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Lab # 9 Mitosis and Meiosis

OBJECTIVES:
After completing this lab exercise, you should be able to:
1. Describe the activities of chromosomes in the cell cycle including all the
stages of mitosis and meiosis.
2. Identify the stages of mitosis in the onion root tip and whitefish blastula cells.
3. Describe the differences in mitosis and cytokinesis in plant and animal cells.
4. Describe the differences between mitosis and meiosis.
5. Prepare and observe giant salivary gland chromosomes in Drosophila.
6. Observe human chromosomes in leukocytes and demonstrate human
karyotyping.
7. Explain crossing over and describe how this can bring about different
arrangements of ascospores in the fungus Sordaria.

INTRODUCTION:

ll cells come from previously existing cells. New cells are formed by the process of
cell division, which involves both division of the cells nucleus (karyokinesis) and
division of the cytoplasm (cytokinesis).
There are two types of nuclear division: mitosis and meiosis. Mitosis usually
results in the production of two daughter cells, which are genetically identical to each
other and to the parent cell. Formation of an adult organism from a fertilized egg, asexual
reproduction, regeneration, and maintenance or repair of body parts are all accomplished
by mitotic cell division. Meiosis, which occurs during the formation of gametes, reduces
the chromosome number in daughter cells to half that of the parent cell. Gametes in
animals and spores in plants are both produced by meiotic division. The egg and sperm,
though usually unequal in size, donate an equal number of chromosomes to the
developing organism: each contributes the haploid number of chromosomes. The
resulting zygote therefore contains a diploid number of chromosomes. In humans, for
example, the zygote contains the full complement of 46 chromosomes while the egg and
sperm contain the haploid number of 23 chromosomes.
In this lab, you and your partners will first explore mitosis in onion root tip and
whitefish blastula. Then, you will observe giant chromosomes in the salivary glands of
fruit fly larva, and prepare slides of human chromosomes. Finally, your team will study
meiosis and crossing over in two strains of the fungus Sordaria.

Exercise 9.1 The Cell Cycle and Mitosis

The complex series of events that encompasses the life span of an actively
dividing cell is called the cell cycle. It includes an interphase during which the cell
appears to be outwardly quiet and an M phase (for mitosis) during which the cell is
actively dividing.

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Even though it is a continuous process, the cell cycle can be divided into several
stages (see Figure 9.1). After a cell divides, it enters interphase, which consists of three
stages: G1, S, and G2. Interphase is frequently referred to as a resting stage, but the cells
are actually quite active and are preparing for the next division.
During interphase, DNA, with its chromosomal proteins, exist in a highly
uncoiled state. Thus, when cells are stained during interphase, distinct chromosome
structures are not visible. Chromosomes appear, instead, as a granular material called
chromatin within the nucleus.
Events during G1: (G for Gap). The cell approximately doubles in size and its
organelles and enzymes double in number. Centrioles also begin to divide during G1.
Cells that normally do not divide remain in G1. Actually some cells like muscle and nerve
cells will never divide again and are said to be in G0 while others such as liver cells have
the potential to divide again if the liver is damaged and pass out of G1 and into S.
Research indicates that a restriction point, R, exist in G1. Passing beyond this point seems
to require a certain concentration of a particular protein (MPF) produced in small
quantities. The R point might also be affected by cell density; when enough cells are
present in a given area division stops. One of the characteristics of cancer cells is that
they seem to disregard the signals that restrict division in normal cells. Thus, discovering
the exact mechanism of the R point is a subject of intense interest and research.
Events of S: Before the S phase, each chromosome consists of a double-stranded
helix of DNA. During the S phase, the two strands of the DNA helix unwind, separate,
and duplicate by the process of replication. By the end of the S phase, each chromosome
is composed of two helices of DNA called chromatids, joined at a region of the
chromosome called the centromere. Distinct chromosomes are not yet visible during
this phase. The DNA molecules are intact, but are largely uncoiled and dispersed as
chromatin. Chromosomal proteins, also synthesized during S phase, will eventually
associate with DNA to help it coil into tightly packed chromosomes prior to the
beginning of mitosis.
Events of G2: During this phase
structures directly involved in cell division
are synthesized. Spindle fibers begin to
assemble. These will become attached to
chromosomes and guide their movement
during mitosis. In animal cells, a pair of
centrioles divides to form two pairs of
centrioles. These will also play a role in the
movement of chromosomes during the
mitotic process. Cells of higher plants have
spindle fibers but usually lack centrioles.

