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BIOLOGY 2060
A LABORATORY MANUAL
FOR CELL BIOLOGY
(Part 1)
Winter 2014
Biology Department
Memorial University of Newfoundland
023-393-12-03-110
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Biology 2060
Cell Biology
TABLE OF CONTENTS
Page
Part 1:
Table of Contents ...............................................................................................................................
2
Laboratory Rules ................................................................................................................................
3
Hazard Symbols and Classes .............................................................................................................
4
WHMIS and the Student ....................................................................................................................
5
Material Safety Data Sheets (MSDS) ................................................................................................
6
Disposal of Chemicals .......................................................................................................................
7
Laboratory Notebooks .......................................................................................................................
8
LAB 1: Microscopy ...........................................................................................................................
10
LAB 2: Cell Structure and Measurement...........................................................................................
28
LAB 3: Isolation and Function of Chloroplasts .................................................................................
37
LAB 4: The Scientific Method ..........................................................................................................
47
LAB 5: Isolation and Function of Mitochondria ...............................................................................
57
APPENDIX I: Critical Illumination...................................................................................................
68
APPENDIX II: Principles of Colorimetry .........................................................................................
69
Part 2:
LAB 6: Cell Membrane .....................................................................................................................
4
LAB 7: Cell Division .........................................................................................................................
16
LAB 8: Muscle Contraction: Actin-Myosin Based Motility .............................................................
27
LAB 9 and 10: Localization of Macromolecules by Cytochemical Methods ...................................
38
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LABORATORY RULES
1.
Come to the lab on time since instructions are given at the beginning of the period.
2.
Read the laboratory exercises BEFORE coming to the lab so you will know what to do.
Read pertinent information in the text or in lecture materials to gain a better
understanding of what is done and the type of results to expect. .
3.
Attend every lab period. There will be no make-up labs. If you miss a lab period, notify
your instructor as soon as possible.
4.
Do not eat or drink in the laboratory.
5.
Report any accidents to the lab instructor immediately.
6.
Forceps, scalpels, etc. will be provided.
7.
Lab coats should be worn during laboratory sessions.
8.
Familiarize yourself with the laboratory, especially the locations of the first aid kit,
eyewash station and the nearest fire exit. If the fire alarm sounds, leave the building
immediately by the most direct route. Do not use the elevators in the event of a fire
alarm.
9.
Students who wear contact lenses should refrain from wearing their lenses to lab.
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5
WHMIS and the Student
WHMIS, the Workplace Hazardous Materials Information System, is a Canada-wide
information system for ensuring that workers are informed about the chemicals and other
hazardous materials they use. Students attending laboratory periods are not included in the
legislation, but the Memorial University Safety Office is promoting the idea of making the same
kind of information available to graduate and undergraduate students as to employees of the
university.
Hazard Classifications
A controlled product is a material that may have characteristics which would put it into
one or more of the hazard classes on the attached table - Hazard symbols and classes.
A controlled product can be recognized if its label:
- has any of the WHMIS hazard symbols,
- has the WHMIS hatched border, or
- makes reference to a material safety data sheet (MSDS).
WHMIS Labels
There are two basic types of WHMIS labels:
- supplier labels, and
- workplace labels.
Supplier labels are attached to all packages of controlled products by suppliers. These
labels give the identity of the product and its supplier, risk phrases, precautionary measures, first
aid measures, hazard symbols, and reference to a material safety data sheet (MSDS). The labels
have the WHMIS hatched border.
Workplace labels are produced in the workplace and are attached to containers of
controlled products which do not have supplier labels such as when products are decanted from
supplier containers, old containers which have been around since before WHMIS became
effective, or other containers which do not have supplier labels for whatever reason. Workplace
labels need only a product identifier, safe handling procedures and reference to an MSDA.
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Material Safety Data Sheets (MSDS)
A material safety data sheet is a technical document relating the health effects of
exposure to a product, hazard evaluation, protective measures and emergency procedures.
MSDS’s are sent by suppliers of controlled products or may be generated in the workplace.
MSDS’s have nine categories of information:
- Name of product and its use, supplier address and phone.
- Name and concentration of all hazardous ingredients.
- Physical characteristics of the product.
- Fire or explosion hazard.
- Reactivity hazards.
- Toxic hazards.
- Actions required to prevent injury or accident.
- First aid procedures.
- Identity of organization which prepared MSDS and date it was prepared.
Students should be aware that MSDS’s are available for the controlled products being
using in laboratories and that these may be consulted for specific information on the controlled
products.
In some cases, MSDS’s will not be sent by suppliers of laboratory chemicals when the
appropriate safety information is given on the container labels.
Exemptions
There are other products used in various workplaces, including laboratories, which might
be considered hazardous but are exempted from WHMIS regulations. These include
manufactured articles, products made of wood or tobacco, products packaged for consumer use,
hazardous waste, and products governed by the federal acts for: explosives, food and drugs, pest
control products, and radioactive materials.
Memorial University Safety Office (09-91)
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CHEMICAL DISPOSAL
Many of the chemicals you will use in these laboratories can have deleterious effects on YOU
particularly if they are not handled properly. Make sure that you read the Material Safety Data
Sheets (MSDS) provided in the lab which will alert you to the specific dangers of individual
chemicals.
Disposal of Chemicals
1.
Fixatives and Acids: e.g. methanol, acetic alcohol, hydrochloric
acid, etc. - discard in the bottle labelled “Fixatives and Acids”
located in the fumehood.
2.
Ethanols and Aqueous Stains: e.g. 70% ethanol, giemsa, schiff reagent - discard in the
sink, letting cold water run for 5 minutes.
3.
Xylene:
NOTE:
discard in bottle labelled “Xylene” located in the fumehood. Xylene is
immiscible with water therefore make sure that ONLY XYLENE goes in this
bottle.
XYLENE IS NEVER USED OUTSIDE THE FUMEHOOD!
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LABORATORY NOTEBOOKS
Laboratory notebooks are used by scientists to record the day to day happenings in their
research laboratories. It is essential that all experiments be documented as clearly as possible
such that another individual could, from the lab notes, carry out the same experiment and obtain
the same results. This means that everything from initial preparation and planning through
execution and interpretation should be in a permanently bound lab notebook. The keeping of
such laboratory notebooks is very skill dependent and require lots of practice.
