you know which conversion value to use) and the length of the object
in ocular units. Therefore, if you were observing a bacillus bacterium with the 100x objective
lens, and its length was 32 graduations of the ocular micrometer, the length of that bacterium
would be 32 x 0.001 mm = 0.032 mm, or 32 µm.
76
APPENDIX D: ASEPTIC TECHNIQUE
When working with bacteria, it is of utmost importance to practice certain aseptic techniques.
This ensures that the culture being examined is not contaminated by organisms from the
environment and that organisms being studied are not released into the environment.
Although the bacteria used in these exercises are not pathogenic, it is still important to
develop and practice good aseptic technique and work with care.
•
At the beginning of the lab, wash your hands with soap and water, then wipe the lab bench
with the disinfectant provided. Repeat these procedures at the end of the exercise.
•
Use a Bunsen burner to flame all nonflammable instruments used to transfer or manipulate
bacteria. If you have long hair, tie it back. Long, floppy sleeved garments should be
removed if possible or sleeves rolled away from your hands and wrists. When not in use,
the Bunsen burner should be turned off.
•
Working with cultures requires efficient and careful work. Preread protocols so that you
know what you need to do with the bacterial material. When culture tubes or plates are
open, slant them away from you. Never lift a culture by the lid. Never lay culture caps or
Petri plate tops on the bench and always recap cultures as soon as possible.
•
Always dispose of your prepared, used bacterial slides and plastic transfer pipettes in the
disposal trays provided. Never place materials that have come into contact with bacteria
in the broken glass box or common garbage.
•
If you spill a liquid culture on the bench top or floor, spray the area with disinfectant,
place paper towels on the spill to contain it, and notify your instructor. The contaminated
towels should be placed in the orange biohazard bag for autoclaving and disposal.
77
APPENDIX E: HOW TO USE A SPECTROPHOTOMETER
Many procedures for the quantitative analysis of compounds in biological fluids are based on the
fact that such compounds will selectively absorb specific wavelengths of light. For example, a
solution that appears red to us (such as blood) absorbs the blue, yellow and green colors of light,
while the red is reflected to our eyes. However, the eye is a poor quantitative instrument and
what appears bright red-orange to one person may appear dull red-purple to another. A
spectrophotometer is one instrument that will objectively quantify the amounts and
wavelengths (kinds) of light that are absorbed by molecules in a solution. A source of white
light is focused on a prism to separate it into its individual bands of radiant energy (Figure 1).
One particular wavelength is selected to pass through a narrow slit and then through the sample
being assessed. The sample, usually dissolved in a solvent, is contained in an optically selected
tube or cuvette, that is standardized for wall thickness and has a light path exactly one
centimeter across. These tubes are therefore expensive and must be handled carefully.
Figure 1. Representation of a photoelectric spectrophotometer.
After passing through the sample, the selected wavelength of light strikes a photoelectric tube.
If the substance in the cuvette has absorbed any of the light, the light transmitted out the far
side will be reduced in total energy content. When it hits the photoelectric tube, it generates
an electric current proportional to the intensity of the light energy striking it. By connecting
the photoelectric tube to a device that measures electric current (a galvanometer), a means of
directly measuring the intensity of light is achieved. The galvanometer has two scales: one
indicates the percent transmittance and the other is a logarithmic scale with unequal division
graduated from 0.0 to 2.0 indicating absorbance.
Since most biological molecules are dissolved in a solvent prior to measurement, a source of
error can be the absorption of light by the solvent. To ensure that the spectrophotometric
measurement will reflect only the light absorption of the molecules being studied, a
mechanism of ‘subtracting’ the absorbance of the solvent is required. To achieve this a
reagent blank (the solvent) is first inserted into the instrument and the scale is set to read 0.0
absorbance (100% transmittance) for the solvent. The sample containing solute plus solvent
78
is then inserted. Any reading on the scale that is greater than 0.0 absorbance (less than 100%
transmittance) is considered to be due to the absorbance by the solute only.
The transmittance scale is a percent number; a ratio of the light exiting the sample tube to
the light entering the tube. However, this number is not a linear reflection of the
concentration of the solute molecules (Figure 2). The absorbance scale, on the other hand,
does reflect a linear relationship. Although you do not necessarily know the exact
concentration of the solute molecules in your sample, you do know that if the absorbance
value doubles, the concentration of solute in your sample has doubled. Absorbance has no
units, but the wavelength of the light is usually indicated using a subscript following the
symbol A, e.g.: A580.
