LABORATORY
1
3
Mitosis and
Meiosis
TEACHER’S MANUAL WITH STUDENT GUIDE
74-6450
74-6451
74-6455
8-Station Kit
1-Station Kit
Replacement Set
Units of Measure Useful in AP® Biology
Property Measured
Unit
Symbol
Description
Length
*meter
m
100 cm = 10 2 cm
centimeter
cm
0.01 m = 10 –2 m
millimeter
mm
0.001 m = 10 –3 m
micrometer
µm
10 –6 m = 10 –3 mm
nanometer
nm
10 –9 m = 10 –3 µm
*kilogram
kg
1000 g
gram
g
1000 mg
milligram
mg
0.001 g = 10 –3 g
microgram
µg
10 –6 g
Amount of Substance
*mole
mol
6.02 x 1023 particles (atoms, ions, or molecules)
Concentration of a Solution
mass percentage
%
Mass % = mass of solute/total mass of soln. × 100
parts per million
ppm
ppm of solute = mass of solute/total mass of soln. × 10 6
or 1 ppm = 1 mg solute/L soln.
Mass
Volume (gases and liquids)
molarity
M
Molarity = moles solute/L soln.
kiloliter
kL
1000 L
liter
L
1000 mL = 1 dm3 = 10 –3 m3
milliliter
mL
mL = cm3 = 10 –3 L
microliter
µL
10 –6 L = 10 –3 mL
Temperature (thermodynamic)
*kelvin
K
K = °C + 273
Temperature (common)
Celsius
°C
0 K = –273°C
Force
newton
N
kg•m/s2
Heat or Energy
joule
J
N•m
**calorie
cal
4.184 J
**Calorie (food)
Cal
1000 calories = 1 kcal
*second
s
60 s = 1 min
millisecond
ms
10 –3 s
pascal
Pa
N/m2 = kg/m•s2
**atmosphere
atm
101,325 Pa = 101.325 kPa = 760 torr = 14.7 lb/in2
Bar
bar
105 Pa
**Torr
torr
mm Hg = 133.3 Pa
Time
Pressure
* SI Base Unit
**Non-metric
The materials and activities in this kit meet the guidelines and academic standards of the Advanced Placement
(AP®) Program® and have been prepared by Carolina Biological Supply Company, which bears sole
responsibility for kit contents. Permission is granted to reproduce the Student Guide blackline masters at the
end of this manual for use with the materials provided in the accompanying CarolinaTM AP® Biology kit or
replacement set.
For complete listings of CarolinaTM AP® Science materials, including the Advanced Placement® Biology
Laboratory Manual for Teachers (RN-74-6681) and the Advanced Placement® Biology Laboratory Manual for
Students (RN-72-6682), log on to www.carolina.com/ or refer to the current CarolinaTM Science & Math
catalog or the current CarolinaTM Biotechnology & AP® Biology catalog.
Advanced Placement Program and AP are registered trademarks of the College Entrance Examination Board.
©2005 Carolina Biological Supply Company
Printed in USA
Laboratory 3. Mitosis and Meiosis
Overview
This lab consists of four parts. Activities A and C are the primary activities
and should be the focus of student efforts.
Activity A (Observing Mitosis): an observation-based introduction to mitosis
Activity B (Estimating the Relative Lengths of Mitotic Phases): estimating
and graphing the relative lengths of the stages of mitosis
Activity C (Simulating Meiosis): an introduction to meiosis using Carolina’s
Chromosome Simulation BioKit®
Activity D (Crossing-Over and Map Units): a study of crossing-over in meiosis
Objectives
• Observe mitosis in plant and animal cells
• Compare the relative lengths of the stages of mitosis in onion root tip cells
• Simulate the stages of meiosis
• Observe evidence of crossing-over in meiosis using Sordaria fimicola
• Estimate the distance of a gene locus from its centromere
Content
Standards
This kit is appropriate for Advanced Placement® high school students and
addresses the following National Science Content Standards:
Unifying Concepts and Processes
• Systems, order, and organization
• Evidence, models, and explanation
• Constancy, change, and measurement
Science as Inquiry
• Abilities necessary to do scientific inquiry
• Understandings about scientific inquiry
Life Science
• The cell
• Matter, energy, and organization in living systems
Time
Requirements
Activity A:
45 minutes
Activity B:
45 minutes
Activity C:
45 minutes
Activity D:
45 minutes
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Mitosis
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Meiosis
Safety
Use this kit only in accordance with prudent laboratory safety precautions,
including approved safety goggles, lab aprons or coats, and gloves. Know and
follow all school district guidelines for lab safety and for disposal of
laboratory wastes.
Preparation and
Presentation
Photocopy the blackline master Student Guide for each student or
group of students.
Note on Activity C: Activity C requires Carolina Biological Supply’s
Chromosome Simulation BioKit® (item RN-17-1100) or a similar product, not
included with this kit or replacement set. If you do not have the Chromosome
Simulation BioKit® or similar materials, obtain them before beginning Activity
C. If you already have the kit materials, you may need to order only the
Replacement Meiosis SG Pad, item RN-17-1102.
Ordering the Sordaria Demonstration Cross Plate
Return the request form for shipment of the Sordaria Demonstration Cross
Plate two weeks prior to the requested delivery date. Cultures are shipped on
Fridays only, and should arrive early the following week. They are ready for use
the following Wednesday, Thursday, or Friday. Note that this item is not
included with the 8-Station Replacement Set (RN-74-6455) and must be
ordered separately if you are replacing kit materials; order item RN-15-5846.