The laboratory manual for this course is designed to help you start developing your lab
note-taking skills. While not in the traditional form of a laboratory notebook the pages of the lab
manual are to be kept in a folder (or binder) that allows you to easily add and remove pages. This
folder/binder MUST contain all of your lab written work and MUST be submitted at the end of
the semester for final assessment.
The following are the broad headings that the lab personnel will use to assess (mark) your
lab written work. In the earlier labs examples of each pre-lab section will be given but as the
semester progresses you will be required to do these.
General Guidelines
1.
2.
3.
All notes should be in ink with the exception of figures (drawings and graphs) which
should be in pencil. Handwriting must be legible.
Do not erase or scribble over errors, simply cross out the incorrect text with a single line.
Always make sure to write the date for each entry associated with a given lab.
Guidelines for each Lab
1. Pre-lab section;
-Date of entry
-Background notes; includes only information from your text and/or lecture notes that is
directly related to the purpose(s) of the lab.
-Purpose of the lab. State the problem/ question from each exercise clearly and concisely.
-Prediction of expected results. Based on your background reading, what do you expect the
answer to the problem(s) to be?
-Methods and Materials. Briefly explain the general principle(s) behind the method. This is
also a good place to note any steps in the procedure that you do not understand the purpose of so
as to remind you to ask lab personnel for clarification.
UP TO AND INCLUDING THE METHODS AND MATERIALS SHOULD BE COMPLETED
BEFORE YOUR LAB PERIOD. You must get the completed pre-lab signed by one of the lab
staff prior to beginning the lab; this ensures that you will be given credit for having prepared for
lab.
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2. In Lab section:
-Date of entry
-Notes from lab introductory talk (if any).
-Note any deviation from procedure, both intentional and accidental( goes in Method
section)
-Results. Here you record any and all observations made during the lab including titled Tables,
Figures (graphs, sketches or biological drawings) and written description. Note: Tables are titled
at the top of the Table while Figures (including graphs) are titled at the bottom of the figure. Raw
data must be processed and presented in a concise fashion that clearly show the outcome of the
observations or experiment. In this section you will also include a concise, clear summary
statement of the major trend illustrated in the results obtained.
-Discussion. Here you state conclusions based on the results obtained (i.e. what is the answer to
the question posed in the purpose? Is this the answer you expected?) and you briefly discuss the
rationale that supports these conclusions.
______________________________________________________________________________
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Lab 1
MICROSCOPY
Before lab read: Appendix A1-A29 in your textbook, Becker et al. 8th edition.
Microscopy is essential to cell biology because cells and their components are too small
to distinguish with the unaided eye. The microscopes most commonly used by cell biologists to
examine cell structure are those that use direct imaging methods. These microscopes use a source
of illumination (either photons or electrons) focussed via a system of lenses on a specimen to
produce actual images of the specimen.
Throughout the Biology 2060 laboratory sessions you will be using the compound light
microscope. In this laboratory session you will determine:
(i)
(ii)
(iii)
(iv)
(v)
(vi)
how images are formed
the factors that influence resolution
the relationship between magnification and image size
How immersion oil contributes to the high resolving ability of the oil immersion
lens.
the relationship between contrast and specimen thickness
compare the resolution of the compound light microscope with the transmission
electron microscope
LAB EXERCISE 1: Determination of Objective Lens Resolving Ability.
1.
Obtain a microscope from the cabinet at the back of the room, sign the appropriate space
(corresponding to the number on the arm of the microscope) on the card pasted to the
front of the cabinet. This will be the microscope you will use for Biology 2060
laboratories during the semester.
Familiarize yourself with the location and function of:
a)
the ocular and objective lenses,
b)
the stage,
c)
the mechanical stage adjustment knobs,
d)
the knob to adjust focus of the condenser lens,
e)
the iris diaphragm and the lever or knob to adjust the iris diaphragm,
f)
the field diaphragm,
g)
the light source; select a setting near 8.
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The compound microscope you will be using has 4 objective lenses and 2 ocular lenses.
The names of the objective lenses, with the magnification indicated parenthetically, are low (4x),
medium (10x), high dry (40x) and oil immersion (100x). The ocular lenses have a magnification
of 10x. One ocular lens is adjustable and can be focussed by turning the ocular base, the other is
fixed (cannot be focussed). What is the total magnification for each objective?
Low power = ______ Medium Power = _______
High Power = _________
Oil immersion = ________
When using the oil immersion objective (100x) always put a drop of immersion oil
between the specimen and the objective lens. Immersion oil should not be used with viewing
under the high dry objective (40x). Both high dry and oil immersion objectives require that the
condenser lens is engaged and focussed. Clean the lenses frequently only with lens paper and
lens cleaning solution which are supplied.
NOTE: Compound light microscopes are expensive pieces of equipment that require proper
cleaning and handling. Students will be responsible for the care of their assigned
microscope.
Any problems with your microscope should be reported immediately.
2.
Examine the objective lenses and Figure 1.1 (pg. 12).
3.
The line of inscribed letters and a number closest to the base of each lens will read either
EA or D Plan followed by the magnifying ability 4, 10, 40 or 100.
4.
The next inscribed line of numbers is the numerical aperture (NA) of the lens. When light
hits a specimen, some light passes through straight while some is at an angle, bent by the
specimen. The finer the specimen details, the greater the angle of bending. These bent
rays are known as the image-forming rays. Numerical aperture is an expression of the
ability of an objective lens to collect these image-forming rays of light. The more the lens
can collect and then focus these rays, the clearer the image. Thus, the larger the N.A., the
greater the ability of the objective lens to collect the rays and therefore, the better its
resolving power. Record the NA for each of the lenses:
Low:________, Medium: ________, High dry: ________ and Oil:________
5.
The third line of inscribed numbers on all of the objective lenses (except low power) will
read 160/0.17. The 160 means that the distance from the objective flange and eyepiece
seating (i.e. the mechanical tube length) is 160mm and that the thickness of the coverslip
over the specimen is 0.17mm. Both of these conditions have to be met for the inscribed
NA to be accurate.
6.
A blue (why blue?) filter is contained just below the iris diaphragm in the condenser.
