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StudentGuide
AP'Biology Laboratory3
Mitosis and Meiosis
Objectives
Observe mitosis in plant and animal cells
Compare the reladve lengths of the stagesof mitosis in onion root tip cells
Simulate the stagesof meiosis
Observe evidence of crossing-over in meiosis using Sor&triafimicoln
Estimate the distance of a gene locus from its centromere
Background to Activities A and B
Cells are the basicunits of life, and ceil divisionis the basicevent that perpetuateslife. Individual cells
do not live forever; millions of the cells in your body die every day.You keep on living becausenew cells
arisingfrom cell divisionreplacethosethat die. One-celledorganismsreproducetheir kind through cell
division,while multicellularorganismsgrow from singlecellsby repeatedcell divisions.This event-the
reproductionof cells-is a fundamentalcharacteristicof life.
A cell'sabihty to exist and function is largelydependentupon the proteinsit is ableto make.Each
protein molecuieconsistsof a long chain or chainsof amino acids.The amino acidsusedto producethe
chainsand their sequencein the chainshelpsdeterminethe structureand function of the protein.One
amino acid chain might go into the formation of a moleculeof catalase;anotherinto a moleculeof
tubulin. The amino acidsusedto producea chain and their sequencewithin the chain is determinedby
the sequenceof bases(C, G, A, T) within the cell'sDNA. The lossof even a small amount of DNA can
be fatal. For example,if a human zygote(the single cell that growsinto an embryo) is missingthe DNA
necessary
to make hemoglobin,it will never be ableto developinto a fetus.Therefore,when a cell
divides,it is essentialthat it provideeachnew cell (daughterceli) with a completeset of DNA. To do
this, the cell must replicate(producea duplicatecopy of) its DNA beforeit divides.When division
Becausethe DNA hasbeen replicated,
begins,the DNA is packagedinto bodiescalledchromosomes.
identical
chromatids.
As the cell divides,the
halves,the sister
eachchromosomeconsistsof two
identicalhalvesof eachchromosomeare separated.One half goesinto one daughtercell and its twin
goesinto the other daughtercell. As a result,both daughtercellshave a completeand identicalset of
DNA. This processof dividing doublechromosomesto achievean equaldistributionof DNA to the
daushtercellsis termedmitosis.l
1 Th. t-o identical halves o{ a chromosome are called chromatids as iong as they are attached to one another, but become
thernselves chromosomes once they are separated. This is simply a matter of terminology and does not involve some magical
transformation of the chromatids themselves.
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Activity A: ObservingMitosis
Materials
Preparedmicroscopeslidesof onion mitosis and whitefish mitosis, microscopes.
Introduction
In this activity, you will observecells that were undergoingmitosis when they were killed and stained.
Although it is really a continuous process,mitotic cell division is usually describedin four stages:
prophase,metaphase,anaphase,and telophase.A fifth stage,interphase,describesthe nondividingcell.
You will observemitosis in preparedslidesof onion root tips and whitefish blastodisc.You are likely to
find mitotic cellsthroughout the blastodisc.In the root tip, examinethe areaadjacentto the root cap.
These are rapidly growing tissues,and you should seemany cells in each stageof mitosis.
Procedure
Use the illustrationson the "Plant Cell Mitosis and Cytokinesis"pageto help you identify the stagesof
mitosis that you find on your slides.On the pagesfollowing, make your own detailed drawingsand make
notesthat will help you understandwhat is happeningduring each stageof mitosis.Rememberthat the
phasesof mitosis flow into each other, so you will likely seeintermediate stagesthat are not shown in the
diagrams.Referto your textbook or other resourcefor detaileddescriptionsof the stages,but remember
that your goal is to understandmitosis as a process.Once you understand the process,you can
concentrateon the details.
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Plant Cell Mitosis and Cvtokinesis
Interphase
Prophase
Prometaphase
Late Metaphase
EarlyAnaphase
Late Anaphase
Interphase
(DaughterCells)
Early Telophase
Late Telophase
A. Nucleolus
D. Cell Wall
B. Centromere
E. Gell Plate
C. Cell Membrane
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Mitosis Observation: Interphase Cells
In the spacesprovidedbelow,on the basisof your observations,draw a plant cell and an animal cell in
interphase.Use the lines undemeatheachillustration to recordnotesabout what is happeningduring
interphase.
Plant Cell
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AnimalCell
9-4
Mitosis Observation: Prophase Cells
In the spacesprovidedbelow,on the basisof your observations,draw a plant cell and an animal cell
in prophase.Use the lines underneatheachillustration to recordnotesabout what is happening
during prophase.