Ordering Onion Bulbs
If you plan to conduct the Optional Activity, Onion Mitosis, Squash Method,
return the request form for shipment of onion bulbs at least two weeks prior to
the requested delivery date.
The Mitosis CD
The mitosis CD contains images of onion cells and whitefish cells undergoing
mitosis. You can project these for simultaneous classroom viewing or set up a
computer monitor to display the images for students who are having difficulty
recognizing the phases of mitosis. The images on the CD may also be used to
visually quiz students’ ability to recognize the stages of mitosis.
Activity A: Observing Mitosis
It is easy for students to become so mired in the minutia of mitosis that they
lose sight of its overall function, which is to equally distribute to each daughter
cell a full set of chromosomes, and therefore a full set of DNA. Thus, this
activity emphasizes mitosis as a process. Trying to reconstruct the process of
mitosis from observing several static stages can be a bit like attempting to
reconstruct a play from a series of photographs. Although not required,
consider conducting the “Mitosis” exercise from the Chromosome Simulation
BioKit® as an enrichment exercise before students view the prepared slides of
onion and whitefish mitosis. A number of Internet sites now post animations
or video clips of mitosis.
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Although unlikely, it is possible that all stages of mitosis may not be seen on
each individual slide. However, all stages will be seen within the set of slides.
Students may need to share slides in order to see all stages of mitosis.
Some authors add a stage, prometaphase, between prophase and metaphase.
Prometaphase is usually defined by the breakdown of the nuclear envelope
and attachment of microtubules to the kinetochores. If your textbook adds
this intermediate stage, consider including it as part of Activity A.
Activity B: Estimating the Relative Lengths of Mitotic Phases
The onion root tip slides used in Activity A are also used for this activity.
Students must be able to recognize all stages of mitosis to complete the
activity. Each group should count the mitotic stages in at least three
nonoverlapping fields of view. High dry power (400× on most microscopes)
should be used when making the counts.
Make a table similar to the one shown below on the chalkboard or an
overhead to record data from the groups. Students should copy the data from
the “Class Totals” column into the corresponding column of data in Table 2 of
their Student Guide.
Group 1
Group 2
Group 8
Class Totals
Interphase
Prophase
Metaphase
Anaphase
Telophase
Activity C: Simulating Meiosis
Conduct Activity C using the materials included with the Chromosome
Simulation BioKit® and its accompanying Meiosis Student Guide. An
understanding of meiosis is crucial to understanding genetics and evolution.
Alleles segregate because they are located on homologous chromosomes. It is
the independent segregation of homologous chromosomes in meiosis and their
recombination in the zygote that produces most of the genetic variability in
populations. Natural selection works on this variability to transform a species.
An understanding of meiosis will form an important background for AP®
Biology Lab 6: Molecular Biology, Lab 7: Genetics of Organisms, and Lab 8:
Population Genetics and Evolution.
Activity D: Crossing-Over and Map Units
As noted above, you should allow at least two weeks notice for shipment of
your Sordaria Demonstration Cross Plate. Your plate will be shipped on a
Friday and should arrive the following Monday or Tuesday. Remove the tape
and incubate the plate at 25°C until used.
The cross has been set up so that the asci are ready for analysis
Wednesday–Friday of the week that the plate arrives; use dates are indicated
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Meiosis
on the plate. However, if the weather has been very cold, the asci may not be
mature until Friday. If this happens and you cannot use the plate on Friday,
refrigerate it over the weekend to keep the spores from discharging and use it
on Monday.
Check the culture each day to see if it is ready for classroom use. To determine
whether or not the asci are mature, place a few perithecia (the black spheres)
on a microscope slide, add a drop of water, and press down on them gently
with a coverslip. The perithecia should break open with gentle pressure and
you should see asci with black and pale brown (tan) spores. If it takes a lot of
pressure to break the perithecia, they are not mature. If the spores are
greenish, they are not mature. If you do not see asci with spores, but instead
see only dark, sac-like perithecia, they are not yet mature. Continue to
incubate the plate, repeating this process until the asci mature. Once the asci
mature, use the culture. If you delay, the asci will discharge their spores. The
culture can be refrigerated at this stage to arrest its development. See the
instructions included with your cross plate for more specific instructions.
The best place on the plates to find heterozygous asci is along the “X” where
the two strains come together, near the outside of the plate.
Make a table similar to the one shown here on the chalkboard or an overhead
to record data from the groups. All groups should copy the Class Totals into
Table 3 of their Student Guide. Notice that only hybrid asci are counted. A
total of 200 hybrid asci should be scored, and more is better. If you have a small
class, each group may have to count more that the recommended 50 asci.
Group 1
Group 2
Group 8
Class Totals
No. of MI Asci
No. of MII Asci
Students should understand that the map units they calculate are not defined
units of length as are inches or meters. They are relative measures, as “C is
farther from A than is B.” With the advent of molecular biology techniques, it
is possible to determine the actual physical distance between genes in terms of
base pairs. When a chromosome is studied by physical methods and genes are
located by restriction sites and numbers of base pairs, the resulting map is
called a physical map. When the order of genes and their relative distances
apart are studied by performing genetic crosses and analyzing frequency of
crossing-over, the resulting map is called a genetic map or linkage map.
Genetic maps have been around longer than physical maps, because
geneticists could perform crosses and analyze the results before physical
mapping methods were discovered. In the few cases in which detailed physical
maps have become available for comparison with detailed genetic maps of the
same organism, the correspondence has been good. The physical length of a
map unit has been found to vary between organisms, because overall rates of
crossing-over vary between organisms.