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7.Using the above information and equation below (called the Abbé equation),
r
0 .61
NA
determine and record the resolving ability of the low, medium, high dry and oil objective lenses
on your microscope. Show all calculations.
Low Power:
Medium Power:
High Power:
Oil immersion:
As can be seen in the Abbé equation, because N.A. is the denominator, it is the factor that affects
resolution the most because the larger the N.A., the smaller the resolution value which means
that objects can be closer together and still be resolved as distinct separate objects.
LAB EXERCISE 2: Calibration of the Microscope for Measurements
Measurement of microscopic specimens is relatively simple using an ocular micrometer.
A series of parallel lines is inscribed on a glass disc, the ocular micrometer, which is set into one
of the microscope eyepieces. The distance between the parallel lines can be determined using a
stage micrometer that has inscribed lines separated by a known distance. (See Figure 1.2, p. 14)
1.
Locate the micrometer scale in one ocular lens.
2.
Place the stage micrometer on the stage and focus on this scale using the medium power
objective while looking through the fixed ocular only.
3.
While looking through the adjustable ocular only adjust the ocular focus so the stage
micrometer is in focus. Now looking through both oculars the scales (eyepiece and stage)
should appear sharply defined.
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4.
Turn the ocular lens containing the micrometer scale (without moving the focus) so that
the scale lines lie parallel to the stage micrometer scale lines.
5.
Align them at the left hand side so that a scale line on both the stage and ocular
micrometers overlap then find a point along the scale where they align exactly again as in
Figure 1.1. Count the number of divisions of each.
Low Medium
6.
High
Oil
Record the number of ocular micrometer divisions:
_____ ______ _____
_____
Record the number of stage micrometer divisions:
_____ ______ _____ _____
Calculate the width inm of each ocular division for all four objective lenses.(N.B. each
smaller division on stage micrometer = 0.01 mm). Show all calculations and reasoning.
Low power objective:
Medium power objective:
High power objective:
Oil immersion objective:
You will use these ocular micrometer calibrations for all measurements during the
semester so make sure you record the width of each micrometer division for each objective.
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LAB EXERCISE 3: Comparison of images produced with the compound light and the
transmission electron microscopes
The preparation of specimens for microscopy depends very much on which aspect of the
cell the cell biologist is interested. In all cases the specimen must be thin enough for light waves,
when using light microscopy, or electrons, when using electron microscopy, to penetrate through
the specimen.
Living cells that are coloured or are big enough to readily cause perturbations in the wave
form of photons can be easily viewed with brightfield microscopes. Placing such specimens in a
drop of appropriate liquid on a glass slide and covering with a cover slip is all that is required to
view. Cells that do not have these properties can be prepared the same way but viewed using
either the phase-contrast or the differential interference contrast microscope, both of which
enhance contrast by exaggerating differences in refractive indices within the cell or at the surface
edges of the specimen respectively. Brightfield microscopes can also be used to view such
uncolored, small cells provided the contrast is enhanced by staining prior to microscopic
examination. The cellular detail of large specimens, that are too opaque to use in light
microscopy, can be discerned if the specimen is first sectioned, macerated or smeared to produce
a monolayer. This monolayer is essential if the structural detail of individual cells is to be
resolved upon microscopic examination. These monolayers can be viewed immediately once
placed on a glass slide and a coverslip added or can be further processed to impart more contrast
within the specimen and then viewed.
The preparation of cells for viewing with the transmission electron microscope is a much
more complex process. Cells must be fixed and embedded in a material that will not disintegrate
in a vacuum, yet can be easily sectioned into ultrathin sections using glass or diamond knives.
The cells are then impregnated with electron dense stains and placed on a copper grid in the
specimen chamber of an electron microscope. All air is removed from the chamber and a beam
of electrons is then focussed on the specimen using electromagnetic lenses that create the image.
Procedure:
1.
Prepare a wet mount of the chicken blood cells provided. To do this use a graduated
pipette to transfer 0.1ml of chicken blood to a test tube. Add 1.5 ml of physiological
saline, cover with parafilm and mix by gentle inversion. Use a Pasteur pipette to transfer
a drop of the resulting mixture to the center of a labelled glass slide. Holding a coverslip
at a 45˚ angle bring the edge of the coverslip to the edge of the drop then gently lower the
coverslip. Using a toothpick or the end of a dissecting needle rather than your finger to
hold the free edge of the angled coverslip will allow you to do this gently.
2.
Examine with the compound light microscope and sketch these small cells in the space
provided on page 22. Now critically illuminate (see Appendix I on page 68) the specimen
and sketch the critically illuminated cells in the space provided on page 22.
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3.
Make cross sections of soybean seed cells as follows:
Obtain a new single edge razor blade.
a)
Place a soybean seed in a watchglass and cover with distilled water.
b)
Remove the membranous seed coat and discard it. Press the seed gently between
thumb and forefinger. The seed will separate into two equal halves.
c)
Take one half and hold it between thumb and forefinger. Use the razor blade,
which is held in your other hand, to make small, thin cross sections (See Fig. 1.3,
p. 17). Dip the razor blade in the distilled water in the watchglass. Sections
should float off the razor blade into the water. Do not let the sections dry out.
The sections should appear translucent. Be careful not to cut your finger(s)
during the sectioning.
4.
Place a small drop of distilled water on a glass microscope slide and using forceps select
a thin, translucent section from the watch glass and place in the distilled water on the
microscope slide. Using the same procedure as outlined for animal cells and cover with a
coverslip.
5.
Examine with the compound light microscope, critically illuminate and find an area
where these large cells overlap and make a sketch in the space provided on page 23. Now
find a monolayer of cells and sketch these cells in the space provided on page 23.
6.
Locate the transmission electron micrographs of chicken red blood cells and soybean
cells in the binder on your bench top. Sketch these cells in the space provided on page 24.
Why is there more detail evident in these cells than the cells you saw with the compound
light microscope?
NOTE: while you are doing the sketches for this exercise keep in mind that you are trying to;
find the relationship between contrast and specimen thickness (purpose iii) and compare
resolution of the compound light and the transmission electron microscopes (purpose iv)! In
other words when observing these cells look critically at the cells and compare, paying close
attention to the amount of detail that is visible in each. Also, why were the chicken blood cells
harder to see than the soybean seed cells?