A. Mitosis in Plant Cells

The onion root tip is one of the most
widely used materials for the study of
mitosis because it is readily available,
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Figure 9.1: Stages of the Cell Cycle.

G1 and G2 stand for the first and second gaps of
interphase. S stands for the synthesis of DNA.

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preparation of the dividing cells is easy and the chromosomes are large and few in
number. The root tips are regions of active cell division so chances are pretty good that
in root tip specimens, you can find every stage of mitosis.
As you examine prepared and student-made slides of Allium and later of whitefish
blastulas, refer to the following descriptions and diagrams (Figure 9.2(b), page 9-8) and
use your textbook in order to learn more about each stage.

Interphase So named because early scientists thought it was resting phase. In

reality, the cell is actively undergoing synthesis of DNA and proteins and
preparing for mitosis. Most of the cells you observe will be in this mode.

Prophase It is the longest of the four stages of mitosis. The chromosomes

shorten and thicken and become progressively visible. The double stranded
chromosomes now turned chromatids are clearly seen. The spindle forms and the
centrioles move to opposite poles. By the end of prophase, the nuclear membrane
and nucleolus have disappeared.

Metaphase This phase is relatively quick and features the chromatids attaching
to the mitotic spindle at the centromeres. By the end of metaphase the chromatids
have lined up along the metaphase plate.

Anaphase The most rapid stage of mitosis features the splitting of the
centromeres and the pulling of the chromatids by the microtubules to opposite
poles of the cell. This stage can be recognized in the onion cell by two groups of
V-shaped chromosomes on opposite sides of the cell.

Telophase This stage is nearly the reverse of prophase. The chromosomes at the
poles begin to uncoil and resume the chromatin form. The spindle fibers
disappear and a nuclear membrane forms around each chromatin mass. Nucleoli
also appear in each of the daughter nuclei.

Materials:
Clean microscope slides and cover slips
Onion bulb with roots
Dissecting needle and razor blade
Fixative solution (HC1-95% ETOH)
Preservative solution (HAc-70% ETOH)
Aceto-orcein stain
Prepared slides of Allium (Onion root tip) and whitefish blastulas
Procedure
1.
Read pages 9-4 through 9-6 about chromosomes and the discoveries that
established the chromosome theory of inheritance.

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2.

Follow steps 1 through 26 on pages 9-7 and 9-8 in order to make your own onion
root tip slides. Go to Step 4 on page 9-11 while the slide is being stained.
(Pages 9-4 to 9-10 are from Prof. Bernard Fritzes original write-up)

LABORATORY INVESTIGATION NINE (D: Cytogenetics, C:

Chromosomes, Copyright 1993 by Bernard H. Fritze,
INTELPROP)
A. Chromosomes are the Microcassettes of Genetic
Instructions Parents Pass to their Progeny in Cells.
Survival of the fittest individuals in each generation in the struggle for life is a
necessary but insufficient criterion of success. To survive and then parent the new
generation, to be a participant in perpetuating the species, defines fitness and success in
the context of evolutionary change.
The most singular property that sets a living being apart from all inanimate objects is
the capacity to parent, to create copies of one self. This essential character exists even
in the smallest globules capable of maintaining the living state, cells. Though measured
in thousandths a dimes thinness (micrometers), cells reproduce by becoming two. Cell
reproduction is no blob of bubble gum dividing in half as some imagine.
Reproduction begins within the nucleus, the cells library of building and operation
instructions recorded digitally along the double ribbon molecule, deoxyribonucleic acid
(DNA). All the recording tape is first duplicated. The tape is then packaged with protein
as a cassette, the chromosome, and each kind of organism has a specific and countable
number. Human body cells have 46 chromosomes, cats 38, corn 20, fruit flies 8, a horse
roundworm 4, etc. This is before they are duplicated.
To survive each newly divided cell must have a complete library of operating
instructions, meaning all the chromosomes. With all the chromosomes now in duplicate
the next sequence of phases is a very precise separation of the twin copies from one
another and their assembly into two identical sets in each forming nucleus. The
remainder of the cell, the protoplasm, is then divided approximately in half. This is
cytokinesis, the nuclear process being called mitosis.
Obviously, large multi-cellular creatures, such as whales or even worms, cannot
multiply by dividing. Here, too, the process of reproduction is essentially cellular with the
formation of specialized sex cells (gametes), egg and sperm, in special organs (gonads),
ovaries and testes.
In the gonads (sex organs) cells that will produce gametes (sex cells) duplicate their
chromosomes. Matching chromosomes named homologues of maternal and paternal
origin mysteriously find each other and align lengthwise, an event called synapsis. This
event only occurs during this kind of division, meiosis, not during ordinary mitosis. Since
each homologue is twinned, a bundle of four chromatids, a tetrad, forms.
Homologous (non-sister) chromatids often cross-splice equivalent segments,socalled crossing over. New gene sequences are formed. Each tetrad of four chromatids
is independently maneuvered to the cells equatorial plane, creating a large variety of
random assortments in the many cells engaged in the process.

