Mitosis and Meiosis - BiologyAlive.com 1/13... · Laboratory 3. Mitosis and Meiosis Safety Preparation and Presentation. Although unlikely, it is possible that all stages of mitosis

TEACHER’S MANUAL WITH STUDENT GUIDE

LABORATORY 1

Mitosis andMeiosis

LABORATORY 3

74-6450 8-Station Kit74-6451 1-Station Kit74-6455 Replacement Set

©2005 Carolina Biological Supply Company Printed in USA

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 soleresponsibility for kit contents. Permission is granted to reproduce the Student Guide blackline masters at theend of this manual for use with the materials provided in the accompanying CarolinaTM AP® Biology kit orreplacement set.

For complete listings of CarolinaTM AP® Science materials, including the Advanced Placement® BiologyLaboratory Manual for Teachers (RN-74-6681) and the Advanced Placement® Biology Laboratory Manual forStudents (RN-72-6682), log on to www.carolina.com/ or refer to the current CarolinaTM Science & Mathcatalog or the current CarolinaTM Biotechnology & AP® Biology catalog.

Advanced Placement Program and AP are registered trademarks of the College Entrance Examination Board.

Property Measured Unit Symbol Description

Length *meter m 100 cm = 102 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

Mass *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. × 106

or 1 ppm = 1 mg solute/L soln.

molarity M Molarity = moles solute/L soln.

Volume (gases and liquids) 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

Time *second s 60 s = 1 min

millisecond ms 10–3 s

Pressure 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

* SI Base Unit

**Non-metric

Units of Measure Useful in AP® Biology

C a r o l i n a TM A P® T e c h S u p p o r t : 8 0 0 . 2 2 7 . 1 1 5 0 e x t 4 3 0 4 a n d e x t 4 3 8 1 T e a c h e r ’ s M a n u a l 3

This lab consists of four parts. Activities A and C are the primary activitiesand 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): estimatingand graphing the relative lengths of the stages of mitosis

Activity C (Simulating Meiosis): an introduction to meiosis using Carolina’sChromosome Simulation BioKit®

Activity D (Crossing-Over and Map Units): a study of crossing-over in meiosis

• 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

This kit is appropriate for Advanced Placement® high school students andaddresses 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

Activity A: 45 minutes

Activity B: 45 minutes

Activity C: 45 minutes

Activity D: 45 minutes

Laboratory 3. Mitosis and Meiosis

Overview

Objectives

ContentStandards

TimeRequirements

Use this kit only in accordance with prudent laboratory safety precautions,including approved safety goggles, lab aprons or coats, and gloves. Know andfollow all school district guidelines for lab safety and for disposal oflaboratory wastes.

Photocopy the blackline master Student Guide for each student or group of students.

Note on Activity C: Activity C requires Carolina Biological Supply’sChromosome Simulation BioKit® (item RN-17-1100) or a similar product, notincluded with this kit or replacement set. If you do not have the ChromosomeSimulation BioKit® or similar materials, obtain them before beginning ActivityC. If you already have the kit materials, you may need to order only theReplacement Meiosis SG Pad, item RN-17-1102.

Ordering the Sordaria Demonstration Cross Plate Return the request form for shipment of the Sordaria Demonstration CrossPlate two weeks prior to the requested delivery date. Cultures are shipped onFridays only, and should arrive early the following week. They are ready for usethe following Wednesday, Thursday, or Friday. Note that this item is notincluded with the 8-Station Replacement Set (RN-74-6455) and must beordered separately if you are replacing kit materials; order item RN-15-5846.

Ordering Onion BulbsIf 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 tothe requested delivery date.

The Mitosis CDThe mitosis CD contains images of onion cells and whitefish cells undergoingmitosis. You can project these for simultaneous classroom viewing or set up acomputer monitor to display the images for students who are having difficultyrecognizing the phases of mitosis. The images on the CD may also be used tovisually quiz students’ ability to recognize the stages of mitosis.