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Write-up Section:
Note that the background, purpose, predictions and material/methods provided are
examples only (to give you an idea of the level of work that we expect).
Background:
Resolution is the distance that two objects can be separated yet still distinguished as
separate. In microscopy this distance can be determined by the equation
r
0.61
NA .
If we minimise the numerator, by using the smallest wavelength of light, and maximise
the denominator, by using a lens with a large numerical aperture, then the best resolution
(smallest resolution distance) should be obtained. The resolving ability of a microscope lens is
considered better if two cell elements can be closer together (or in fact smaller) and still be
visible. The imaging beam used in transmission electron microscopy are electrons that have a
much shorter wavelength than light photon so the resolution of the electron microscope will be
much greater than a light microscope.
Magnification is the ability of lenses to create an image that appears to be proportionally
larger than the actual specimen while the area viewed is proportionally smaller.
Images in microscopy are formed by the specimen interfering with the light waves when
the specimen is placed in the light path. This interference creates an image as the light passes
through a series of lenses. Specimens that are too thick will not allow the light to pass through
and either no image will form or an image that has too much contrast. Specimens however can be
too small such that they do not cause perturbations in the light (little or no interference) and will
not provide enough contrast. In both cases the detail visible will be reduced due to poor contrast.
Purpose:
To determine:
(i)
(ii)
(iii)
(iv)
(v)
(vi)
how images are formed
the factors that influence resolution
the relationship between magnification and image size
How immersion oil contributes to the high resolving ability of the oil immersion
lens.
the relationship between contrast and specimen thickness
compare the resolution of the compound light microscope with the transmission
electron microscope
20
Predictions:
1.
If resolution is affected by wavelength of light and the numerical aperture then with a
constant wavelength the resolution will increase as the numerical aperture of the lens
increases.
2.
If image size increases as magnification increases then the width of the ocular
micrometer divisions will decrease as magnification increases since the image of the
stage micrometer will be increasing.
3.
If images are created by interference in the light waves passing through specimens then
specimens that are too thin or too thick will not provide good contrast so good detail and
clarity will not be evident in these specimens.
4.
If electrons have a shorter wavelength than light photons the transmission electron
micrographs should show greater detail than the images formed by the compound light
microscope.
Material and Methods:
The theoretical resolving ability of each objective lens is determined by using the
equation:
0.61
r
NA .
The wavelength () is known to be 450 nm; since a blue filter is contained in the light
path of the microscope and the numerical aperture (NA) that is inscribed on each lens are used.
The relationship between magnification and image size is determined by measuring the
width of divisions in an ocular micrometer against a calibrated stage micrometer at four different
magnifications; 40X, 100X, 400X and 1000X.
The relationship between contrast and specimen thickness is determined by preparing
temporary wet mounts of diluted chicken red blood cells, which will give a monolayer of very
small cells, and sections of soybean seed cells, which are large cells that will be in a monolayer
only at the edges of the section. The differences in the cell sizes and in monolayer vs. nonmonolayer will be visually compared to determine the amount of detail observable. These results
will also be compared with the amount of detail present in transmission electron micrographs of
the same cells.
Detailed procedures are on pages 10 to 18 of the Biology 2060 lab manual (2014).
21
Results:
Date: __________________
Construct a Table that clearly summarise the results from exercises 1 and 2.
22
Figure 1:Sketches of Chicken Erythrocytes to illustrate the effect of critical illumination on the
amount of detail visible in cells.
Note: viewed at 400X
23
Figure 2:
Sketches of Soybean Seed cells to compare the amount of detail visible in
monolayers and non-monolayers.
Note: viewed at 400X
24
Figure 3:
Sketches of Soybean Seed cells and Chicken Erythrocytes to illustrate the amount
of detail present in transmission electron micrographs.
25
Result summary statements:
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________.
Discussion:
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
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_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________.
_______________________________________________________________________
Reference: ______________________________________________________________
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Lab 2
Cell Structure and Measurement
This lab will not follow the usual write-up style since the purpose is to give you practice
at making microscopic observations and measurements and to become familiar with whole cell
structure. The biological drawings will be submitted at 4:50PM today.
One method used by cell biologists to record results is to make a biological drawing of
the cell in question. The more often you practice this skill the better your drawings will get and
the better honed your microscopic observational skills will become. The most difficult part of
making accurate biological drawings is to stop the “control” part of your brain from telling you
what you should be seeing and to let the part of your brain that makes accurate observations
move into dominance. To do this you must really concentrate focussing on the cell, moving
through all the different planes of focus, looking at not just the proportion of cell length to cell
width but also the relative proportion of all the internal cell structures/areas to each other and to
the cell. This can easily be achieved by measuring (remember you calibrated your eyepiece
micrometer last week) and recording the cells’ length and width as well as the size of internal
structures. Once this is done you can easily figure out the proportions, select how big you want
to make your drawing and then lightly draw the cell framework on unlined paper. Then with
constant back and forth between viewing the cell and drawing, you can fill in the cell detail. This
should give you a reasonably accurate representation of the cell.
Some other points to keep in mind when using biological drawings to record results:
- The drawing should be big enough to allow you to fit in all the cellular detail but not
so large that you exaggerate detail (usually 2 drawings per page)
- The long axis of drawing should be drawn on the long axis of the left-hand side of the
page (this leaves room for labels)
- Labels are placed to the right of the drawing at the end of ruler drawn lines and are
placed directly underneath one another.
- The drawing must be in scale i.e. the width and length of drawing must be same
proportion as actually exist and the organelles must be drawn in proportion. The scale
(in m/cm) must be included usually at the bottom right hand corner of the drawing
Some formulae you will use are: (also show calculations for actual sizes)
Scale= Actual length of specimen (µm)
Drawing length (cm)
Drawing width = actual width (µm)
Scale (µm
29
Organelle drawing length (State which organelle you are calculating!)
= actual organelle length (µm)
Scale (µm/cm)
Organelle drawing width (State which organelle you are calculating!)
= actual organelle width (µm)
Scale (µm/cm)
- Figure titles are placed at the bottom of each drawing. Each title, like all figure titles,
should contain enough concise information to allow the drawing to be understandable
by somebody else.
Materials: Prepared slides of the following:
Dicot leaf x.s. (only draw 1 palisade parenchyma cell (the main chloroplast
containing cell)).