Figure 2. The relationship between percent transmittance and solution concentration (left)
and absorbance and solute concentration (right).
Instructions for Use of the Spectronic 20TM (see Figure 3):
•
•
•
•
The spectrometer should be turned on prior to the start of your lab period. If not, use
the power switch (D, Fig. 3) to turn on the spectrophotometer, and then let it warm up
for at least five minutes prior to reading your samples.
Adjust the wavelength to the appropriate setting using the wavelength control knob (C,
Fig. 3).
Zero the spectrophotometer by adjusting the zero control knob (same knob as the
power switch) so that the needle on the transmittance scale is set to 0%. There should
be nothing in the sample compartment and the lid should be closed.
Calibrate the spectrophotometer by inserting the reagent blank, a solution containing
all of the components of the sample being measured except for the molecule of
interest, into the sample compartment and closing the lid. Ensure that the cuvette is
clean and dry before placing it in the sample compartment. Cuvettes must be oriented
in the sample compartment such that the vertical line near the top of the cuvette is
opposite the raised notch in the sample compartment. Rotate the absorbance control
knob (E, Fig. 3) until the needle on the transmittance scale reads 100% (or 0 on the
absorbance scale). Remove the reagent blank from the sample compartment.
79
•
Transfer your sample to a cuvette, insert it into the sample compartment, and read the
absorbance value. Your absorbance reading should be between 0.0 and 2.0.
Figure 3. The Spectronic 20™ spectrophotometer. (A - Sample compartment, B Transmittance and absorbance scales, C - Wavelength control knob, D - Power switch / zero
control knob, and E - Transmittance / absorbance control knob.)
80
APPENDIX F: SCIENTIFIC INQUIRY
Background Reading
As background for this material, you will find it useful to read pages 19 to 24 in
Campbell and Reece (2005). You may also find the Science Toolkit
(http://home.uleth.ca/bio/toolkit/) section regarding scientific method helpful.
The Scientific Process
Today’s scientific methodology has its roots in the methodology of Aristotle. The
scientific method consists of a progression of steps that include a preliminary information
gathering phase (observations), the generation of a question based on initial observational
data, the development of a hypothesis, the listing of a prediction of experimental outcome
based on the hypothesis, the design of an experiment to test the hypothesis, the collection
and analysis of experimental data, and the conclusions drawn from the experiment
regarding the original hypothesis (Figure 1). During the experimental portions of the
course, you will be asked to apply the scientific method, and know the terminology
associated with it.
A. Hypothesis
Observations of some natural phenomena lead you to formulate a question. When a
question is asked, researchers attempt to answer it by proposing a hypothesis, a logical,
testable, falsifiable explanation for the observed phenomenon. The hypothesis suggests
an answer to the question. A hypothesis need not be extremely complex and should
strive to be the simplest explanation. The data collected from an investigation cannot
prove a hypothesis, but the data may support the hypothesis. Although a hypothesis can
never be proven with 100% certainty (because we can never be certain that we have not
overlooked an alternative explanation), repeated tests provide greater and greater support
for a hypothesis, giving us more and more confidence that it is correct.
Typically, scientific studies require two hypotheses that are mutually exclusive. For
statistical reasons, we must contrast the hypothesis we wish to test, the alternative
hypothesis (Ha), with a null hypothesis (Ho), a hypothesis in which we indicate that the
treatment will have no effect on the variable that we are measuring. The null hypothesis
permits us to make a statement that can be proven false by data.
B. Experimental Design
The best way to test a hypothesis is with an experiment -- a replicated, controlled
situation in which only one factor is manipulated. The factor being tested is varied in a
known way, while all other factors are held as constant as possible. Critical to any
experiment are aspects of experimental design. An experiment must be carefully
designed so that appropriate materials are selected, all of the variables are identified,
81
Figure 1. Rigorous version of the scientific method. (In practice not all of these steps
may be rigidly followed.)
external influences are controlled, and appropriate unbiased measurements are collected
and analyzed.
All of the variables with the experiment must be explicitly defined. In this course we will
focus on experiments in which only a single factor is being examined at a time. The
independent variable is the factor that is being manipulated or varied during the
experiment; it is the cause. The dependent variable is the variable that shows some
response or change to the manipulated independent variable; it is the effect. Controlled
variables are factors that are held constant between treatments during the course of the
experiment.