A frequently asked question is, “Why is the percent of crossing-over divided
by two to get map units for Sordaria? This is not done when determining map
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units for Drosophila.” Remember that in crossing-over, only two of the four
chromatids exchange material. In Sordaria, one crossover event produces one
crossover ascus; that is, all products of the crossover are counted as one. In
Drosophila, as in most organisms that have been used to generate genetic maps
from crossover frequencies, meiosis results in the production of four gametes
from one parent cell. Two of the gametes will carry the crossover and two will
not. Therefore, it is necessary to halve (divide by 2) the percent of crossover
asci of Sordaria to make the results comparable to those determined for
Drosophila, corn, and most other organisms.
Station Setup
Following is a list of the materials needed for one group of students to perform
the activities in this lab. Prepare as many setups as needed for your class.
Materials needed for the Optional Activities, Onion Mitosis, Squash Method
and Performing Sordaria Crosses, are listed with their respective protocols.
Activity Activity Activity Activity
D
A
B
C
microscope slide: onion root tip
1
microscope slide: whitefish blastodisc
1
*microscopes
2–4
1
2–4
2–4
mature Sordaria cross plate
1
slides and coverslips
4
pipet and water in plastic cup
1
*Chromosome Simulation BioKit ®
(beads, bags, string, and Student Guide
instructions; see kit for specifics)
1 set
*scalpel (for handling perithecia)
1
*Not supplied.
Sample Answers
to Questions in
the Student Guide
Activity A: Observing Mitosis
Analysis of Results
Instruction Note: Because different textbooks give slightly different details of
the events of mitosis, you may wish to alter or add to these questions. You may
also encounter some differences in terminology. For example, the centrosome
is sometimes referred to as the microtubule-organizing center.
1. Mitosis is much the same in the animal cells and plant cells you have
examined. What can you infer from this about the origins of mitosis?
Mitosis probably originated before the origin of plants and animals. Other
eukaryotic cells (fungi, protists, etc.) probably divide by mitosis also.
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2. List at least two ways that mitosis differs in the cells of animals and
higher plants.
(1) The centrosomes of animal cells have a pair of centrioles.
(2) In animal cells, cytokinesis results from the formation of a cleavage furrow.
In plant cells, cytokinesis occurs through the formation of a cell plate and growth
of a cell wall.
3. Describe what happens to each of the following during mitosis. Indicate
the phase(s) in which the changes occur.
a. nuclear envelope:
Fragments during prophase; reforms during telophase.
b. mitotic spindle:
Begins to form during prophase; some microtubules of the spindle attach to
the kinetochores of the chromosomes. During anaphase, microtubules
attached to kinetochores shorten, pulling apart the halves of the
chromosomes. During telophase, the spindle is disassembled and disappears.
c. chromatin:
Coils to form the chromosomes during prophase. Begins to uncoil during
telophase. Uncoiling continues into interphase.
d. centrosomes:
Move apart during prophase, possibly pushed by elongating spindle
microtubules. By metaphase, the centrosomes have reached opposite poles of
the cell.
e. nucleolus:
Disappears during prophase; usually reappears during telophase.
4. List the subphases of interphase and describe the important events that
occur during each.
G1 ; cell growth, including protein synthesis and production of cell organelles
S; cell growth continues and chromosomes are replicated
G2 ; cell growth continues
5. List at least two ways that prokaryotic cell division is similar to eukaryotic
cell division.
Answers may include: DNA replicates before division; a complete set of DNA is
distributed to each daughter cell; cytokinesis divides the cytoplasm.
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Activity B: Estimating the Relative Lengths of Mitotic Phases
Sample Table 2: Class Data
Class Totals
Decimal
Fraction of
Total Count
Estimated
Time Spent
in Phase
Interphase
2100
0.9
21.6
Prophase
117
0.05
1.2
Metaphase
47
0.02
0.48
Anaphase
23
0.01
0.24
Telophase
49
0.02
0.48
Total Cells
Counted
2336
Instruction Note: If desired, the estimated times can be converted from hours
to minutes by multiplying by 60.
Analysis of Results
1. Using the data from Table 2, construct a pie graph of the onion root tip
cell cycle showing the percent of time spent in each stage. Provide a title
and key for your graph.
Sample Pie Graph: Percent Time in Each Phase
of the Cell Cycle for Onion Root Tip Cells
Interphase 90%
Prophase 5%
Metaphase 2%
Anaphase 1%
Telophase 2%
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2. On the basis of your data, rank the stages of mitosis in order of time spent
in each phase.
Answers should reflect the data collected.
Results of the sample data:
1. interphase
2. prophase
3. metaphase/telophase (tie)
4. anaphase
3. On the basis of your observations in Activity A and information on the
events of mitosis from your textbook, explain why some phases are longer
than others. Refer specifically to each stage.
• Interphase is longest because there must be time to replicate the chromosomes
and to replace the cytoplasm, cytoplasmic organelles, etc.
• Prophase is next in length, due to the many events that take place: coiling
of chromosomes, disassembly of nuclear envelope, formation of the spindle
and asters.
• Metaphase: chromosomes are ready to divide.
• Telophase is largely the reverse of prophase. Chromosomes uncoil, the nuclear
envelope reforms, and cytokinesis begins.
• During anaphase, sister chromosomes move rapidly toward the poles.