Soybean seed x.s.
Chicken blood w.m. smear
Starfish oocytes w.m.
Sea urchin sperm w.m.
Procedure:
1.
Each of the slides presents a representative cell with various organelles of particular
interest. Observe each cell.
2.
Measure the appropriate dimensions of each cell and the main organelles. Record these
dimensions on pages 30 and 31.
3.
Make a biological drawing, following the guidelines given, of each cell type. Label the
drawings and indicate the magnification used to view each.
30
Results:
31
Results:
32
Results:
33
Results:
34
Results:
35
36
CELL FRACTIONATION (information for Lab 3 and 5)
Cell fractionation is used by cell biologists to investigate the structure and function of
organelles outside the complex environment of the intact cell. The general procedure consists of
disrupting cell boundaries to release cell contents and then centrifuging to separate the
organelles. The relative purity of each fraction can be confirmed by microscopic examination.
To prepare tissues for cell fractionation they must first be minced and then homogenized
to disrupt cell boundaries. This releases the cell constituents into an appropriate medium, usually
a buffered, isotonic salt or sugar solution. Why buffered and why isotonic?
The resulting suspension is called the homogenate.
The homogenate is then centrifuged repeatedly at successively higher speeds. As the
speed (measured in revolutions per minute = rpm) increases so does the centrifugal force (the
force of the earth's gravity exerted on a mass = g). Each cell component is of different size and
density so that with each increase in rpm (and subsequently g) progressively smaller particles
form a sediment.
The first centrifugation at low speed and short time usually sediments the nuclear
fraction. This pellet contains nuclei, intact cells and tissue debris. For further separation the post
nuclear supernatant, which consists of the suspended particles and liquid above the nuclear
pellet, is transferred to another centrifuge tube. This is spun at a higher speed for a longer time
to obtain the next cell constituent fraction. This procedure is repeated until the organelle of
interest is obtained.
You will use cell fractionation procedures in Lab 3 to obtain the chloroplast fraction from
pea seedlings and in Lab 5 to obtain the mitochondrial fraction from rat liver.
37
Lab 3
ISOLATION AND FUNCTION OF CHLOROPLASTS
Before lab read: pp. 293-305 in your text, especially Fig. 11.9.
LAB EXERCISE 1: Isolation of Chloroplasts
Chloroplasts are one of the largest organelles in plant cells. They can be isolated from
plant tissue with relative ease. Tissues are homogenated in a blender to release cell contents,
then filtered to remove larger plant tissue fragments. The homogenate is then centrifuged at low
rpm to form a pellet containing cell components heavier than the chloroplast (e.g. unbroken cells,
nuclei and large membrane fragments). The supernatant contains all other cell inclusions and is
centrifuged a second time at a higher rpm. The pellet formed during this centrifugation will
contain the chloroplasts. Where will organelles lighter than the chloroplasts be located at the end
of the second centrifugation?
Materials:
refrigerated centrifuge
1-2 week old pea seedlings
top loading balance
cheese cloth
0.05M phosphate buffer pH 7.3 (made to 0.4M sucrose and 0.01M KC1)
30 ml centrifuge tubes
blender
glass stirring rod
microscope slides
cover slips
Pasteur pipettes
150 ml beakers
50 ml graduated cylinders
containers of crushed ice
Procedures: Work in teams of four.
1.
Obtain 10g pea seedling leaves and homogenize in 50 ml ice-cold buffer (WHY ice
cold?) for 60 sec. in blender.
2.
Filter the suspension through 5 layers of cheese-cloth into a 150 ml beaker.
3.
Divide the homogenate equally between two 30 ml centrifuge tubes.
4.
Centrifuge for 5 min. at 1500 rpm.
38
5.
Decant the postnuclear supernatant into clean 30 ml centrifuge tubes (check to make sure
it contains chloroplasts and that they are intact).
6.
Balance the tubes by volume and centrifuge for 10 min. at 2000 rpm.
Pour off and discard the supernatant.
7.
Add 10 ml of ice-cold buffer to each centrifuge tube and resuspend the pellet using a
Pasteur pipette. Keep ice-cold!
8.
With a Pasteur pipette remove a small drop of chloroplast suspension and place on a glass
slide, cover with a cover slip. Under high-power magnification observe the suspension
for intact chloroplasts.
9
From the suspension sketch three chloroplasts that are of different orientations. In your
discussion explain what information your drawings give with respect to both the isolation
procedure and chloroplast structure.
10
Sketch a chloroplast from a transmission electron micrograph. Label the structural
features and indicate on the drawing where the catalytic activities in the following
experiment occur.
LAB EXERCISE 2: The Catalytic Properties of Isolated Chloroplasts
Photosynthesis is an oxidation-reduction process during which light energy is converted
to chemical energy. Light photons excite electrons in chlorophyll molecules; these electrons are
then used to reduce carbon dioxide to carbohydrates. Electrons are replaced in the chlorophyll
by a process called photolysis when an assembly of proteins and manganese ions, the oxygenevolving complex, catalyzes the oxidation of water to produce molecular oxygen, electrons and
protons.
Photolysis can be demonstrated by adding a redox dye to a suspension of isolated
chloroplasts providing the proper experimental controls are used. The dye simply acts as an
electron acceptor and changes color as it becomes reduced so to determine exactly why the dye is
being reduced the experiment must be set up so that results with the two major components that
affect the rate of electron production during photolysis eliminated are compared with results
obtained when these two components are functioning. You can determine these two components
by carefully reading the assigned text pages. The amount of color change can be measured using
a spectrophotometer (see Appendix II, p.69).
Materials:
chloroplast suspension
10 ml pipettes and 1 ml pipettes
10 ml test tubes
100oC water bath
0.05M phosphate buffer, pH 7.3
(made to 0.4M sucrose and 0.01M KC1)
0.1% 2,6-dichlorophenol-indophenol dye
light source and spectrophotometer
39
Procedure: Work in teams of two.
1
Turn on the spectrophotometer and set the wavelength to 620nm
2.
Pipette a 2.0 ml sample of the chloroplast suspension prepared in Exercise 11 into a glass
test tube. Place in a boiling water bath for 1.0 min. Cool immediately by placing in a
beaker of tap water.
3.