Activity A: Observing MitosisIt is easy for students to become so mired in the minutia of mitosis that theylose sight of its overall function, which is to equally distribute to each daughtercell a full set of chromosomes, and therefore a full set of DNA. Thus, thisactivity emphasizes mitosis as a process. Trying to reconstruct the process ofmitosis from observing several static stages can be a bit like attempting toreconstruct a play from a series of photographs. Although not required,consider conducting the “Mitosis” exercise from the Chromosome SimulationBioKit® as an enrichment exercise before students view the prepared slides ofonion and whitefish mitosis. A number of Internet sites now post animationsor video clips of mitosis.

4 T e a c h e r ’ s M a n u a l C a r o l i n a TM A P® T e c h S u p p o r t : 8 0 0 . 2 2 7 . 1 1 5 0 e x t 4 3 0 4 a n d e x t 4 3 8 1

L a b o r a t o r y 3 . M i t o s i s a n d M e i o s i s

Safety

Preparation andPresentation

Although unlikely, it is possible that all stages of mitosis may not be seen oneach 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 envelopeand attachment of microtubules to the kinetochores. If your textbook addsthis intermediate stage, consider including it as part of Activity A.

Activity B: Estimating the Relative Lengths of Mitotic PhasesThe 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 theactivity. Each group should count the mitotic stages in at least threenonoverlapping 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 anoverhead to record data from the groups. Students should copy the data fromthe “Class Totals” column into the corresponding column of data in Table 2 oftheir Student Guide.

Activity C: Simulating MeiosisConduct Activity C using the materials included with the ChromosomeSimulation BioKit® and its accompanying Meiosis Student Guide. Anunderstanding of meiosis is crucial to understanding genetics and evolution.Alleles segregate because they are located on homologous chromosomes. It isthe independent segregation of homologous chromosomes in meiosis and theirrecombination in the zygote that produces most of the genetic variability inpopulations. 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 UnitsAs noted above, you should allow at least two weeks notice for shipment ofyour Sordaria Demonstration Cross Plate. Your plate will be shipped on aFriday and should arrive the following Monday or Tuesday. Remove the tapeand incubate the plate at 25°C until used.

The cross has been set up so that the asci are ready for analysisWednesday–Friday of the week that the plate arrives; use dates are indicated



Group 1 Group 2 Group 8 Class Totals

Interphase

Prophase

Metaphase

Anaphase

Telophase

on the plate. However, if the weather has been very cold, the asci may not bemature 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 iton Monday.

Check the culture each day to see if it is ready for classroom use. To determinewhether 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 gentlywith a coverslip. The perithecia should break open with gentle pressure andyou should see asci with black and pale brown (tan) spores. If it takes a lot ofpressure to break the perithecia, they are not mature. If the spores aregreenish, they are not mature. If you do not see asci with spores, but insteadsee only dark, sac-like perithecia, they are not yet mature. Continue toincubate the plate, repeating this process until the asci mature. Once the ascimature, use the culture. If you delay, the asci will discharge their spores. Theculture can be refrigerated at this stage to arrest its development. See theinstructions included with your cross plate for more specific instructions.

The best place on the plates to find heterozygous asci is along the “X” wherethe two strains come together, near the outside of the plate.

Make a table similar to the one shown here on the chalkboard or an overheadto record data from the groups. All groups should copy the Class Totals intoTable 3 of their Student Guide. Notice that only hybrid asci are counted. Atotal of 200 hybrid asci should be scored, and more is better. If you have a smallclass, each group may have to count more that the recommended 50 asci.

Students should understand that the map units they calculate are not definedunits of length as are inches or meters. They are relative measures, as “C isfarther from A than is B.” With the advent of molecular biology techniques, itis possible to determine the actual physical distance between genes in terms ofbase pairs. When a chromosome is studied by physical methods and genes arelocated by restriction sites and numbers of base pairs, the resulting map iscalled a physical map. When the order of genes and their relative distancesapart are studied by performing genetic crosses and analyzing frequency ofcrossing-over, the resulting map is called a genetic map or linkage map.Genetic maps have been around longer than physical maps, becausegeneticists could perform crosses and analyze the results before physicalmapping methods were discovered. In the few cases in which detailed physicalmaps have become available for comparison with detailed genetic maps of thesame organism, the correspondence has been good. The physical length of amap unit has been found to vary between organisms, because overall rates ofcrossing-over vary between organisms.