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The first segregation parts the twinned homologues from each other and the second
separates the identical twin chromatids.
Crossing over re-assorts the genes along the chromosomes length, random
alignment at the cells equator creates myriad chromosome assortments and two
divisions reduces their number to half that of body cells.
Fertilization of egg by sperm restores the double chromosome number in the first
body cell of the new life, the zygote or fertilized egg. But more than, that each new
individual has a unique assortment of genetic instructions pooled by the union of sex
cells produced by the special nuclear phases of meiosis.
And then the random circumstances of the environment, Nature, select those
individuals with the best combination of genetic traits to survive and parent another
generation. Generation after generation this repeating process has caused biological
change, which is evolution.

B. Discoveries Establishing the Chromosomes Theory of

Inheritance.
1665 Hooke observes microscopically the porous construction of various plant tissues
and names the tiny boxes cells.
1833 Brown discovers the nucleus as a consistent organelle in a great variety of plant
cells.
1839 Schwann writes, The most frequent and important basis for recognizing the
existence of a cell (plant or animal) is the presenceof the nucleus.
1839 Von Mohl reports cell division is easily seen in plant root tips.
1854 Newport is the first to prove the sperm cell penetrates the frog egg cell to begin
fertilization.
1855 Virchow states that new cells originate only from older cells and in no other way.
1866 Mendels cross breeding experiments with peas establish that factors determining
inheritance are paired particles that segregate into egg and sperm and recombine
at fertilization. At the time these particles were hypothetical abstractions.
1866 Haekel believes the nucleus controls inheritance.
1873 Schneider in studies of flatworm embryos cells is first to report the appearance of
stainable threads (chromosomes) in place of the nucleus and their segregation into
each of the twin cells resulting from the division.
1876 Hertwig is first to observe the fusion of the sperm nucleus with the egg nucleus 15
minutes after penetration in sea urchin fertilization and the appearance of carmine
stainable filaments (chromosomes) during the following first cell division.
1882 Flemming in studies of living and stained salamander embryo cells details the
condensation, duplication count and segregation of chromosomes. He names the
process mitosis after the Greek word for strings or threads.

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1884 Hertwig, Weismann and others conclude inheritance is passed on by the
chromosomes of the nucleus.
1887 Weismann theorizes chromosomes occurring as equal parental sets (male and
female) whose number must be halved before the doubling of their count that
occurs at fertilization.
1887 Van Beneden establishes the chromosome number in all body cells is the same but
varies with each species. He shows the number doubling before egg and sperm
cells are formed. Two subsequent cell divisions reduce the number to half.
1888 Boveri in studies of an intestinal roundworm with only 4 chromosomes observes
the number reduced to 2 in the formation of egg and sperm (meiosis), paired again
at fertilization, duplicated and segregated at the first division (mitosis) of the
embryo.
1895 Wilson theorizes that stainable chromatin in the nucleus is identical to nuclein, a
compound first isolated by Meischer in 1869, and that inheritance might be caused
by a specific chemical.
1902 Sutton correlates in grasshopper sperm formation the matching of maternal and
paternal chromosomes and their random segregation with Mendels rediscovered
paper on the duplicate nature and segregation of the hypothetical elements that
control heredity.
1909 Janssens studying the synapsis (pairing) of homologous (matched) chromosomes
during meiosis suggests they might break and rejoin, a process called crossing
over.
1910 Morgan locates in fruit flies the gene for white eye color on the X chromosomes, a
sex chromosome because it determines gender.
1913 Sturtevant maps five gene loci (locations) on the X chromosome of fruit flies based
on the frequency of crossing over, fewer if the genes are close together, more if far
apart.
1931 McClintock and Creighton prove experimentally crossing over occurs in corn
among the 4 chromatids of chromosome 9.
1931 Stern using abnormal X chromosomes of fruit flies establishes microscopically the
reality of crossing over.