Plant Cell
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AnimalCell
9-5
Mitosis Observation:MetaphaseCells
In the spacesprovidedbelow,on the basisof your observations,dtaw a plant cell and an animal cell
in metaphase.Use the lines undemeatheachillustration to recordnotesabout what is happening
during metaphase.
Plant Cell
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AnimalCell
9-6
Mitosis Observation:AnaphaseCells
In the spacesprovidedbelow,on the basisof your observations,
draw a plant cell and an animal cell in
anaphase'Use the lines underneatheach illustrationto record
notesabout what is happeningduring
anaohase.
Plant Cell
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AnimalCell
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Mitosis Observation:TelophaseCells
In the spacesprovidedbeloq on the basisof your observations,draw a plant cell and an animal cell
in telophase.Use the lines undemeatheachillustration to recordnotesabout what is happening
during telophase.
Plant Cell
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AnimalCell
5-b
Mitosis Observation:Daughter Cells
In the spacesprovidedbelow,on the basisof your observations,
draw plant daughtercellsand animal
daughtercells.Use the lines underneatheachillustration to recordnotesabout the characteristicsof
daughtercells.
PlantCell
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AnimalCell
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Analysis of Results,Activity A: ObservingMitosis
Use your observationsof mitosis and your textbook or other sourcesto answerthe following:
1. Mitosis is much the samein the animal cellsand plant cellsyou have examined.'S7hatcan you infer
from this about the origins of mitosis?
2. List at least two waysthat mitosis differs in the cells of animals and higher plants.
3. Describewhat happensto eachof the followingduring mitosis.Indicatethe phase(s)in which the
changes occur.
a. nuclear envelope:
b. mitotic spindle:
c. chromatin:
d. centrosomes:
e. nucleolus:
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4. List the subphasesof interphaseand describethe important events that occur during each.
5. List at leasttwo waysthat prokaryoticcell divisionis similarto eukaryoticcell division.
Activity B: Estimating the Relative Lengths of Mitotic Phases
Materials
Prepared microscope slides of onion mitosis, microscopes.
Introduction
In this activify,you will estimatethe relativeduration of eachphaseof mitosisin onion root tip cells.
The assumptionis that the number of cells observedto be in a phaseis related to the amount of time
spentin that phase.For example,if phaseA laststwo minutesand phaseB lasrcone minute, the ratio of
observedA to observedB wouldbe 2:1,
Procedure
1. Using the low-powerobjective (10x), locate the areaof cell division.Shift to the high power
objective(40x;, and count the number of cellsthat are in eachstageof mitosis(interphase,
prophase,metaphase,anaphase,and telophase).
2. Repeatthis count in at leasttwo more nonoverlappingfieldsof view.Recordyour data in Table1.
Table 1: Group Count
Number of Cells
Field1
Field2
Field3
Total 1-3
Interphase
Prophase
Metaphase
Anaphase
Telophase
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Table 2: Glass Data
ClassTotals
3. Recordthe classtotals for eachphasein Table2. Calculatethe decimalfraction of the total counted
for each phaseand record it in Thble 2 under Decimal Fraction of Total Count.
4. Given that it takeson average24 hoursfor onion root tips to completethe cell cycle,calculatethe
averagetime spent in each phaseasfollows and record answersin Thble 2.
Fractionof cellsin phasex24 hrs : EstimatedTime Spentin Phase
Analysis of Results, Activity
B: Estimating
the Relative Lengths of Mitotic
Phases
1. Using the data from Gble 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:
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2. On the basisof your data, rank the stagesof mitosisin order of time spentin eachphase.
1.
(mosttime)
7.
3.
4.
5.
(least time)
3. On the basisof your observationsin Activity A and information on the eventsof mitosisfrom your
textbook,explainwhy somephasesare longerthan others.Referspecificallyto eachphase.
Background to Activity C
As you have seen,mitosismaintainsthe chromosomenumber (and DNA content) from one
generationof cellsto the next. A secondtype of nucleardivision is requiredin the life cyclesof
sexuallyreproducingorganisms.