Activity C: Simulating Meiosis
Analysis of Results
1. Returning to our example of a diploid organism with chromosomes,
A and B (n = 2), how many different combinations of these chromosomes
are possible in the gametes? (If necessary, use the figures below to diagram
the division that would give rise to the gametes.)
Four (4) combinations of the two chromosomes are possible.
2. Using your answer to 1 above, and given the following,
n (chromosome number)
Number of possible
combinations in the gametes
3
8
4
16
5
32
state a formula for calculating the number of possible chromosome
combinations in the gametes based on the value of n.
Number of chromosome combinations = 2n
Students may state this relationship in different forms, but the outcome of the
calculation should be the same.
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3. For humans, n = 23. Using your formula and a calculator, how many
possible combinations of chromosomes are there for human gametes?
8,388,608
4. For our hypothetical organism with two chromosomes, A and B, when two
members of the species reproduce, how many possible combinations of
chromosomes are there for the offspring?
16
5. Looking back at your answers to 1–4, what is the relationship of meiosis to
variation in populations (including human populations)?
Meiosis increases variation by reshuffling the chromosomes. Meiosis decreases
the likelihood that two offspring will have the same genetic makeup.
6. List at least three ways that meiosis differs from mitosis.
(1) Mitosis maintains chromosome number while meiosis reduces it.
(2) Mitosis produces two cells; meiosis produces four cells.
(3) Mitosis consists of one division; there are two divisions in meiosis.
Activity D: Crossing-Over and Map Units
Analysis of Results
Sample Table 3
Class Data
No. of
MI Asci
(4:4)
No. of
MII Asci
(2:4:2 or
2:2:2:2)
172
177
Total Asci
%MII Asci
(No. of
MII/Total)
Gene-toCentromere
Distance
(%MII/2)
349
51
26
1. Does crossing-over increase or decrease genetic variation?
Support your answer.
Crossing-over increases genetic variation by producing new combinations of the
genetic material.
2. A city creates a new lake for its water supply system. The lake is colonized
by two water plants, species A and species B. Species A reproduces
exclusively by means of buds that grow from rhizomes (runners). Species B
reproduces by budding but also reproduces by seeds, which involves sexual
reproduction. Given that for both species n = 7, would you expect to find
more genetic variation in the population of species A or species B? Explain
your answer.
Species B will have more genetic variation. Species A is reproducing asexually,
which means there is no recombination of genetic material from parent plant to
offspring. Species B is reproducing sexually, which means that there is a
recombination of chromosomes due to meiosis and fertilization of gametes from
parent to offspring. Crossing-over in meiosis will also produce new combinations
of genetic material that is linked on the chromosomes. [Note: It is possible to
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argue that not enough information is given to frame an answer. For example, the
ratio of asexual to sexual reproduction is not given for species B. If the ratio is
very low (1 in 10,000 for example), it may make no difference. It could also be
that the lake was colonized by one seed of species B and that seed came from a
highly inbred population.]
3. Suppose your Class Data from Table 3 showed 397 MI asci and 0 MII asci.
What would you conclude from this?
That the gene for spore color is too near its centromere for crossing-over to
occur.
Optional
Activities
If you wish to have students perform their own Sordaria crosses, see
“Performing Sordaria Crosses.” If you wish to have students prepare their own
slides to view mitosis in onion cells, see “Onion Mitosis, Squash Method.” If
you wish students to observe slides of meiosis, see “Observing Meiosis.”
Blackline masters of the student instructions for the latter activities can be
found following this section of the Teacher’s Manual, before the Student
Guide blackline masters. Photocopy and distribute these instructions as
needed.
Performing Sordaria Crosses
You can have students perform their own Sordaria crosses. All necessary
materials, including parent Sordaria cultures, media, and instructions, are
included in the Sordaria Genetics BioKit® (Carolina item RN-15-4847).
Onion Mitosis, Squash Method (Photocopy page OA-1)
Preparation
Rooting the onions: Twenty-four hours before class, peel six small pearl
onions, removing the dry, brown membrane and the outer layer of white onion
tissue. Cut off any green shoots. Wrap the onions in wet paper towels and
place them in an open plastic bag. Store them in a warm, dark place and keep
the paper towels moist. Use root tips that are about 1 mm long.
Instruction Note: The smaller the onion tip, the more mitotic cells your
students are likely to find.
Materials and Equipment
4 pearl onion bulbs
0.5% toluidine blue
1 M HCl
2 plastic transfer pipets
8 slides and coverslips
clothespin (for holding slides)
razor blade
*Bunsen burner, alcohol burner, or hot plate
* Not supplied.
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Station Setup
Distribute the 1 M HCl and 0.5% toluidine blue into six labeled containers
each, one per group. The 60-mL cups, labels, and solutions are supplied with
this kit.
Observing Meiosis (Photocopy page OA-2)
If you wish students to observe slides of meiosis, see Carolina’s Lily
Megasporogenesis Slide Set (item RN-29-3056), or individual slide listings for
lily ovulary cross sections. Several methods for producing meiotic smears from
the anthers of flowers have been published. The following method is adapted
from “Teaching Meiosis with Rhoeo discolor,” Carolina Tips, March 1, 1972,
and from “Spider Plant Meiosis,” Carolina Tips, November 1, 1973. You will
need immature anthers from flower buds, not open flowers. Once the flower
opens, most of the pollen will be mature and few if any cells will be undergoing
meiosis. In addition to Rhoeo discolor and Spider Plant, flower buds of
Wisconsin Fast PlantsTM, or flower buds from a florist can be used.