C. Mitosis and Cytokinesis are Observed in Onion Root

Tip Cells Using Squash Technique.

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Proceed to
step 4 on
page 11
viewing
the
prepared
onion root
slide.

1. Use cleanser at the sink to clean 2 microscope

slides. Both will be processed together.
2. Lift the onion bulblet and foam float from the water
in the plastic glass. With your fingers pluck an
entire root from its base. Keep track of the
tapered tip from the torn base end.
3. Place a white root on each of the 2 cleaned slides
over the black surface of your desk. Do not allow
the root cells to dry out during the rest of this
procedure.
4. With a sharp blade cut off 2 mm only of the tip
no more. Be sure youve not cut the wrong end.
Discard the rest of the root, (2 mm is the size of a
capital letter in this text).
5. With the tip of a dissecting needle move the root
tip to the far right of the slide. Cover it with
several drops of fixative. This instantly kills the
cells. They are stopped in the middle of what they
are doing, such as dividing.
6. Keep the root tip covered with the fixative for 5 full
minutes as it becomes whiter and more opaque.
The fixative contains concentrated acid. Keep it
all on the slide.
7. At the end of five minutes move the root tip with
the dissecting needle to the far left of the slide.
8. Cover it with preservative for 5 minutes.
9. At the end of five minutes use the needle to move
the root tip to the center of the slide.
10. Absorb with a piece of paper towel both the
fixative and preservative from the ends of the
slide, keeping them off your skin.
11. Cover the root tip with the red stain aceto-orcein
for the next 25 - 30 minutes, replacing stain as
necessary from drying.
12. While the root tips are staining use the time to
study the same material (onion) prepared by
different technique and using multiple stains rather
than one.
13. Obtain from the supply a prepared slide.
14. Hold it over a white background and note the
Fig. 9.2(a): Mitosis in
tapered dark streaks, usually 3, under the coverPlants
slip. Each streak is a very thin lengthwise slice of
an onion root tapering to the tip.
15. Place the slide on the stage of the microscope and with the scanning objective
(lowest power) observe each of the three root tip preparations. Pick the best of the
three to study at high powers.
16. Study the tapered tip at scanning power and move up to 100X. Most of the nuclei
look like grainy red dots. Find those few that appear to be stringy or show red
threads against the green-stained background.
17. Center the cell in the field and swing to high power (40X objective), locking the
objective in alignment and increasing the light.
18. Since this root tip has been so thinly sliced, parts of the chromosomes may be
missing, making some of the cells difficult to place into the correct stage of division.

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19. As many as 95 percent of the cells are in interphase (red dots) and not in the
process of mitosis.
20. Cells whose nuclei appear as loose balls of red yarn
are in prophase, lasting up to 70 percent of mitosis.
21. Metaphase (chromosomes on the equator), anaphase
(two chromosome clumps being pulled apart), and
telophase (two stringy red nuclei with a partition wall
forming between them) are the rarest but most
distinctive phases of mitosis.
Here the stringy
chromosome threads are most easily distinguished
from the grainy blobs of the much more common
interphase nuclei.
22. After thirty minutes place each of the two slides on a
paper towel.
Gently lower a new, unscratched
coverslip on to the root tip in the stain. The pressure
of the coverslip should squash the root tip into a single
layer of cells that appears as a purple-red smudge
over the white towel.
23. Fold one end of the paper towel over your slide and
apply pressure with your thumb (squash technique)
directly over the coverslip. This spreads the root tip
cells and absorbs excess stain squeezed from under
the coverslip. Removing excess stain makes the
stained nuclei and chromosomes clearer.
24. Tapping firmly on the coverslip with a pencil eraser
over the squashed root tip should further spread the
cells into a single layer. An ideal slide will show no air
spaces formed from this treatment.
25. Use the 10X objective to scan the field for those few
cells among the many whose nuclei have become
strings (chromosomes). Dont use high power to scan
the field. Use it only for a closer look at promising
prospects. Avoid fields of cells layered upon one
another.
26. It is not uncommon to find all the phases of mitosis in a
single field even at the highest magnification.