All of the experimental treatments must be defined. A treatment is a test group of
individuals subjected to the same levels or amounts of the independent variable. The
experiment may also have a control treatment (please note this is NOT the same as a
controlled variable!). The control treatment is a group in which the independent variable
82
is held at an established level or omitted. The control treatment serves as a reference for
comparison of the experimental treatments and allows the researcher to determine
whether the predicted effect is actually due to the independent variable.
The experiment must be replicated (repeated) so that the conclusions are based on more
than a single trial. This reduces the probability that the results we see are based just on
chance and allows us to estimate this probability. The number of replicates used in each
treatment is referred to as sample size, or n. The results from replicated experiments are
usually averaged. Further analysis may be conducted using statistical tests. The type of
data that you collect, how you will collect them, and how the data will be analyzed (the
type of statistical tests you will use) must all be determined prior to conducting the
experiment.
Following a great deal of careful planning, the experiment can be carried out. The
procedure is generally organized in a sequence of progressive steps. When conducting an
experiment, precise notes should be taken and any exceptions or deviations from the
original design must be noted.
C. Collecting and Analyzing Results
The data recorded from an experiment may be collected as discrete data, in which each
value represents a whole, separate, complete unit (e.g.: the number of bean seeds) or as
continuous data, in which each value is taken from a continuous range using some type of
measuring instrument (e.g.: bean seed mass).
Raw data collected from scientific experiments are never presented in assignments,
scientific papers, or published works. We must now include an assessment of these data
by computing descriptive statistics, values such as the mean, which summarizes the
central tendency and standard deviation or variance, which summarizes the spread of
data (dispersion) around the mean. Descriptive statistics alone however will NOT permit
us to directly compare the means and draw conclusions. The following formulae can be
used to calculate the mean and standard deviation (however, most calculators will
determine the mean and standard deviation for you):
! xi
Mean (X) =
n
where Σ(xi) is the sum of
all individual, measured
values (xi); n is the total
number of measurements
taken.
2
Standard deviation (s.d.) = (! (xi )) -
(! xi )2
n-1
n
where Σ(xi2) the sum of all individual squared measurements;
Σ(xi)2 is the sum of all individual measurements squared; n is
the total number of measurements taken.
OR
s.d. =
!(Xi - X)2
n-1
where Σ(xI - X)2 is the sum of the difference between each
measurement and the mean squared; n is the total number of
measurements taken.
83
D. Reporting Results
The descriptive statistics from experiments are generally presented in tables or figures.
Tables and figures serve two main functions. They are used to help you analyze and
interpret your results and to enhance the clarity with which you present the work to a
reader. Tables are columns and rows of numbers, whereas figures may be graphs,
schematic diagrams, pencil drawings or even photographs. The format that you use to
display your data depends on the data. If the data show a clear trend, a graph will
accentuate and highlight this trend. If, however, the absolute data values are of
importance then a table may be a better format choice for your data. Choose either a
table or graph format to display your data but never show the same data in both formats.
TABLES
Tables are used to present results that range from a few to many data points. They are
also useful for displaying the data from several dependent variables. For example, Table
1 shows the heart rate of frogs subjected to five temperature treatments. Notice that
because each treatment had a different sample size (n) the sample sizes are also recorded
in the table itself. The table is also accompanied by a table caption that appears above
the table.
The following guidelines will help you construct a table:
• All values of the same kind should read down the column, not across a row.
• The headings of each column should include the units of measurement
• Each table presented is numbered consecutively (starting with Table 1) and is
accompanied by a caption that is placed above the table. The caption briefly and
clearly describes the content of the table. It makes reference to the organism (or
cell, tissue type etc.) used in the study and its complete scientific name, the
variables that were assessed, the treatments that were used, the types of values
reported, and the sample sizes for the treatments.
• Information and results not essential (e.g.: test-tube numbers, simple calculations)
should NOT be included.
• If the table is part of a report, the text must contain a least one reference to every
table. Summarize the data and refer to the table; for example, “As the
temperature increased from 5 to 25°C, there was a corresponding increase in frog
heart rate from 10 to 80 beat per minute (Table 1)”. DO NOT write “See the
results in Table 3.” It is not necessary to repeat each and every number in the
table; refer to the trend only.