Label four test tubes and add the appropriate buffer/chloroplast suspension as follows:
Buffer (ml)
Chloroplast suspension (ml)
Chloroplast in light
9.0
1.0
Chloroplasts in dark
9.0
1.0
Boiled chloroplasts in light
9.0
1.0 (boiled)
Reagent Blank
9.2
1.0
4.
Transfer the contents of the reagent blank tube to a spectrophotometry tube, use this to set
the spectrophotometer at zero Absorbance.
5.
Pipette 0.2 ml of dye into the chloroplasts in light suspension, IMMEDIATELY transfer
this solution to a clean dry spectrophotometer tube, measure and record the absorbance
and time. Place the tube in a test-tube rack about 18 inches in front of a 100-watt
incandescent lamp.
6.
Repeat step 5 with boiled chloroplasts in light suspension, noting the time of adding the
dye and placing the tube in the same light.
6.
Repeat step 5 with chloroplasts in dark suspension, but after taking the initial absorbance
and time place this tube immediately in the dark.
7.
Measure the absorbance of each suspension at 5 min. intervals for 30 min. Before
each spectrophotometer reading, be sure that you shake the tube vigorously to
resuspend the chloroplasts uniformly and zero the reagent blank.
8.
When you have completed all measurements graph the results of your experiment and
explain these results in terms of photolysis.
40
WRITE UP:
Date:_____________
Background: (hint: read the purposes and predictions BEFORE you start, and explain the
structure of chloroplast and what is photolysis?)
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Purpose:
41
To determine: If chloroplasts can be isolated from cells and if so will these organelles be intact.
If isolated chloroplasts can carry out photolysis.
Predictions:
-If chloroplasts have a mass and density different than other cell organelles then
after cellular fractionation the resulting suspension will contain only chloroplasts.
- If these organelles are intact then they will be bean-shaped.
-If isolated chloroplasts can carry out photolysis then electrons should be
produced and the redox dye reduced by chloroplasts in the light but not by
chloroplasts in the dark or boiled chloroplasts in the light.
Material and Methods:
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Results:
Date: _________________________
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Results:
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Result Summary Statements:
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Discussion:
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47
Lab 4
The Scientific Method
As Science students, you are all aware that the scientific method is a procedure used to
test scientific hypotheses through observation and theory. This usually involves following
several steps with the goal to justify that the hypothesis formulated is supported. Because
Scientists generally think of the scientific method as a way of thinking rather than just following
a set of specific steps, not all scientists agree on the specific steps that should be taken.
Therefore, the outline below provides a brief description of one way to do this. Keep in mind that
other Science courses, including other Biology courses, may expect a different way of writing up
laboratory reports using the scientific method besides the one provided here.
The purpose of today’s lab is to help you understand and use the scientific method
outlined below in writing your Cell Biology lab reports.
Background: (also known as the Introduction or part of the Introduction).
This section of the scientific method usually includes a brief description of the organism,
organelle or process/principles which is being studied. The idea is to provide information to aid
in the understanding of the experiment and therefore should be directly related to the purpose(s)
of the experiment or lab exercise. The background must demonstrate that you understand the
context for the experiment or exercise that you're completing. For example, if you were looking
at how a specific drug affects metabolism in a particular species of fish, you may give a brief
description of what metabolism is, a description of the drug that’s being used and how it can
affect metabolism, specifically in fish of that species.
Purpose:
Generally this is just one sentence which gives a brief statement as to what you want
accomplished or what it is you want to find out from each exercise. An example could be:
“The purpose of the exercise is to determine if and how Drug X will affect the metabolism of
Fish (Species “Y”)”.
Prediction:
This is a brief statement indicating the expected results that may occur based on the
hypothesis or on the background information you have read.
Materials and Methods:
Completion of this section involves explaining the basic principle(s) involved in the
method. You DO NOT repeat the procedure from the lab manual, rather, a description of how the
results are going to be obtained and measured, if applicable, should be provided. This may
involve a certain technique, procedure or equipment which should be described. You must also
give the page numbers that the procedure is on in the lab manual as well as any changes to the
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procedure. For example, in the experiment looking at the effect of Drug X on the fish’s
metabolism, in the methods you may explain how you were going to be able to observe or
measure the effect of that drug on metabolism through the counting of opercular beats/minute as
compared to control fish and then how the use of opercular beats counts will be a method of
determining metabolism.
NOTE: The Background, Purpose, Prediction and Methods and Material sections are
completed BEFORE the lab period.
Results:
Rough or Raw Results may first be recorded on raw data sheets but the important
information from this data should then be recorded in a clear, concise manner that is easy to
interpret. Make sure the results are in order and that if you are doing more than one experiment,
the results from one experiment is all together and not mixed up with the results from another
experiment. Once that data is processed in some way, (for example, converting raw data to
percents, putting data into a nicely organized table, graphing raw data or using it in other
formulae, etc) it is placed under a “Processed Results” heading. If that Processed data is further
manipulated, say for example in constructing a graph of calculations, constructing a graph
comparing the differences between % changes between two different data sets or if the slope of a
graph line is determined, then this is called Analyzed Results.
Depending on what you are observing or measuring, there are several ways to record the
results. Generally, quantitative results and most qualitative results are tabulated. Ensure that the
tables are properly constructed, with explanatory titles (at the top of the table) and column
headings that are both easy to understand. Quantitative results can be further analyzed using
graphs. If so, ensure that the graph is easy to follow, has labeled axes and a descriptive title (Any
figures, including graphs, are titled at the bottom) which explains what the graph is representing.
If applicable, include a key that explains the different lines, bars, etc. of the graph (which must
be on proper graph paper).
Biological drawings and sketches are also used to record observations on biological
structure, both microscopically and macroscopically. It is important with drawings that you draw
what you can actually see from the specimen provided and not what you expect to see. Drawings
should be as accurate a representation of the specimen as possible. A specific title and
magnification are required, along with a size estimate or scale. Label as much on the drawing as
possible.
Results Summary or Statement:
The results summary/ statement is usually in the form of a statement where the general
trends seen by the results are given. You simply state in clear, concise words what the data is
showing. DO NOT interpret or explain the results here or go into any detail. Example: “The
results recorded in Table 1 show that fish exposed to Drug X had an increase in opercular beat
counts per minute as compared to fish not exposed to Drug X”.