Fig. 9.2(b): Phases of

Mitosis

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D. The Sequence of 8 Spores in a Sordaria Ascus Reveals
Either Segregation or Crossing Over.
(Note: This section is for Exercise 9.4. It is described here to maintain the integrity of
Prof. Fritzes original write up.)
1. Observe the Petri plate labeled Sordaria. Keep the lid on. Note the inoculation site
of the two parent strains: + is the black spore parent (normal or wild-type) and O is
the tan spore parent (mutant).
2. The fungal filaments (hyphae) have grown rapidly over
the fresh agar surface, radiating in all directions from
the point of inoculation.
3. Within a couple of days or so the hyphae of both
fungus strains meet and fertilization occurs as the
nuclei fuse.
4. In that zone, roughly the midlines of the plate, tiny
pear-shaped reproductive bodies called perithecia
form at the agar surface. Their color changes from
amber to black as they mature in 5 to 7 days.
5. Within each perithecium are cluster of test tub-like
asci, each containing 8 ascospores, the products of a
meiosis (plus a mitosis) sequenced in a column in the
transparent tube. The row of spores in an ascus can
be observed by squashing an ascus cluster (like
fingers or bananas) out of a perithecium by applying
pressure.
6. Remove the cover of the Sordaria Petri plate and
place it on the stereo microscope stage, choosing the
best lighting.
7. At the lowest power focus your attention on the zone
where the black spore parent and the tan spore parent
fungal filaments have grown together, crossed (fused
nuclei, fertilized) and have produced the dark pearshaped perithecia.
8. In the center of a clean slide place a large drop of
methyl cellulose.
9. Moving to higher power if needed, use a sharp, angled
dissecting needle to select 10 to 15 dark, mature
perithecia from a midline of the cross and place them
in the methyl cellulose, well separated.
10. Drop a new, unscratched coverslip on the preparation
and place the slide on the stereo scope at low power.
11. As you watch, with the tip of the dissecting needle
apply pressure on the coverslip to each perithecium,
rupturing it and squeezing out the cluster of asci.
12. Over-ripe asci will scatter their spores, but favorable
asci will maintain the 8 spores aligned within the tube.
Concentrate on those.
13. Scan the slide you have prepared using the 10X
Fig. 9.3: Meiosis in a
objective with the microscope. Ignore asci that are all
black or all tan. These are not a cross between wildtype (black) and mutant (tan).
14. An ascus with 4 black spores in one half the tube and 4 tan spores in the other half
visualizes the fact of gene segregation (separation of alleles) when sex cells form.
15. Two black, two tan, two black, two tan results from a crossing over between chromatid
2 (paternal homologue) and 3 (maternal homologue).

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16. Two tan, two black, two tan, two black is a crossing over of chromatids 1 (paternal)
and 4 (maternal).
17. A two tan, 4 black, two tan spore sequence means a crossing over between
chromatids 1 (paternal) and 3 (maternal).
18. A two black, 4 tan, two black pattern
shows crossing over has occurred
between chromatids 2 (paternal) and 4
(maternal).
19. Chromosomes occur as matched pairs
(homologues) that may first exchange
equivalent segments (crossing over) and
then segregate separately into the sex
cells, thus reducing their number to half.
The half sets are put together and
fertilization in
an entirely unique
assortment in each zygote (first body cell).
Such shuffling of the genetic material,
genes and chromosomes, generates the
variety of life forms from which selection
by nature creates the process of evolution,
a progression of origination and extinction
that still continues even now.