A frequently asked question is, “Why is the percent of crossing-over dividedby two to get map units for Sordaria? This is not done when determining map



Group 1 Group 2 Group 8 Class Totals

No. of MI Asci

No. of MII Asci

units for Drosophila.” Remember that in crossing-over, only two of the fourchromatids exchange material. In Sordaria, one crossover event produces onecrossover ascus; that is, all products of the crossover are counted as one. InDrosophila, as in most organisms that have been used to generate genetic mapsfrom crossover frequencies, meiosis results in the production of four gametesfrom one parent cell. Two of the gametes will carry the crossover and two willnot. Therefore, it is necessary to halve (divide by 2) the percent of crossoverasci of Sordaria to make the results comparable to those determined forDrosophila, corn, and most other organisms.

Station SetupFollowing is a list of the materials needed for one group of students to performthe activities in this lab. Prepare as many setups as needed for your class.Materials needed for the Optional Activities, Onion Mitosis, Squash Methodand Performing Sordaria Crosses, are listed with their respective protocols.

Activity A: Observing Mitosis

Analysis of ResultsInstruction Note: Because different textbooks give slightly different details ofthe events of mitosis, you may wish to alter or add to these questions. You mayalso encounter some differences in terminology. For example, the centrosomeis sometimes referred to as the microtubule-organizing center.

1. Mitosis is much the same in the animal cells and plant cells you haveexamined. What can you infer from this about the origins of mitosis?Mitosis probably originated before the origin of plants and animals. Othereukaryotic cells (fungi, protists, etc.) probably divide by mitosis also.



ActivityD

ActivityC

ActivityB

ActivityA

microscope slide: onion root tip

microscope slide: whitefish blastodisc

*microscopes

mature Sordaria cross plate

slides and coverslips

pipet and water in plastic cup

*Chromosome Simulation BioKit ®

(beads, bags, string, and Student Guide instructions; see kit for specifics)

*scalpel (for handling perithecia)

1

1

2–4

1

2–4

1

1 set

1

1

2–4

4

*Not supplied.

Sample Answersto Questions inthe Student Guide

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 growthof a cell wall.

3. Describe what happens to each of the following during mitosis. Indicatethe 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 tothe kinetochores of the chromosomes. During anaphase, microtubulesattached to kinetochores shorten, pulling apart the halves of thechromosomes. During telophase, the spindle is disassembled and disappears.

c. chromatin: Coils to form the chromosomes during prophase. Begins to uncoil duringtelophase. Uncoiling continues into interphase.

d. centrosomes: Move apart during prophase, possibly pushed by elongating spindlemicrotubules. By metaphase, the centrosomes have reached opposite poles ofthe cell.

e. nucleolus: Disappears during prophase; usually reappears during telophase.

4. List the subphases of interphase and describe the important events thatoccur during each.G1; cell growth, including protein synthesis and production of cell organellesS; cell growth continues and chromosomes are replicatedG2; cell growth continues

5. List at least two ways that prokaryotic cell division is similar to eukaryoticcell division.Answers may include: DNA replicates before division; a complete set of DNA isdistributed to each daughter cell; cytokinesis divides the cytoplasm.



Activity B: Estimating the Relative Lengths of Mitotic Phases

Sample Table 2: Class Data

Instruction Note: If desired, the estimated times can be converted from hoursto minutes by multiplying by 60.

Analysis of Results

1. Using the data from Table 2, construct a pie graph of the onion root tipcell cycle showing the percent of time spent in each stage. Provide a titleand key for your graph.