Submerge the buds in FAA fixative (RN-86-3593) or Carnoy fixative (RN-853253). Leave the buds in fixative for two days. After that, transfer the buds
into 70% ethanol (RN-86-1261) and store in a refrigerator at 0–4°C until
you are ready to use them. Although written for use with aceto-carmine
(RN-84-1423) or aceto-orcein (RN-84-1451) stains, you could use toluidine
blue with HCl as described in Onion Mitosis, Squash Method. Students may
have to make several slides before they master the technique.
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Optional Activity for
AP Biology Laboratory 3
®
Onion Mitosis, Squash Method
Introduction
You will prepare your own stained slides of onion root tips and then observe mitotic figures. Your teacher
has rooted onion bulbs in water. Growth of new roots is due to the production and elongation of new
cells; mitotic divisions are usually confined to the cells near the tip of the root. Follow the procedure
below to make your own root tip preparation.
Procedure
1. Obtain an onion bulb that is just beginning to show the emergence of roots. Cut off a root and lay it
on a microscope slide. Cut off the first 1–2 mm of the root tip; a dot-sized piece of root tip is all you
need. Discard the rest of the root. The mitotic cells are in the tip, so extra root tissue will only
interfere with finding mitotic cells.
2. Cover the root tip with two or three drops of 1 M HCl. Using a clothespin to hold the slide, warm
the slide by passing it back-and-forth over the flame of a Bunsen burner, (or an alcohol burner, or
over a hot plate) for five seconds. Wear safety glasses and gloves. You might smell a faint aroma of
cooking onion. If the onion turns brown or if the liquid boils away, stop and start over.
3. Use the edge of a paper towel to blot around the root and remove excess HCl. Cover the root tip
with 0.5% aqueous toluidine blue (use caution when handling HCl and toluidine blue). Wearing
safety glasses and gloves, pass the slide over the heat source again, two times, without boiling the
liquid. Let the slide stand for one minute.
4. Carefully blot around the root to remove excess stain. Add one drop of fresh toluidine blue stain
to the slide and then apply a coverslip. Place the slide, coverslip-side-up, between two layers
of paper towel on your laboratory bench. Using a pencil eraser or your finger, firmly but carefully
apply pressure to the coverslip in order to squash and spread the root tip tissue. Caution: Do not
break the coverslip.
5. Using your microscope (l0×), locate the meristematic region of the root tip. Examine the slide at
40× magnification and identify chromosomes at the various stages of mitosis.
6. Locate cells in prophase, metaphase, anaphase, telophase, and interphase. Look for evidence
of cytokinesis. If the slide is not satisfactory, repeat the procedure. Make sketches of these stages
of mitosis.
©2005 Carolina Biological Supply Company
OA-1
Optional Activity for
AP Biology Laboratory 3
®
Observing Meiosis
Procedure
1. Place a flower bud in a drop of 70% ethanol in the bottom of a petri dish. Transfer the dish to the
stage of a stereomicroscope. Use dissecting needles to tease apart the bud, exposing the anthers.
Dissect out 2–3 intact anthers, being careful that you do not allow them to dry out. Transfer the
anthers into a drop of aceto-carmine stain on a clean microscope slide.
2. Using a dissecting needle, break open the anthers and squeeze out the cells (developing pollen)
contained in the pollen sacs into the stain. Remove as much of the ruptured anther walls as possible
with the needle tip and discard them.
3. Place a clean coverslip over the drop of aceto-carmine stain.
4. Caution: Wear safety glasses and gloves for this step. Use a clothespin to hold the slide. Warm the slide
by passing it back-and-forth over the flame of a Bunsen burner (or alcohol burner or hot plate) for
five seconds. Do not allow the stain to boil.
5. Place some paper towel or other absorbent paper over the coverslip. Use your thumb or a pencil
eraser to press down on the paper. Press firmly, but be careful not to break the coverslip. Do not
push the coverslip sideways. If you do, the cells will roll up into tight bundles. You want to flatten
the cells so the chromosomes are more easily seen.
6. Examine the slide under a microscope. If you see cells with chromosomes, switch to high power. You
may not be able to identify every stage of meiosis, but you may see several stages. Depending on the
species of plant you are using, you may also see some abnormal divisions in which the homologous
chromosomes do not separate properly.
OA-2
©2005 Carolina Biological Supply Company
Name/Group #
Date
Student Guide
AP® Biology Laboratory 3
Mitosis and Meiosis
Objectives
• Observe mitosis in plant and animal cells
• Compare the relative lengths of the stages of mitosis in onion root tip cells
• Simulate the stages of meiosis
• Observe evidence of crossing-over in meiosis using Sordaria fimicola
• Estimate the distance of a gene locus from its centromere
Background to Activities A and B
Cells are the basic units of life, and cell division is the basic event that perpetuates life. Individual cells
do not live forever; millions of the cells in your body die every day. You keep on living because new cells
arising from cell division replace those that die. One-celled organisms reproduce their kind through cell
division, while multicellular organisms grow from single cells by repeated cell divisions. This event—the
reproduction of cells—is a fundamental characteristic of life.