Fig. 9.4: Chromosome continuity

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3.
After observing the slides you made, make drawings of each stage. Remember to
draw only one large cell in each circle.
In the circles below, draw onion cells you observed from your squashed root tips that
illustrate the important stages of the cell cycle. Name each stage. Draw one cell in each
circle.

4.

Using the high power objective, observe a prepared slide of the onion root tip.
Examine a single field in the actively growing region, count and record the phases
of the first 50 cells you observe. Record your results in Table 9.1 below. Repeat
this count of the two other remaining root tips on your slide. Use Table 9.1 to
collect and calculate your results.
Example of Table 9.1 Percent of Cells in Each Phase of the Cell Cycle
Phase

Field 1

Field 2

Field 3

Totals

Percent of Grand Total

Interphase

30

33

27

90

90/120 = 75%

Prophase

10

12

30

30/120 = 25%

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Table 9.1: Percent of Cells in Each Phase of the Cell Cycle
Cell Stage
Interphase
Prophase
Metaphase
Anaphase
Telophase
Grand Total
5.

Field 1 Field 2 Field 3 Total % Grand Total (total/grand x 100)

The duration of mitosis varies for different tissues in the onion. However,
prophase is always the longest phase (1-2 hours) and anaphase is always the
shortest (2-10 minutes). Metaphase (5-15 minutes) and telophase (10-30
minutes) are also relatively short in duration. Interphase may range from 12-30
hours. Consider that it takes, on average, 16 hours for the onion root tip cells to
complete the cell cycle. You can calculate the amount of time spent in each phase
of the cell cycle from the percent of cells in that stage (Note: enter results and
calculations in the space below).
Percent of cells in stage x 960 min. = ______ min. of cell cycle spent in stage
(16 hr.)
(convert to hours and minutes)

Using the information presented above, calculate the following:

(a) Time spent in Prophase
Time spent in Metaphase
Time spent in Anaphase
Time spent in Telophase

_____hr ___min
_____hr ___min
_____hr ___min
_____hr ___min

(b) What was the total time spent in mitosis? ____________

What was the total time spent in interphase? __________
(c) How do your results compare with what is known about the Allium cepa
cell cycle?

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6.

Mitotic Index. In order to get some idea of how many cells are undergoing
mitosis at any one time, select at random, the actively dividing zone of two
different root tips. In each area, count 100 cells at random and record how many
of these cells are undergoing mitosis. Record your data in Table 9.2 and then
calculate the percentage of dividing cells and average your results.
Table 9.2: Mitotic Index for Onion Cells
Area 1 Area 2 Average
Total # of Cells
# Cells in Mitosis
Percentage of Cells in Mitosis

B. Mitosis in Animal Cells

Mitosis is easily observed using a prepared slide of whitefish blastula (an early
stage of development formed by successive mitotic divisions after the egg has been
fertilized by the sperm). Obtain a prepared slide of the whitefish blastula and study it
under high power. Identify all stages of mitosis. You may have to examine more than
one blastula in order to observe all stages. Draw these stages. Do you notice any
differences between mitosis in whitefish blastulas as compared to mitosis in onion root
tips? Make a list of these differences.

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Exercise 9.2 Giant Chromosomes of Drosophila

What happens if DNA duplicates in preparation for mitosis but cytokinesis does
not occur? This actually occurs in some tissues of the fruit fly, Drosophila, during the
early stages of larval development. The cells of the salivary glands increase in size but
do not divide and as a result, the number of strands making up the chromosome continues
to increase. The chromosomes become multi-stranded and are called polytene
chromosomes. The DNA content of a polytene chromosome is approximately 1000X
greater than the normal DNA content of a regular chromosome.
The many DNA strands of a polytene chromosome condense and fold in the same
manner as a single strand of DNA found in chromosomes of other organisms. Highly
condensed or folded areas stain darkly and give chromosomes a banded appearance.
Human chromosomes also have a banded appearance. The patterns of bands as you
will see in Exercise 9.3 can be used to identify genetic abnormalities including
chromosomal or gene deletions or rearrangements.
The banded chromosomes are easily seen under low power. This effect is further
magnified because homologous chromosomes also pair up. Thus the 8 chromosomes in a
fruit fly cell are seen as 4 thick polytene chromosomes.
Materials Needed:
Prepared slide of Drosophila polytene chromosome
Drosophila larva (live)
Clean glass slide and cover slip
Aceto-orcein stain
Dissecting needles and probe
Bottle of 0.7% saline solution
Procedure: (Do only steps one and two. Steps four onwards should be done at the end of
the lab if time permits, or if larvae are available.)
1.
Obtain a slide of Drosophila polytene chromosomes and
observe under low power.
2.
Locate a giant polytene chromosome and show it to your
partner and the instructor. Draw one chromosome.
a. Do you see any bulges along the length of the chromosome?
__________
What do you think they represent? _______________________
b. How many chromosomes does each actually represent?
__________________________
Explain your answer:

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3.
4.

5.

6.
7.
8.
9.

**************************
Obtain a clean slide and place a drop of 0.7% saline toward one end.
Use the dissecting microscope and place a Drosophila larva in the saline. Place
one needle at the middle of the larva and the other just behind the head (see
Figure 9.5) Use a blunt probe to hold the larva at the posterior end and a
dissecting needle to remove the head at the first segment behind the mouth.
Use a blunt probe to gently press on the anterior part of the larva just behind the
third and fourth segments. Roll out and expose the salivary glands. The glands
are elongated and semi-transparent. Remove the fat bodies and digestive tube
adhering to the pear-shaped glands.
Place a drop of aceto-orcein stain next to the drop of saline and with a dissecting
needle transfer the glands from the saline to the stain.
Stain for 10 minutes. Make sure the drop of stain does not dry up.
Place a cover slip on the preparation.
Place the slide between the folds of a paper towel and press down on the cover
slip firmly. Examine the slide under low power (100X) to locate the
chromosomes. Examine the chromosomes to observe the banded pattern. Make a
sketch of a banded chromosome in the previous page.

Figure 9.5: Procedure for removing salivary chromosomes from the larva of the fruit fly,
Drosophila. The structure of a typical polytene chromosome is also shown.

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Exercise 9.3 Human Chromosomes Analysis

INTRODUCTION
The genetic information for all organisms is encoded in the form of one or more
DNA molecules. DNA is associated with proteins that function to protect the DNA,
facilitate its replication prior to cell division and mediate the expression of the
information carried in the DNA nucleotide code; each DNA-protein complex is termed a
chromosome. Human chromosomes, like most chromosomes of other eukaryotes
(animals, plants, fungi and protists), each contain a single, long, linear molecule of DNA.
The chromosomes of these types of organisms are contained in cellular organelles called
nuclei. In this lab you will prepare and analyze chromosomes from human tumor cells
that have been grown in culture and we will compare them to the standard normal set of
human chromosomes with respect to their number, size, proportions and banding
patterns.
(The procedure for this section starts on page 24)

CHROMOSOME NUMBER AND HOMOLOG PAIRING

The nuclei of most human cells contain 46 chromosomes: 2 chromosomes are
involved in sex determination and are termed sex chromosomes; the other 44
chromosomes are termed autosomes. The autosomes occur in pairs (1-22). Both copies
of each chromosome carry the same genes although often different alleles (forms) of
those genes; hence, members of a pair are termed homologs. One member of each pair
was originally inherited from the individuals mother and the other from the individuals
father; cells that contain two sets of chromosomes are referred to as diploid. The gross
morphology of the chromosomes of a given pair is generally identical although they
differ at the level of nucleotide sequence. (See Figure 9.6 for the standard human
chromosome map or karyotype) The two types of sex chromosomes also pair but, in
contrast to the autosomal pairs, sex chromosomes are quite different from one another in
morphology and carry different genes. The two types of human sex chromosomes are
designated X and Y; the X and Y chromosomes have regions of similar nucleotide
sequence (termed pseudo-autosomal regions) as well as their distinctive and different
genes. The X chromosome carries a large number of genes that are critical to the normal
development and function of both females and males. The Y chromosome carries many
fewer genes. Among them is the gene that codes for male sex determination. An
individual with two X chromosomes is a female, and an individual with an X and a Y is a
male because of the critical nature of the genes carried on the X chromosome. All
humans must have at least one X chromosome in their karyotype (there are no viable YY
individuals).