Sample Pie Graph: Percent Time in Each Phase

of the Cell Cycle for Onion Root Tip Cells



Estimated

Time Spent

in Phase

Decimal

Fraction of

Total Count

Class Totals

Interphase

Prophase

Metaphase

Anaphase

Telophase

Total Cells

Counted

2100

117

47

23

49

2336

0.9

0.05

0.02

0.01

0.02

21.6

1.2

0.48

0.24

0.48

Interphase 90%

Prophase 5%

Metaphase 2%

Anaphase 1%

Telophase 2%

2. On the basis of your data, rank the stages of mitosis in order of time spentin each phase.Answers should reflect the data collected.Results of the sample data:

1. interphase 2. prophase3. metaphase/telophase (tie)4. anaphase

3. On the basis of your observations in Activity A and information on theevents of mitosis from your textbook, explain why some phases are longerthan 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 spindleand 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 chromosomesare possible in the gametes? (If necessary, use the figures below to diagramthe 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,

state a formula for calculating the number of possible chromosomecombinations 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 thecalculation should be the same.



n (chromosome number)Number of possible

combinations in the gametes

3

4

5

8

16

32

3. For humans, n = 23. Using your formula and a calculator, how manypossible combinations of chromosomes are there for human gametes?8,388,608

4. For our hypothetical organism with two chromosomes, A and B, when twomembers of the species reproduce, how many possible combinations ofchromosomes are there for the offspring? 16

5. Looking back at your answers to 1–4, what is the relationship of meiosis tovariation in populations (including human populations)?Meiosis increases variation by reshuffling the chromosomes. Meiosis decreasesthe 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

1. Does crossing-over increase or decrease genetic variation? Support your answer.Crossing-over increases genetic variation by producing new combinations of thegenetic material.

2. A city creates a new lake for its water supply system. The lake is colonizedby two water plants, species A and species B. Species A reproducesexclusively by means of buds that grow from rhizomes (runners). Species Breproduces by budding but also reproduces by seeds, which involves sexualreproduction. Given that for both species n = 7, would you expect to findmore genetic variation in the population of species A or species B? Explainyour 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 tooffspring. Species B is reproducing sexually, which means that there is arecombination of chromosomes due to meiosis and fertilization of gametes fromparent to offspring. Crossing-over in meiosis will also produce new combinationsof genetic material that is linked on the chromosomes. [Note: It is possible to



Gene-to-

Centromere

Distance

(%MII/2)

%MII Asci

(No. of

MII/Total)

Total Asci

No. of

MII Asci

(2:4:2 or

2:2:2:2)

No. of

MI Asci

(4:4)

Class Data 172 177 349 51 26

argue that not enough information is given to frame an answer. For example, theratio of asexual to sexual reproduction is not given for species B. If the ratio isvery low (1 in 10,000 for example), it may make no difference. It could also bethat the lake was colonized by one seed of species B and that seed came from ahighly 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 tooccur.

If you wish to have students perform their own Sordaria crosses, see“Performing Sordaria Crosses.” If you wish to have students prepare their ownslides to view mitosis in onion cells, see “Onion Mitosis, Squash Method.” Ifyou wish students to observe slides of meiosis, see “Observing Meiosis.”Blackline masters of the student instructions for the latter activities can befound following this section of the Teacher’s Manual, before the StudentGuide blackline masters. Photocopy and distribute these instructions asneeded.

Performing Sordaria Crosses You can have students perform their own Sordaria crosses. All necessarymaterials, including parent Sordaria cultures, media, and instructions, areincluded in the Sordaria Genetics BioKit® (Carolina item RN-15-4847).

Onion Mitosis, Squash Method (Photocopy page OA-1)

PreparationRooting the onions: Twenty-four hours before class, peel six small pearlonions, removing the dry, brown membrane and the outer layer of white oniontissue. Cut off any green shoots. Wrap the onions in wet paper towels andplace them in an open plastic bag. Store them in a warm, dark place and keepthe paper towels moist. Use root tips that are about 1 mm long.