GRAPHS
Graphs permit trends and patterns that arise from a relationship between the independent
and dependent variables to be emphasized. A graph is a visual summary of the results.
Data may be represented in different formats depending on the type of data collected.
You must determine whether your experimental variables (dependent and independent)
are discrete or continuous. Discrete variables are those in which observations are placed
into one of several mutually exclusive categories (e.g.: flower color, organelle type,
inhibitor name, number of organelles with a cell). Continuous variables result from
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quantitative observations in which the data may be any value in a continuous interval of
measurement (e.g.: tissue sample weight, reaction time, amount of carbon dioxide
exhaled, growing temperature, solution concentration).
Table 1. The effect of temperature on the heart rate of Rana pipiens after a one-hour
exposure to fresh water. The values represent the mean + the standard deviation; n is
the sample size at each temperature.
Water Temperature
(°C)
Sample Size (n)
Hear Rate (beat min-1)
5
10
15
8
9
12
10 + 2
20 + 3
35 + 8
20
25
11
7
55 + 8
80 + 10
The following guidelines will help you construct a graph:
• Use graph paper and a ruler to plot the values accurately.
• Always print labels and use a sharp, hard-lead pencil. NEVER use a pen.
• You must decide which variable is the dependent variable and which variable is
the independent variable. The dependent variable is always placed on the y axis
(vertical or ordinate axis), while the independent variable is graphed in the x axis
(horizontal or abscissa axis) (Figure 3).
• Label the axes with a few words describing the variable, and put the units of the
variable in parentheses after the variable description (Figure 3).
• The numerical range (scale) for each axis should be appropriate so that all data
(including standard deviation or standard error values) can easily be plotted on the
graph paper. Select your intervals and range to maximize the available graphing
space. Choose intervals that are logically spaced and therefore permit easy
interpretation of the graph (e.g.: intervals of 5s or 10s). Do not select intervals
such as sixth, eighths, fourteenths, etc. If there are no data points at the low end
of the scale, it is not necessary to begin the scale at zero. Otherwise you may
have a large block of unused space on your graph. Similarly, it is not necessary to
label every subunit of the scale; label every second, fifth, or tenth. It is important,
however, that every subunit of the scale is of the same value. For example, you
would not start with intervals of two at the lower end of the scale and then switch
to intervals of ten at the upper end.
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Y axis
Dependent variable label here
(variable units)
25
Legend:
= high dose
= low dose
20
15
10
5
0
0
15
20
25
30
35
X axis
Independent variable label here
(variable units)
Figure 3.This is the figure caption. It always appears below the
figure and describes the graph. You must include all variables,
treatments, types of values reported, symbols, sample sizes amd
organism, tissues or cell studied in the figure caption.
•
•
•
•
•
5
10
Several data sets can be placed on a single graph, however they must be clearly
labeled, usually by a legend, and the points of each data set should be given
different symbols. Colored graphs are rarely used in scientific publications.
Select a graph type that best describes your data. Line graphs, bar graphs and
scatter graphs are most frequently used in scientific presentations. The choice of
graph type depends on the nature of the variables being graphed. (Please see
below.)
All graphs are given a caption which is located below (not above) the graph. Each
graph is labeled as a Figure (NOT graph) and given a number (e.g.: Figure 1). The
first figure referred to in the text is Figure 1, the second, Figure 2, and so on. A brief
description of the information shown in the graph (all variables, treatments, types of
values reported, symbols, sample sizes, and organisms, tissues or cells studied)
follows the figure number. Capitalize the first word in a figure title and place a
period at the end (Figure 3).
If the figure is part of a report, the text must contain at least one reference to every
figure. Summarize the data and refer to the figure. For example, “The 0.01 µM
solution of the plant growth substance promoted plant height in canola (Figure 4)”.
DO NOT write “The results are shown in Figure 4”. It is not necessary to repeat
every data point shown in the figure, but do refer to the trend.
Computer-generated graphs may be used instead of hand-drawn graphs provided
that they conform to the proper format. If you do not know how to manipulate the
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various attributes of the graph to generate a properly labeled graph, it may be
advantageous to hand-draw your graph.
Line graphs are generally used when the independent variable is continuous, one or two
dependent variables are reported, or several data points are reported. Line graphs show
changes in the quantity of the selected variable and emphasize changes of the values over
their range. Figure 4 shows the relationship between the application of two
concentrations of a substance to canola plants during a 30 day incubation period.