Considera sexuallyreproducinganimal with two chromosomes,
A and B. An animal of this specieswill
possess
two copiesof eachchromosome.This is becauseit receivesone chromosomeA and one
chromosomeB from eachparent.Thus, it would have chromosomes
A1A2 and BlB2.An organismwith
(2n)
two setsof chromosomes
is saidto be diploid in chromosomenumber or, simpiy,dipioid. The
chromosomesof a pair are saidto be homologous;that is, highly similar to each other.If chromosomeA1
has the DNA neededfor the production of catalase,chromosomeA2 will have the same(or highly
simiiar) DNA. Reproductivecells (gametes,eggand spermin animals;sporesin plants) resultfrom
meiosis,a type of cell divisionthat reduceschromosomenumber by separatingthe homologues.Meiosis
accomplishes
this reductionin an orderlymanner such that our hypotheticaldiploid animal with
chromosomes
A1A2 and Bl82 producesgametesthat areAB and not AlA2 or Bl82. Thus, reproducrive
cellshave one set of chromosomesand are haploid (n) in chromosomenumber.(Rareeventscalled
chromosomalnondisjunctionscan alter this pattern, producing,usingour example,gameteswith
A1AZB;A; B; or AB1B2chromosomes.)
Meiosisinvolvestwo nucleardivisions,designatedmeiosisI (or MI) and meiosisII (or MII). The
reductionof chromosomenumber occursin meiosisI. MeiosisII is essentiallya mitotic division.
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Activity C: Simulating Meiosis
Materials
Simulation
BioKlt@.
Chromosome
setsand MeiosisStudentGuidefrom the Chromosome
Introduction
modelsto simulatemeiosis.
Followthe instructions
in the MeiosisStudent
Youwill usechromosome
Guide to complete this activiry.
Analysisof Results,Activity C: SimulatingMeiosis
A and B (n : 2), how many
1. Returningto our exampleof a dlploid organismwith chromosomes,
different combinationsof thesechromosomesare possiblein the gametes?(lf necessaryusethe
figuresbelow to diagram the division that would give rise to the gametes.)
combinationsof the two chromosomesare possible.
A1
82
B1
A2
fl} n
1i l 1
fifl]
nt$$Iil,|
\1
w ffi K d b
Figure1
2. Usingyour answerto 1 above,and given the following,
n (chromosomenumber)
Number of possible
combinations in the gametes
3
8
4
16
5
32
statea formula for calculatingthe number of possiblechromosomecombinationsin the gametes
basedon the valueofn.
Number of chromosomecombinations:
3. For humans,n: 23. Usingyour formula and a calculator,how many possiblecombinationsof
chromosomesare there for human gametesl
4. For our hypothetical organism with two chromosomes, A and B, when two members of the species
reproduce,how many possiblecombinationsof chromosomesare there for the offspring?
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A1
A2
81
A3
82
A4
83
84
ffiwww ffiffiffiw
Fi gure 2
5. Looking back at your answers to 1-{, what is the relationship of meiosis to variation in populations
(including human populations)I
6. List at least three ways that meiosis differs from mitosis.
Background to Activity D
pair in prophaseI of meiosis,they can exchangeparts,which is called
When homologouschromosomes
exchangeparts,geneticmaterialis ffansferredfrom one chromosome
chromosomes
crossing.over.'When
has four
to another.This altersexpectedinheritancepatterns.For example,the fruit fly,Drosophiia,
Chromosome2 carriesa genefor eyecolor.The normal form of this gene (Pr, the "wildchromosomes.
type") producesflieswith red eyes,but there is anotherform or "allele" of the gene (pr) that produces
purple eyes.On the samechromosomeis another gene that effectswing form. The normal wild-type
allele (Vg) producesnormal wingswhile the altemateform (o'g)producesvestigialwings,which are
smallerand uselessfor flight. Becausethesegenesare locatedon the samechromosome,they are
considered"linked" and are inherited together.For example,if one chromosomeof a homologouspair
carriesthe allelesfor red eyesand normal wingsand its homologuecarriesthe allelesfor purpleeyesand
vestigialwings,we would expecthalf the gametesto have the linked allelesfor red eyesand normal
wings and half the gametesto have the linked allelesfor purple eyesand vestigial wings.
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If crossoveroccursbetweenthe locations(geneloci) of the genesfor eyecolor and wing rype,there will
be two new combinationsof allelesin the gametes:red eyeswith vestigialwings,and purple eyeswith
normal wings (Fig.3).
pr
Vg
pr
vg
edt==:
Vg
7,-.-\-*"=*"----f
l_!
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 crossing.
over for a spore color gene. You will collect data on the frequency of crossover for this gene and
calculate the relative distance of the eene locus from its centromere.
Activity D: Crossing.Overand Mup Units
Introduction
Sordrtria
fimicolnis a common speciesof ascomycetethat growson the dung of herbivores.Eight spores
(ascospores)
are producedin an ascus.Many asciare groupedtogetherwithin a vase-shaped
structure
calleda perithecium.Jwo nuclei within a developingascusfuseto producea diplold (2n) nucleus.This
diploid nucleusthen undergoesmeiosis,followedby a mitotic divisionto produceeight ascospores
in a
linear serieswithin the ascus(Figure4).