49
Discussion:
This section is where you explain all results, being sure to refer to them, stating any
interpretations you have made on these results. A common mistake that many inexperienced
students make is to just restate the results as their discussions. You have to explain what the
results indicate and why the results are the way they are! Be sure to discuss the reasoning
behind your interpretations using appropriate terminology. You should state the answer to the
Purpose and tell if the results were or were not what you expected and why. Also, if a control
was used, include an explanation of any control(s) results and what the control(s) indicate.
References cited:
Throughout the lab report, including the Background, Discussion, etc, if you use
information from a textbook, scientific paper, the lab manual, etc, you must cite it within your
text using the Author, date method (as in Smith, 2012). Also, at the end of your lab report,
properly list your references, giving all pertinent information.
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WRITE UP:
Date:_____________
Background:
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Purpose:
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Predictions:
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Material and Methods:
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Results:
Date: _________________________
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Results:
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Result Summary Statements:
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Discussion:
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Lab 5
ISOLATION AND FUNCTION OF MITOCHONDRIA
Before the lab read: pp. 252-264, especially Figure 10-8 and pp. 267-275 in your text.
LAB EXERCISE 1: Isolation of Mitochondria
Mitochondria are found in almost all eukaryotic cells including both plants and animals.
In this lab rat liver tissue will be homogenized using a homogenising buffer containing EDTA
which inhibits autolytic enzymes. The homogenate will be centrifuged at 2000 rpm to obtain the
nuclear fraction. The postnuclear supernatant will be centrifuged at 10,000 rpm to obtain the
mitochondrial fraction.
Materials:
refrigerated centrifuge
pre-weighed portions of fresh rat liver
sucrose/EDTA/buffer pH 7.5
30 ml centrifuge tubes
blender
glass stirring rod
microscope slides
cover slips
Pasteur pipettes
50 ml beaker
50 ml graduated cylinders
Styrofoam containers of crushed ice
Janus Green B (0.01% in buffer)
Aceto -orcein stain
Procedure: Work in teams of four.
1.
Obtain 2g of pre-weighed liver portions. Cut the liver into small pieces using scissors
and decant off the coloured supernatant.
2.
Add 10 ml of fresh, cold (essential this time to slow reaction rates of autolytic enzymes)
homogenising buffer to the tissue pieces.
3.
Pour the contents of the beaker into a cold Blender cup. Rinse the beaker using 5 ml of
fresh, cold buffer.
4.
Homogenize for 1 min. at top speed.
5.
Transfer the homogenate to a cold 30 ml centrifuge tube. Rinse the blender cup several
times using a total of 10 ml of fresh, cold buffer. Add each rinse to homogenate in
centrifuge tube.
58
6.
Balance the tube by volume with that of another team and Centrifuge both at 2000 rpm
for 10 min.
7.
Pour the post nuclear supernatant into a clear centrifuge tube. Save the nuclear pellet to
make a smear in step 11.
8.
Centrifuge the supernatant (after balancing by volume with another team) at 10000 rpm
for 20 min. Decant the supernatant.
9.
Add 15 ml of ice-cold assay buffer to the pellet and resuspend the mitochondria using a
Pasteur pipette. Store on ice. Note: assay buffer does not contain EDTA! WHY NOT?
10.
Place a few drops of mitochondrial suspension in a clean test tube and add an equal
number of drops of Janus Green B solution. Shake and let stand for 10 minutes. Place a
drop of this solution on a microscope slide, add a coverslip and examine with the
microscope. Record the relative number and size of the mitochondria in your field of
view.
11.
Add 1.5 ml of homogenising buffer to the nuclear pellet and resuspend. Place a small
drop of the nuclear suspension on a clean microscope slide and add a drop of aceto-orcein
stain, cover with a coverslip and observe with the microscope. Compare the number and
size of the nuclei in your field of view with the previous mitochondria slide.
12.
Explain what these observations tell you about the isolation procedure and about the
structure of mitochondria and nuclei. Sketch a mitochondrion from a transmission
electron micrograph (your textbook has some good micrographs). Label the structural
features and indicate on the drawing where the catalytic activities in the following
experiment occur.
LAB EXERCISE 2: Catalytic Activity of Mitochondria
Cellular respiration is a complex series of reactions that break down organic molecules
(glucose, fatty acids etc.) to release cellular energy. The initial sequence of reactions, called
glycolysis, occurs in the cytoplasm. These reactions involve the breakdown of glucose to
pyruvate. In the presence of oxygen, pyruvate is decarboxylated to yield acetyl coenzyme A.
A second series of reactions, called the Krebs or TCA cycle, occur in the mitochondria.
These reactions oxidize acetyl coenzyme A to carbon dioxide and water. Electrons are generated
during this series of reactions that reduce coenzymes NAD+ and FAD to NADH and FADH2.
59
The final series of reactions, called electron transport, also occurs in the mitochondria.
These reactions remove the electrons from the reduced coenzymes transferring them to oxygen
and generating ATP in the process.
One of the best understood enzymes of aerobic respiration is succinate dehydrogenase.
The oxidation of succinate to fumarate in the Krebs cycle is catalyzed by this enzyme. Succinate
dehydrogenase is covalently bound to FAD so that the oxidation of succinate is coupled to the
reduction of FAD. The reduced FAD can then pass its electrons directly to the respiratory chain
where they are eventually transferred to molecular oxygen.
When supplied with succinate, isolated mitochondria are capable of carrying out
oxidation of succinate to fumarate. If the electron transport chain is blocked by sodium azide,
which inhibits cytochrome oxidase, an artificial dye may serve as an acceptor for the electrons in
reduced FAD. The use of an artificial dye as an electron acceptor is analogous to its use in
photolysis of photosynthesis (see Lab 3), however since many electron generating reactions can
occur in mitochondria more controls are necessary to eliminate all of the variables.
Materials:
mitochondria suspension
5 ml pipettes
1 ml pipettes
10 ml test tubes
100oC water bath
sucrose/phosphate buffer pH 7.5
0.1% 2, 6-dichlorophenol-indophenol dye
spectrophotometer
spectrophotometry tubes
succinate
Sodium azide
Procedure: Work in teams of two.
Note: turn on spectrophotometer and set wavelength to 620nm.