Instruction Note: The smaller the onion tip, the more mitotic cells yourstudents are likely to find.

Materials and Equipment

4 pearl onion bulbs0.5% toluidine blue1 M HCl2 plastic transfer pipets8 slides and coverslipsclothespin (for holding slides)razor blade*Bunsen burner, alcohol burner, or hot plate

* Not supplied.



OptionalActivities

Station SetupDistribute the 1 M HCl and 0.5% toluidine blue into six labeled containerseach, one per group. The 60-mL cups, labels, and solutions are supplied withthis kit.

Observing Meiosis (Photocopy page OA-2)

If you wish students to observe slides of meiosis, see Carolina’s LilyMegasporogenesis Slide Set (item RN-29-3056), or individual slide listings forlily ovulary cross sections. Several methods for producing meiotic smears fromthe anthers of flowers have been published. The following method is adaptedfrom “Teaching Meiosis with Rhoeo discolor,” Carolina Tips, March 1, 1972,and from “Spider Plant Meiosis,” Carolina Tips, November 1, 1973. You willneed immature anthers from flower buds, not open flowers. Once the floweropens, most of the pollen will be mature and few if any cells will be undergoingmeiosis. In addition to Rhoeo discolor and Spider Plant, flower buds ofWisconsin Fast PlantsTM, or flower buds from a florist can be used.

Submerge the buds in FAA fixative (RN-86-3593) or Carnoy fixative (RN-85-3253). Leave the buds in fixative for two days. After that, transfer the budsinto 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 toluidineblue with HCl as described in Onion Mitosis, Squash Method. Students mayhave to make several slides before they master the technique.



Optional Activity for AP® Biology Laboratory 3

Onion Mitosis, Squash Method

© 2 0 0 5 C a r o l i n a B i o l o g i c a l S u p p l y C o m p a n y OA-1

IntroductionYou will prepare your own stained slides of onion root tips and then observe mitotic figures. Your teacherhas rooted onion bulbs in water. Growth of new roots is due to the production and elongation of newcells; mitotic divisions are usually confined to the cells near the tip of the root. Follow the procedurebelow to make your own root tip preparation.

Procedure1. 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 youneed. Discard the rest of the root. The mitotic cells are in the tip, so extra root tissue will onlyinterfere 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, warmthe slide by passing it back-and-forth over the flame of a Bunsen burner, (or an alcohol burner, orover a hot plate) for five seconds. Wear safety glasses and gloves. You might smell a faint aroma ofcooking 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 tipwith 0.5% aqueous toluidine blue (use caution when handling HCl and toluidine blue). Wearingsafety glasses and gloves, pass the slide over the heat source again, two times, without boiling theliquid. 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 carefullyapply pressure to the coverslip in order to squash and spread the root tip tissue. Caution: Do notbreak the coverslip.

5. Using your microscope (l0×), locate the meristematic region of the root tip. Examine the slide at40× 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 stagesof mitosis.

Procedure1. 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 theanthers 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 possiblewith 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 slideby passing it back-and-forth over the flame of a Bunsen burner (or alcohol burner or hot plate) forfive 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 pencileraser to press down on the paper. Press firmly, but be careful not to break the coverslip. Do notpush the coverslip sideways. If you do, the cells will roll up into tight bundles. You want to flattenthe 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. Youmay not be able to identify every stage of meiosis, but you may see several stages. Depending on thespecies of plant you are using, you may also see some abnormal divisions in which the homologouschromosomes do not separate properly.