Nuclear
Fusion
A*n
Meiosis I
Div.
t\
fJ* il*
n+ n
Meiosis ll
Div.
I\
l,tll
lvu
I
tl
lt
t,
tt
H
2n
\.
I\,/l
it
lol
I N/ J
\i
\/
4P
--->
/4'\ \
at'
Mitotic
Div.
--+
F
-**
l^-1
--+
-*
->,
Figure 4. Development of ascospores
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The order of the ascospores
in the ascusreflecrcthe order in which the
chromosomes
are segregated
during meiosis'This can be clearlvvisualized
if rhe diploid ;r;1"r, is a hybrid fo. tro
Jiff".".,,
,por"
colors'The wild-rypegene (+) producesa
dark ,pore, .hil" th" L.,tu.rt tan gene (r) produces
a
light
spore'If crossing-overdoesnot occur,these
genessegregate
during meiosisI to producea 4:4 sequence
of ascospores
(Figure5)' However,if crossing-orr".
do", o..ur, the genesdo not segregate
until meiosis
il, producing a 2:2:2:2or 2:4:2sequence
of ascospores
(Figure6).
Meiosist Div,
Meiosistt Div.
)G=H
*\i4
MitoticDiv.
OR
-fti{
Figure5. Productionof Mlasci
Meiosis I Div.
t
I-.,
Meiosis il Div.
Mitotic Div.
-\#
*ArL
t
L)€:=
Figure 6. production of Mll asci
Procedure
1' Use a scalpelto removeseveralperithecia
from either areaA or areaB of the crossplate
culture
(Figure7).
2' Make a wet mount of the peritheciaand gently
pressthe coverslipwith your thumb or
an eraser
until the peritheciaare crushed.This wilirelease
clustersor"r.t.
3' Using the low power of a-microscope,
searchfor hybrid asci (light and dark spores)
and determinein
which areaof the crossplate they ur" found.
4. After locatinghybrid asci,usehigh dry magnification
to count the number of MI and MII asci.
5. count at least50 hybrid asciand record
the resurtsin Table3.
6. calculate the percentMII and gene-to-centromere
distancein map unirs. Recordthis data
in Thble3.
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Figure 7. Sordaria cross plate
Analysis of Results, Activity D: Crossing.Over
and Map Units
Table 3
No. of
MlAsci
14:41
No. of
Mll Asci
(2:4:2or
2:2:2:21
Total Asci
% Ml l A sci
(No. of
Mll/Total)
Gene-toCentromere
Distance
(o/oMlll2l
Group Data
Class Data
1. Doescrossing-over
increaseor decreasegeneticvariation?Supportyour answer.
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Z. A crttycreatesa new lake for its water supply system.The lake is colonizedby two water plants,
speciesA and speciesB. SpeciesA reproducesexclusivelyby meansof budsthat grow from rhizomes
(runners).SpeciesB reproducesby buddingbut alsoreproducesby seeds,which involvessexual
reproducdon. Given that for both speciesn - 7, would you expect to find more genetic variation in
the populationof speciesA or speciesB? Explainyour answer.
'What
3. Supposeyour ClassData from'Iable 3 showed397 MI asciand 0 MII asci.
would you
conclude from this?
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Optional Activity for
AP' Biology Laboratory3
Onion Mitosis, SquashMethod
Introduction
Youwill prepareyour own stainedslidesof onion root tips and then observemitotic figures.Your teacher
has rooted onion bulbsin water.Growth of new roots is due to the production and elongationof new
cells;mitotic divisionsare usuallyconfinedto the cellsnear the tip of the root. Follow the procedure
belowto makeyour own root tip preparation.
Procedure
L. 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 {irst l-7 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 ciothespin to hold the slide, warm
the slide by passingit back-and-forth over the flame of a Bunsen bumer, (or an alcohol burner, or
over a hot plate) for five seconds. \Uear safety glassesand gloves. You might smell a faint aroma of
cooking onion. If the onion tums 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 excessHCl. Cover the root tip
'Wearing
with 0.57o aqueous toluidine blue (use caution when handling HCI and toluidine blue).
safery giassesand gloves, passthe 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 excessstain. Add one drop of fresh toluidine biue 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 pressureto the coverslip in order to squash and spread the root tip tissue. Cautionr Do not
break the couerslip.
5. Usingyour microscope (10x), locate the meristematic region of the root tip. Examine the slide at
40x magnification and identifi' chromosomes at the various stagesof 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.
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