A cell’s ability to exist and function is largely dependent upon the proteins it is able to make. Each
protein molecule consists of a long chain or chains of amino acids. The amino acids used to produce the
chains and their sequence in the chains helps determine the structure and function of the protein. One
amino acid chain might go into the formation of a molecule of catalase; another into a molecule of
tubulin. The amino acids used to produce a chain and their sequence within the chain is determined by
the sequence of bases (C, G, A, T) within the cell’s DNA. The loss of even a small amount of DNA can
be fatal. For example, if a human zygote (the single cell that grows into an embryo) is missing the DNA
necessary to make hemoglobin, it will never be able to develop into a fetus. Therefore, when a cell
divides, it is essential that it provide each new cell (daughter cell) with a complete set of DNA. To do
this, the cell must replicate (produce a duplicate copy of) its DNA before it divides. When division
begins, the DNA is packaged into bodies called chromosomes. Because the DNA has been replicated,
each chromosome consists of two identical halves, the sister chromatids. As the cell divides, the
identical halves of each chromosome are separated. One half goes into one daughter cell and its twin
goes into the other daughter cell. As a result, both daughter cells have a complete and identical set of
DNA. This process of dividing double chromosomes to achieve an equal distribution of DNA to the
daughter cells is termed mitosis.1
1 The two identical halves of a chromosome are called chromatids as long as they are attached to one another, but become
themselves chromosomes once they are separated. This is simply a matter of terminology and does not involve some magical
transformation of the chromatids themselves.
©2005 Carolina Biological Supply Company
S-1
Activity A: Observing Mitosis
Materials
Prepared microscope slides of onion mitosis and whitefish mitosis, microscopes.
Introduction
In this activity, you will observe cells that were undergoing mitosis when they were killed and stained.
Although it is really a continuous process, mitotic cell division is usually described in four stages:
prophase, metaphase, anaphase, and telophase. A fifth stage, interphase, describes the nondividing cell.
You will observe mitosis in prepared slides of onion root tips and whitefish blastodisc. You are likely to
find mitotic cells throughout the blastodisc. In the root tip, examine the area adjacent to the root cap.
These are rapidly growing tissues, and you should see many cells in each stage of mitosis.
Procedure
Use the illustrations on the “Plant Cell Mitosis and Cytokinesis” page to help you identify the stages of
mitosis that you find on your slides. On the pages following, make your own detailed drawings and make
notes that will help you understand what is happening during each stage of mitosis. Remember that the
phases of mitosis flow into each other, so you will likely see intermediate stages that are not shown in the
diagrams. Refer to your textbook or other resource for detailed descriptions of the stages, but remember
that your goal is to understand mitosis as a process. Once you understand the process, you can
concentrate on the details.
©2005 Carolina Biological Supply Company
S-2
Plant Cell Mitosis and Cytokinesis
Interphase
A
Prophase
Prometaphase
Early Anaphase
Late Anaphase
B
C
D
Late Metaphase
Interphase
(Daughter Cells)
Early Telophase
Late Telophase
E
A. Nucleolus
D. Cell Wall
B. Centromere
E. Cell Plate
C. Cell Membrane
©2005 Carolina Biological Supply Company
S-3
Mitosis Observation: Interphase Cells
In the spaces provided below, on the basis of your observations, draw a plant cell and an animal cell in
interphase. Use the lines underneath each illustration to record notes about what is happening during
interphase.
Interphase Cells
Plant Cell
©2005 Carolina Biological Supply Company
Animal Cell
S-4
Mitosis Observation: Prophase Cells
In the spaces provided below, on the basis of your observations, draw a plant cell and an animal cell
in prophase. Use the lines underneath each illustration to record notes about what is happening
during prophase.
Prophase Cells
Plant Cell
©2005 Carolina Biological Supply Company
Animal Cell
S-5
Mitosis Observation: Metaphase Cells
In the spaces provided below, on the basis of your observations, draw a plant cell and an animal cell
in metaphase. Use the lines underneath each illustration to record notes about what is happening
during metaphase.
Metaphase Cells
Plant Cell
©2005 Carolina Biological Supply Company
Animal Cell
S-6
Mitosis Observation: Anaphase Cells
In the spaces provided below, on the basis of your observations, draw a plant cell and an animal cell in
anaphase. Use the lines underneath each illustration to record notes about what is happening during
anaphase.
Anaphase Cells
Plant Cell
©2005 Carolina Biological Supply Company
Animal Cell
S-7
Mitosis Observation: Telophase Cells
In the spaces provided below, on the basis of your observations, draw a plant cell and an animal cell
in telophase. Use the lines underneath each illustration to record notes about what is happening
during telophase.
Telophase Cells
Plant Cell
©2005 Carolina Biological Supply Company
Animal Cell
S-8
Mitosis Observation: Daughter Cells
In the spaces provided below, on the basis of your observations, draw plant daughter cells and animal
daughter cells. Use the lines underneath each illustration to record notes about the characteristics of
daughter cells.
Daughter Cells
Plant Cell
©2005 Carolina Biological Supply Company
Animal Cell
S-9
Analysis of Results, Activity A: Observing Mitosis
Use your observations of mitosis and your textbook or other sources to answer the following:
1. Mitosis is much the same in the animal cells and plant cells you have examined. What can you infer
from this about the origins of mitosis?
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
2. List at least two ways that mitosis differs in the cells of animals and higher plants.
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
3. Describe what happens to each of the following during mitosis. Indicate the phase(s) in which the
changes occur.
a. nuclear envelope: _______________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
b. mitotic spindle: _________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
c. chromatin: ____________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
d. centrosomes: ___________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
e. nucleolus: _____________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
©2005 Carolina Biological Supply Company
S-10
4. List the subphases of interphase and describe the important events that occur during each.
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
5. List at least two ways that prokaryotic cell division is similar to eukaryotic cell division.
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
Activity B: Estimating the Relative Lengths of Mitotic Phases
Materials
Prepared microscope slides of onion mitosis, microscopes.