OA-2 © 2 0 0 5 C a r o l i n a B i o l o g i c a l S u p p l y C o m p a n y

Optional Activity forAP® Biology Laboratory 3

Observing Meiosis

© 2 0 0 5 C a r o l i n a B i o l o g i c a l S u p p l y C o m p a n y S-1

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 BCells are the basic units of life, and cell division is the basic event that perpetuates life. Individual cellsdo not live forever; millions of the cells in your body die every day. You keep on living because new cellsarising from cell division replace those that die. One-celled organisms reproduce their kind through celldivision, while multicellular organisms grow from single cells by repeated cell divisions. This event—thereproduction 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. Eachprotein molecule consists of a long chain or chains of amino acids. The amino acids used to produce thechains and their sequence in the chains helps determine the structure and function of the protein. Oneamino acid chain might go into the formation of a molecule of catalase; another into a molecule oftubulin. The amino acids used to produce a chain and their sequence within the chain is determined bythe sequence of bases (C, G, A, T) within the cell’s DNA. The loss of even a small amount of DNA canbe fatal. For example, if a human zygote (the single cell that grows into an embryo) is missing the DNAnecessary to make hemoglobin, it will never be able to develop into a fetus. Therefore, when a celldivides, it is essential that it provide each new cell (daughter cell) with a complete set of DNA. To dothis, the cell must replicate (produce a duplicate copy of) its DNA before it divides. When divisionbegins, 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, theidentical halves of each chromosome are separated. One half goes into one daughter cell and its twingoes into the other daughter cell. As a result, both daughter cells have a complete and identical set ofDNA. This process of dividing double chromosomes to achieve an equal distribution of DNA to thedaughter 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 becomethemselves chromosomes once they are separated. This is simply a matter of terminology and does not involve some magicaltransformation of the chromatids themselves.

Activity A: Observing Mitosis

MaterialsPrepared microscope slides of onion mitosis and whitefish mitosis, microscopes.

IntroductionIn 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 tofind 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.

ProcedureUse the illustrations on the “Plant Cell Mitosis and Cytokinesis” page to help you identify the stages ofmitosis that you find on your slides. On the pages following, make your own detailed drawings and makenotes that will help you understand what is happening during each stage of mitosis. Remember that thephases of mitosis flow into each other, so you will likely see intermediate stages that are not shown in thediagrams. Refer to your textbook or other resource for detailed descriptions of the stages, but rememberthat your goal is to understand mitosis as a process. Once you understand the process, you canconcentrate on the details.



Plant Cell Mitosis and Cytokinesis

Interphase Prophase Prometaphase

Late Metaphase Early Anaphase Late Anaphase

Early Telophase Late Telophase

Interphase(Daughter Cells)

A

C

D

B

E

A. Nucleolus

B. Centromere

C. Cell Membrane

D. Cell Wall

E. Cell Plate

Mitosis Observation: Interphase CellsIn the spaces provided below, on the basis of your observations, draw a plant cell and an animal cell ininterphase. Use the lines underneath each illustration to record notes about what is happening duringinterphase.

Interphase Cells


Plant Cell Animal Cell

Mitosis Observation: Prophase CellsIn the spaces provided below, on the basis of your observations, draw a plant cell and an animal cellin prophase. Use the lines underneath each illustration to record notes about what is happeningduring prophase.

Prophase Cells



Mitosis Observation: Metaphase CellsIn the spaces provided below, on the basis of your observations, draw a plant cell and an animal cellin metaphase. Use the lines underneath each illustration to record notes about what is happeningduring metaphase.

Metaphase Cells



Mitosis Observation: Anaphase CellsIn the spaces provided below, on the basis of your observations, draw a plant cell and an animal cell inanaphase. Use the lines underneath each illustration to record notes about what is happening duringanaphase.

Anaphase Cells



Mitosis Observation: Telophase CellsIn the spaces provided below, on the basis of your observations, draw a plant cell and an animal cellin telophase. Use the lines underneath each illustration to record notes about what is happeningduring telophase.

Telophase Cells



Mitosis Observation: Daughter CellsIn the spaces provided below, on the basis of your observations, draw plant daughter cells and animaldaughter cells. Use the lines underneath each illustration to record notes about the characteristics ofdaughter cells.

Daughter Cells



Analysis of Results, Activity A: Observing MitosisUse 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 inferfrom this about the origins of mitosis?

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2. List at least two ways that mitosis differs in the cells of animals and higher plants.