1.
Obtain 7.0 ml of cold mitochondria suspension prepared in Exercise 1. Keep on ice.
2.
Prepare five test tubes according to the following table.
60
Table 1: Setup for Succinate Dehydrogenase Assay
Quantity (ml)
Tube &
Contents
Reagent
Blank
Succinate
(the
substrate) +
NaAz (the
inhibitor)
NaAz
only
Succinate
only
boiled
assay buffer
4.0
3.0
3.5
3.5
3.0
succinate
0.5
0.5
-
0.5
0.5
sodium azide
0.5
0.5
0.5
-
0.5
-
1.0
1.0
1.0
1.0
redox dye
3.
Add 0.5 ml of mitochondrial suspension to the reagent blank. Cover with parafilm, invert
to mix. Pour contents into a spectrophotometry tube; use to set the spectrophotometer at
zero absorbance.
4.
Add 0.5 ml of mitochondria suspension to the tube containing the substrate Succinate and
the inhibitor NaAz. Quickly mix, pour into a spec. tube and immediately read and
record the Absorbance.
5.
Repeat step 4 with the NaAz only and the Succinate only setups.
6.
Pipette 1.0 ml of mitochondrial suspension into a clean test tube. Place in 100 o C water
bath for 1 min. Allow to cool then add 0.5 ml to the boiled setup, immediately read and
record the Absorbance.
7.
Read and record the Absorbance of each setup at 3 min. intervals for 21 minutes.
8.
Graph your results. Explain how these results illustrate the functioning of mitochondria.
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Write Up:
Background:
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Predictions:
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Materials and Methods:
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Raw Data
Date:______________
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Results:
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Results summary statements:
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APPENDIX I: CRITICAL ILLUMINATION
Many cell components you will be looking at this semester are near the lower limit of resolution
of the light microscope. Because of this it is essential that you focus the light uniformly on each
specimen that you view using the technique of Critical Illumination. In this process an image of
the light source is projected into the plane of the specimen, thus superimposing the light source
onto the specimen.This cuts down on diffraction and allows the best compromise between
resolution and contrast.
To adjust the compound light microscope for critical illumination:
1.
Focus the specimen at medium power.
2.
Close the field diaphragm (located in microscope base).
3.
Focus the condenser by moving the condenser knob until EITHER:
the outline of the field appears as a sharp hexagon, usually surrounded by
a blue halo;
OR:
the red halo surrounding the field just disappears and is replaced by a blue
halo.
4.
If field is not in center ask for help from laboratory personnel to adjust centering controls.
5.
Open the field diaphragm until the light just covers the full field observed.
6.
Adjust the iris (condenser) diaphragm.
\
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APPENDIX II: PRINCIPLES OF COLORIMETRY
Light is a form of radiant energy, and as such exists in many wavelengths. "White" light
is made up of all colors of the rainbow. These colors have different wavelengths, and when all
the colors are arranged according to wavelength, violet (400 nm) is at one end, it is followed by
blue, green, yellow, and orange to red( 700 nm). When a beam of light passes through a
solution, part of it is absorbed by the components of that solution. In addition, certain portions
(wavelengths) of the light beam are selectively absorbed. If the wavelength and intensity of light
entering a solution and the amount of light passing through, as recorded on a highly sensitive
photoelectric cell, are known, the requirements for quantitative measurements are met. A
mathematical relationship exists between the absorption, or transmission, of light by the solution.
This is the basis of spectrophotometry (colorimetry).
The following formulation can be made:
transmission = Is
Io
........... (2)
in which Is is the intensity of light after it has passed through the solution and Io is the intensity
of the incident beam.
The term transmission has limited application, however, since the beam of light passing
through a test tube or cuvette loses intensity at each of the interfaces between air, glass, and
water. There is also a certain amount of light intensity lost due to the dispersed or dissolved
material and due to the solvent itself. Because the material that is dissolved or dispersed is our
main interest, it is more convenient to use the formulation for transmittance, which describes the
ratio of light passing through the solution to the light passing through a blank:
transmittance = Is
Ib
.......... (3)
or
% T = Is x 100 ...... (4)
Ib
in which Is is the intensity of the light after it has passed through the solution, and Ib is the light
intensity of the beam which has passed through a blank. The blank is a tube that has the same
amount of solvent as the tube containing the solution to be tested, the only difference between
the blank and the unknown being the presence of the dissolved or dispersed material to be
measured. One great advantage of the transmittance, as opposed to transmission, is that the
intensity of the incident beam is not needed for calculations. Equation (4) is used if it is more
convenient to express the transmittance as a percent figure.
The term transmittance provides a quantitative tool for predicting the relationship
between concentration in solution and the behaviour of the light intensity. The plot of
transmittance vs. concentration, unfortunately, does not yield a straight-line relationship;
70
therefore, a logarithmic plot is used in order to obtain the straight line. The use of absorbencies
gets round this problem, as absorbance and concentration are arithmetically related. The
logarithm of the reciprocal of the transmittance is called the optical density (O.D.), or
absorbance, and is expressed as follows:
O.D. = log 1 .......... (5)
O.D. = log Ib .......... (6)
Is
or:
O.D. = log 100
%T
.......... (7)
Notice that when the percent transmittance is 100 (i.e. all light is passing through), the
O.D. is 0. Any decrease in the transmittance thus increases the optical density.
Absorbencies (and transmittances) are measured using a spectrophotometer, an
instrument that allows wavelengths to be selected, over a continuous range, by interposing a
light-dispersing prism or diffraction grating between the light source and the sample (see Figure).
Light of a selected narrow band of wavelength is thus permitted to pass through the sample.
Light not absorbed by the sample material is incident on the photosensitive surface of the
detector, which converts the light energy to an electric signal: this is displayed on the display
meter.
In order to obtain optical density (or transmittance) values for a test solution, it must be
directly measured against a reference (reagent) blank, which is identical to the test solution,
except that it lacks the light absorbing material to be measured. Both solutions must be
measured under identical conditions of wavelength, incident light intensity and light path length.
An alternative method, sometimes used, is to obtain absorbencies of: (a) the test substance
against a distilled water blank, (b) the reference blank, also read against a distilled water blank.
The difference between the test and reference blank absorbance can be calculated from (a) and
(b).