Introduction
In this activity, you will estimate the relative duration of each phase of mitosis in onion root tip cells.
The assumption is that the number of cells observed to be in a phase is related to the amount of time
spent in that phase. For example, if phase A lasts two minutes and phase B lasts one minute, the ratio of
observed A to observed B would be 2:1.
Procedure
1. Using the low-power objective (10×), locate the area of cell division. Shift to the high power
objective (40×), and count the number of cells that are in each stage of mitosis (interphase,
prophase, metaphase, anaphase, and telophase).
2. Repeat this count in at least two more nonoverlapping fields of view. Record your data in Table 1.
Table 1: Group Count
Number of Cells
Field 1
Field 2
Field 3
Total 1–3
Interphase
Prophase
Metaphase
Anaphase
Telophase
©2005 Carolina Biological Supply Company
S-11
Table 2: Class Data
Class Totals
Decimal
Fraction of
Total Count
Estimated
Time Spent
in Phase
Interphase
Prophase
Metaphase
Anaphase
Telophase
Total Cells
Counted
3. Record the class totals for each phase in Table 2. Calculate the decimal fraction of the total counted
for each phase and record it in Table 2 under Decimal Fraction of Total Count.
4. Given that it takes on average 24 hours for onion root tips to complete the cell cycle, calculate the
average time spent in each phase as follows and record answers in Table 2.
Fraction of cells in phase × 24 hrs = Estimated Time Spent in Phase
Analysis of Results, Activity B: Estimating the Relative Lengths of Mitotic Phases
1. Using the data from Table 2, construct a pie graph of the onion root tip cell cycle showing the
percent of time spent in each stage. Provide a title and key for your graph.
Pie Graph
Title: ____________________________________________________________
©2005 Carolina Biological Supply Company
S-12
2. On the basis of your data, rank the stages of mitosis in order of time spent in each phase.
1. ________________________ (most time)
2. ________________________
3. ________________________
4. ________________________
5. ________________________ (least time)
3. On the basis of your observations in Activity A and information on the events of mitosis from your
textbook, explain why some phases are longer than others. Refer specifically to each phase.
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
Background to Activity C
As you have seen, mitosis maintains the chromosome number (and DNA content) from one
generation of cells to the next. A second type of nuclear division is required in the life cycles of
sexually reproducing organisms.
Consider a sexually reproducing animal with two chromosomes, A and B. An animal of this species will
possess two copies of each chromosome. This is because it receives one chromosome A and one
chromosome B from each parent. Thus, it would have chromosomes A1A2 and B1B2. An organism with
two sets of chromosomes (2n) is said to be diploid in chromosome number or, simply, diploid. The
chromosomes of a pair are said to be homologous; that is, highly similar to each other. If chromosome A1
has the DNA needed for the production of catalase, chromosome A2 will have the same (or highly
similar) DNA. Reproductive cells (gametes, egg and sperm in animals; spores in plants) result from
meiosis, a type of cell division that reduces chromosome number by separating the homologues. Meiosis
accomplishes this reduction in an orderly manner such that our hypothetical diploid animal with
chromosomes A1A2 and B1B2 produces gametes that are AB and not A1A2 or B1B2. Thus, reproductive
cells have one set of chromosomes and are haploid (n) in chromosome number. (Rare events called
chromosomal nondisjunctions can alter this pattern, producing, using our example, gametes with
A1A2B; A; B; or AB1B2 chromosomes.)
Meiosis involves two nuclear divisions, designated meiosis I (or MI) and meiosis II (or MII). The
reduction of chromosome number occurs in meiosis I. Meiosis II is essentially a mitotic division.
©2005 Carolina Biological Supply Company
S-13
Activity C: Simulating Meiosis
Materials
Chromosome sets and Meiosis Student Guide from the Chromosome Simulation BioKit®.
Introduction
You will use chromosome models to simulate meiosis. Follow the instructions in the Meiosis Student
Guide to complete this activity.
Analysis of Results, Activity C: Simulating Meiosis
1. Returning to our example of a diploid organism with chromosomes, A and B (n = 2), how many
different combinations of these chromosomes are possible in the gametes? (If necessary, use the
figures below to diagram the division that would give rise to the gametes.)
___________ combinations of the two chromosomes are possible.
A1
A2
B1
B2
Figure 1
2. Using your answer to 1 above, and given the following,
n (chromosome number)
Number of possible
combinations in the gametes
3
8
4
16
5
32
state a formula for calculating the number of possible chromosome combinations in the gametes
based on the value of n.
Number of chromosome combinations = _____________
3. For humans, n = 23. Using your formula and a calculator, how many possible combinations of
chromosomes are there for human gametes? ____________
4. For our hypothetical organism with two chromosomes, A and B, when two members of the species
reproduce, how many possible combinations of chromosomes are there for the offspring? ____________
©2005 Carolina Biological Supply Company
S-14
A1
A2
B1
B2
A3
A4
B3
B4
×
Figure 2
5. Looking back at your answers to 1–4, what is the relationship of meiosis to variation in populations
(including human populations)?