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3. Describe what happens to each of the following during mitosis. Indicate the phase(s) in which thechanges occur.

a. nuclear envelope: _______________________________________________________________

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b. mitotic spindle: _________________________________________________________________

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c. chromatin: ____________________________________________________________________

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d. centrosomes: ___________________________________________________________________

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e. nucleolus: _____________________________________________________________________

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4. List the subphases of interphase and describe the important events that occur during each.

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5. List at least two ways that prokaryotic cell division is similar to eukaryotic cell division.

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Activity B: Estimating the Relative Lengths of Mitotic Phases

Materials Prepared microscope slides of onion mitosis, microscopes.

IntroductionIn 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 timespent in that phase. For example, if phase A lasts two minutes and phase B lasts one minute, the ratio ofobserved A to observed B would be 2:1.

Procedure1. 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


Field 1 Field 2 Field 3 Total 1–3

Interphase

Prophase

Metaphase

Anaphase

Telophase

Number of Cells

Table 2: Class Data

3. Record the class totals for each phase in Table 2. Calculate the decimal fraction of the total countedfor 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 theaverage 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 Phases1. 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: ____________________________________________________________


Estimated

Time Spent

in Phase

Decimal

Fraction of

Total Count

Class Totals

Interphase

Prophase

Metaphase

Anaphase

Telophase

Total Cells

Counted

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 yourtextbook, explain why some phases are longer than others. Refer specifically to each phase.

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Background to Activity CAs 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 willpossess two copies of each chromosome. This is because it receives one chromosome A and onechromosome B from each parent. Thus, it would have chromosomes A1A2 and B1B2. An organism withtwo sets of chromosomes (2n) is said to be diploid in chromosome number or, simply, diploid. Thechromosomes 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 highlysimilar) DNA. Reproductive cells (gametes, egg and sperm in animals; spores in plants) result frommeiosis, a type of cell division that reduces chromosome number by separating the homologues. Meiosisaccomplishes this reduction in an orderly manner such that our hypothetical diploid animal withchromosomes A1A2 and B1B2 produces gametes that are AB and not A1A2 or B1B2. Thus, reproductivecells have one set of chromosomes and are haploid (n) in chromosome number. (Rare events calledchromosomal nondisjunctions can alter this pattern, producing, using our example, gametes withA1A2B; A; B; or AB1B2 chromosomes.)

Meiosis involves two nuclear divisions, designated meiosis I (or MI) and meiosis II (or MII). Thereduction of chromosome number occurs in meiosis I. Meiosis II is essentially a mitotic division.


Activity C: Simulating Meiosis

MaterialsChromosome sets and Meiosis Student Guide from the Chromosome Simulation BioKit®.

IntroductionYou will use chromosome models to simulate meiosis. Follow the instructions in the Meiosis StudentGuide 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 manydifferent combinations of these chromosomes are possible in the gametes? (If necessary, use thefigures below to diagram the division that would give rise to the gametes.)

___________ combinations of the two chromosomes are possible.

Figure 1

2. Using your answer to 1 above, and given the following,

state a formula for calculating the number of possible chromosome combinations in the gametesbased on the value of n.Number of chromosome combinations = _____________

3. For humans, n = 23. Using your formula and a calculator, how many possible combinations ofchromosomes are there for human gametes? ____________

4. For our hypothetical organism with two chromosomes, A and B, when two members of the speciesreproduce, how many possible combinations of chromosomes are there for the offspring? ____________


A1 A2 B1 B2

n (chromosome number)Number of possible

combinations in the gametes

3

4

5

8

16

32

Figure 2

5. Looking back at your answers to 1–4, what is the relationship of meiosis to variation in populations(including human populations)?

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6. List at least three ways that meiosis differs from mitosis.