The following guidelines will help you construct a line graph:
• Follow the general guidelines for making a graph. Be sure to decide on uniform
scales for each of the axes.
• Plot the data as separate points. Use separate symbols to differentiate between different
data sets. Be sure to define these symbols in your legend.
• Deciding whether to connect the data points on a line graph depends on the type
of data and how they were collected. To illustrate trends, draw smooth curves or
straight lines to fit the values for any one data set. To emphasize meaningful
changes in value on the x-axis, connect the points dot to dot. Do not extrapolate
beyond a data set unless you are using it as a prediction technique because you do
not know from your experiment whether the relationship holds beyond the range
tested. Do NOT force the lower end of the line through the origin (0, 0).
Plant
height
(cm)(cm)
Plant
Height
80
60
40
20
0 µM (control)
0.01 µM
0
0
5
10
15
20
25
30
35
Days post-application
Figure 4.The influence of gibberellin, a plant growth substance,
on elongation (plant height) Brassica
of
rapaplants as measured
over a 30 day period following application. The values represent
the means+ s.d., n = 25.
87
Bar graphs are usually used to represent data that represent discrete or discontinuous
groups or non-numerical categories, thereby emphasizing differences between groups. Bar
graphs may represent frequency data, that is, data in which measurements are repeated and
the counts are reported (Figure 5) or data from discrete groups that do not lend themselves
to a linear scale (Figure 6).
8
Frequency
6
4
2
0
1
2
3
4
5
6
7
8
9
Number of Golgi bodies observed
Figure 5.The frequency of Golgi body occurence with Chinese
hamster kidney cells as determine using transmission electron
micrographs, n = 21 cells.
Cell Size µm)
2000
1500
1000
500
0
E.E.coli
coli
Erythrocyte Lymphocyte Sperm cell Human egg
Amoeba
Frog egg
Cell type
Figure 6.Comparison of the sizes of various cell types as
determined from literature values. The values shown represent
the means+ s.d., n = 20 cells for each type.
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E. Communicating Results
Effectively communicating scientific results is just as important as designing and carrying
out the experiments. Scientific findings must be shared and may be communicated either
in oral (spoken presentations) or written (published papers) fashion. A scientific paper
will consist of the following components:
TITLE (a statement of the question posed or nature of the investigation)
ABSTRACT (a short summary of all components of the paper)
INTRODUCTION (a brief literature review of the question being addressed and
statement of the hypothesis being tested and resulting predictions)
MATERIAL AND METHODS (a brief summary of what was done and how)
RESULTS (presentation of data in tabular or graphical format and accompanying
notation of trends and patterns observed)
DISCUSSION (interpretation and discussion of results)
LITERATURE CITED (alphabetical listing of all references used)
Literature Cited
Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings,
San Francisco, CA.
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APPENDIX G: CONVERSIONS AND TAXONOMY
Metric Conversion Values
Below is a list of metric conversion values that you must be familiar with. If these are new to
you, please memorize them. Metric conversion values will NOT be provided for quizzes and
exams.
Volume
1 L (litre) = 1000 mL (milliliter)
1 mL = 1000 µL (microliter)
Length
1 m (metre) = 100 cm (centimeter)
1 cm = 10 mm (millmeter)
1 mm = 1000 µm (micrometer)
1 µm = 1000 nm (nanometer)
1 nm = 1000 pm (picometer)
Mass
1 kg = 1000 g (gram)
1 g = 1000 mg (milligram)
1 mg = 1000 µg (microgram)
Basic Taxonomy
Typically in biology the binomial name (a two part Latin name) and not the common name is
used to refer to an organism. The first word in the binomial designates the genus to which the
organism belongs. Both words together refer to the species name. For example, you examine
a micrograph showing rat liver cells. The rat belongs to the genus Rattus. The first letter of
the genus name is always capitalized and the entire word is italicized. There are several
species of rat that belong in this genus, but the specific organism from which the cells came
from was Rattus norvegicus. You should take note of how the species name is represented.
Scientific convention requires that if a species name is typed, it should be placed in italics.
Since it is difficult to discern italic hand writing from normal hand writing, hand written
species names are always underlined (e.g.: Rattus norvegicus). As above, the first letter of the
genus name is always capitalized. However, the second part of the species name is an
adjective that describes the genus and it is never capitalized and always appears in lowercase
letters. You should get in the habit of properly using the binomial name whenever possible.