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
6. List at least three ways that meiosis differs from mitosis.
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
Background to Activity D
When homologous chromosomes pair in prophase I of meiosis, they can exchange parts, which is called
crossing-over. When chromosomes exchange parts, genetic material is transferred from one chromosome
to another. This alters expected inheritance patterns. For example, the fruit fly, Drosophila, has four
chromosomes. Chromosome 2 carries a gene for eye color. The normal form of this gene (Pr, the “wildtype”) produces flies with red eyes, but there is another form or “allele” of the gene (pr) that produces
purple eyes. On the same chromosome is another gene that effects wing form. The normal wild-type
allele (Vg) produces normal wings while the alternate form (vg) produces vestigial wings, which are
smaller and useless for flight. Because these genes are located on the same chromosome, they are
considered “linked” and are inherited together. For example, if one chromosome of a homologous pair
carries the alleles for red eyes and normal wings and its homologue carries the alleles for purple eyes and
vestigial wings, we would expect half the gametes to have the linked alleles for red eyes and normal
wings and half the gametes to have the linked alleles for purple eyes and vestigial wings.
©2005 Carolina Biological Supply Company
S-15
If crossover occurs between the locations (gene loci) of the genes for eye color and wing type, there will
be two new combinations of alleles in the gametes: red eyes with vestigial wings, and purple eyes with
normal wings (Fig. 3).
pr
vg
pr
vg
pr
vg
pr
vg
Vg
Vg
Vg
vg
pr
Pr
pr
Pr
Pr
vg
Pr
Vg
Pr
Vg
Pr
Vg
Figure 3. Crossing-over involving genes for eye color and wing type
The farther apart two gene loci are on a chromosome, the more likely it is that a crossover will occur
between them. By counting the frequency of crossover events between two gene loci, geneticists can
determine the relative distance between them. In this way, linkage maps have been produced for many
organisms, including Drosophila and even humans. In Activity D, you will observe the results of crossingover for a spore color gene. You will collect data on the frequency of crossover for this gene and
calculate the relative distance of the gene locus from its centromere.
Activity D: Crossing-Over and Map Units
Introduction
Sordaria fimicola is a common species of ascomycete that grows on the dung of herbivores. Eight spores
(ascospores) are produced in an ascus. Many asci are grouped together within a vase-shaped structure
called a perithecium. Two nuclei within a developing ascus fuse to produce a diploid (2n) nucleus. This
diploid nucleus then undergoes meiosis, followed by a mitotic division to produce eight ascospores in a
linear series within the ascus (Figure 4).
Nuclear
Fusion
n+n
Mitotic
Div.
Meiosis II
Div.
Meiosis I
Div.
2n
2n
n
n
n
Figure 4. Development of ascospores
©2005 Carolina Biological Supply Company
S-16
The order of the ascospores in the ascus reflects the order in which the chromosomes are segregated
during meiosis. This can be clearly visualized if the diploid nucleus is a hybrid for two different spore
colors. The wild-type gene (+) produces a dark spore, while the mutant tan gene (t) produces a light
spore. If crossing-over does not occur, these genes segregate during meiosis I to produce a 4:4 sequence
of ascospores (Figure 5). However, if crossing-over does occur, the genes do not segregate until meiosis
II, producing a 2:2:2:2 or 2:4:2 sequence of ascospores (Figure 6).
Meiosis I Div.
Meiosis II Div.
Mitotic Div.
t
t
t
t
t
t
+
+
OR
+
+
+
+
Figure 5. Production of MI asci
Meiosis I Div.
Meiosis II Div.
Mitotic Div.
t
t
t
+
+
+
t
t
t
OR
OR
OR
+
+
+
Figure 6. Production of MII asci
Procedure
1. Use a scalpel to remove several perithecia from either area A or area B of the cross plate culture
(Figure 7).
2. Make a wet mount of the perithecia and gently press the coverslip with your thumb or an eraser
until the perithecia are crushed. This will release clusters of asci.
3. Using the low power of a microscope, search for hybrid asci (light and dark spores) and determine in
which area of the cross plate they are found.
4. After locating hybrid asci, use high dry magnification to count the number of MI and MII asci.
5. Count at least 50 hybrid asci and record the results in Table 3.
6. Calculate the percent MII and gene-to-centromere distance in map units. Record this data in Table 3.
©2005 Carolina Biological Supply Company
S-17
Figure 7. Sordaria cross plate
Analysis of Results, Activity D: Crossing-Over and Map Units
Table 3
No. of
MI Asci
(4:4)
No. of
MII Asci
(2:4:2 or
2:2:2:2)
Total Asci
%MII Asci
(No. of
MII/Total)
Gene-toCentromere
Distance
(%MII/2)
Group Data
Class Data
1. Does crossing-over increase or decrease genetic variation? Support your answer.
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
©2005 Carolina Biological Supply Company
S-18
2. A city creates a new lake for its water supply system. The lake is colonized by two water plants,
species A and species B. Species A reproduces exclusively by means of buds that grow from rhizomes
(runners). Species B reproduces by budding but also reproduces by seeds, which involves sexual
reproduction. Given that for both species n = 7, would you expect to find more genetic variation in
the population of species A or species B? Explain your answer.
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
3. Suppose your Class Data from Table 3 showed 397 MI asci and 0 MII asci. What would you
conclude from this?
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
©2005 Carolina Biological Supply Company
S-19
CarolinaTM AP® Biology Lab Kits
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RN-74-6410
Lab 2. Enzyme Catalysis
RN-74-6430
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RN-74-6450
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RN-74-6470
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RN-74-6490
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pBLU® Colony Transformation
RN-21-1146
Restriction Enzyme Cleavage of DNA
RN-21-1149
Green Gene Colony Transformation
RN-21-1082
Colony Transformation
RN-21-1142
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RN-74-6530
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RN-74-6540
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RN-74-6570
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Lab 11. Animal Behavior
RN-74-6614
Lab 12. Dissolved Oxygen and Aquatic Primary Productivity RN-74-6630
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