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Background to Activity DWhen homologous chromosomes pair in prophase I of meiosis, they can exchange parts, which is calledcrossing-over. When chromosomes exchange parts, genetic material is transferred from one chromosometo another. This alters expected inheritance patterns. For example, the fruit fly, Drosophila, has fourchromosomes. Chromosome 2 carries a gene for eye color. The normal form of this gene (Pr, the “wild-type”) produces flies with red eyes, but there is another form or “allele” of the gene (pr) that producespurple eyes. On the same chromosome is another gene that effects wing form. The normal wild-typeallele (Vg) produces normal wings while the alternate form (vg) produces vestigial wings, which aresmaller and useless for flight. Because these genes are located on the same chromosome, they areconsidered “linked” and are inherited together. For example, if one chromosome of a homologous paircarries the alleles for red eyes and normal wings and its homologue carries the alleles for purple eyes andvestigial wings, we would expect half the gametes to have the linked alleles for red eyes and normalwings and half the gametes to have the linked alleles for purple eyes and vestigial wings.


A1 A2 B1 B2 A3 A4 B3 B4

×

If crossover occurs between the locations (gene loci) of the genes for eye color and wing type, there willbe two new combinations of alleles in the gametes: red eyes with vestigial wings, and purple eyes withnormal wings (Fig. 3).

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 occurbetween them. By counting the frequency of crossover events between two gene loci, geneticists candetermine the relative distance between them. In this way, linkage maps have been produced for manyorganisms, 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 andcalculate the relative distance of the gene locus from its centromere.

Activity D: Crossing-Over and Map Units

IntroductionSordaria 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 structurecalled a perithecium. Two nuclei within a developing ascus fuse to produce a diploid (2n) nucleus. Thisdiploid nucleus then undergoes meiosis, followed by a mitotic division to produce eight ascospores in alinear series within the ascus (Figure 4).

Figure 4. Development of ascospores


prpr pr

Pr Pr Pr

pr pr pr

Pr Pr PrVg Vg Vg

Vg

Vg Vg

vgvg

vgvg

vg

vg

NuclearFusion

Meiosis I

Div.

Meiosis II

Div.

Mitotic

Div.

n + n 2n2n

nn

n

The order of the ascospores in the ascus reflects the order in which the chromosomes are segregatedduring meiosis. This can be clearly visualized if the diploid nucleus is a hybrid for two different sporecolors. The wild-type gene (+) produces a dark spore, while the mutant tan gene (t) produces a lightspore. If crossing-over does not occur, these genes segregate during meiosis I to produce a 4:4 sequenceof ascospores (Figure 5). However, if crossing-over does occur, the genes do not segregate until meiosisII, producing a 2:2:2:2 or 2:4:2 sequence of ascospores (Figure 6).

Figure 5. Production of MI asci

Figure 6. Production of MII asci

Procedure1. 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 eraseruntil 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 inwhich 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.


OR

Meiosis I Div. Meiosis II Div. Mitotic Div.

t

t

t

t

t t

+

++

+

+

+

OR

Meiosis I Div. Meiosis II Div. Mitotic Div.

t

t

t

t

tt

+

+

+

+

OROR

+

+

Figure 7. Sordaria cross plate

Analysis of Results, Activity D: Crossing-Over and Map Units

Table 3

1. Does crossing-over increase or decrease genetic variation? Support your answer.

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Gene-to-

Centromere

Distance

(%MII/2)

%MII Asci

(No. of

MII/Total)

Total Asci

No. of

MII Asci

(2:4:2 or

2:2:2:2)

No. of

MI Asci

(4:4)

Group Data

Class Data

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 sexualreproduction. Given that for both species n = 7, would you expect to find more genetic variation inthe population of species A or species B? Explain your answer.

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3. Suppose your Class Data from Table 3 showed 397 MI asci and 0 MII asci. What would youconclude from this?

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Lab 1. Diffusion and Osmosis RN-74-6410

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Lab 3. Mitosis and Meiosis RN-74-6450

Lab 4. Plant Pigments and Photosynthesis RN-74-6470

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Mitosis and Meiosis - BiologyAlive.com 1/13... · Laboratory 3. Mitosis and Meiosis Safety Preparation and Presentation. Although unlikely, it is possible that all stages of mitosis