CHROMOSOME STRUCTURE
CHROMATIN: Eukaryotic chromosomes are made up of a complex of DNA and
protein that is termed chromatin. Each human diploid nucleus contains a total of two
meters of DNA in the form of 46 individual molecules that are 2 nm in diameter. The
DNA is protected by binding with proteins as shown in Figure 9.7. The diagrams in
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Figure 9.7 are based on images seen with the electron microscope and on the results of
biochemical experiments in which chromatin, extracted from cells was analyzed. During
interphase, the period between cell divisions, the DNA-protein complex is relatively
extended. The long fragile fibers of DNA are protected by complexing with the histone
proteins to form nucleosomes. The beads-on-string structure of the nucleosomes is
condensed to form a solenoid (a thicker fiber) that, in turn is locally condensed in the
form of loops. Experimental evidence indicates that during interphase these loops are
probably stabilized by binding to the matrix proteins present in the nuclei. Interphase
chromosomes are organized as shown in the first four levels of Figure 9.7. In living cells,
chromosomes at this stage are relatively diffuse, thin (300 nm), tangled-appearing threads
that fill the nuclei of the cells.

Figure 9.6: Human chromosome map with G-bands.

From Barch (Ed.). The ACT Cytogenetics Laboratory Manual.

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Figure 9.7: Model for the stages in the
condensation of DNA (a) as it
complexes with proteins to form
chromatin (b-e) and metaphase
chromosomes (f). From Barch (ed.),
THE ACT Cytogenetics Laboratory
Manual.

When cells prepare to divide, their

chromosomes are duplicated and
later a copy of each chromosome is
inherited by each of the daughter
cells that result from the division.
Somatic (body) cells, such as the
cell line we will use in lab, divide by
a process termed mitosis (see Figure
9.8). During mitosis, the duplicated
chromosomes progressively
condense to become relatively
compact X-shaped structures. Each
has characteristic proportions and
banding patterns on its arms if
appropriately stained. In Figure 9.8
note particularly the late prophase
and metaphase stage chromosomes.
In the lab you will see that these
chromosomes are each made up of
two sister chromatids, which are
held together at a constricted region,
the centromere. The ends of the
chromosome arms are termed
telomeres. There is experimental
evidence that indicates that the
coiled looped structure of a
chromosome in a dividing cell (see Figure 9.8) is stabilized by binding of its chromatin
fiber to an internal, X-shaped scaffold of protein.

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Figure 9.8: Diagram of a cell in interphase and mitosis. Note especially the changes in
cell structure during late prophase. (From Avers, Cell Biology.)
CENTROMERES: Human chromosomes, like those of other eukaryotes, differ in total
length and centromere position (see Figure 9.9). In the human karyotype, centromeres of
several pairs of chromosomes are positioned so that the two arms of the chromosomes are
approximately equal in length (chromosomes 1-3, 19 and 20); these chromosomes are
designated as metacentric. Others (4-12, 16-18, and X) have a short arm and long arm,
these are submetacentric. In the third type of chromosome (13-15, 21, 22 and Y), the
centromere is positioned very close to one end so that one arm is very short relative to the
other, these are acrocentric. For all types of chromosomes the shorter arms are termed
the p (petite) arms and the longer are the q arms. In Figure 9.10, which are the p arms
and which are the q arms? How would you classify this chromosome in terms of
centromere position?

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Figure 9.10: Whole mount of human

chromosome 12 photographed with
microscope.
Figure 9.9:a transmission
Chromosomeelectron
Classification.
(From Avers,
Cell Biology.)
(a) Metacentric
(b) Submetacentric

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TELOMERES: Telomeres cap the ends of the chromosome arms. The DNA in these
regions consists of a short (six nucleotides) repeated sequence that is added to each end
of the chromosomal DNA molecule by an RNA-protein complex termed a telomerase.
Because telomerase activity has been found to be present in germ line cells (cells that
produce eggs or sperm) but has not been detected in normal somatic cells, it has been
suggested that telomeric sequences are normally added to the ends of chromosomal DNA
only in germ line cells. Telomeric sequences are thought to function in protecting the
informational content of the chromosomes and to be in the same way involved in the
ability of the chromosomes to be replicated. During the successive cell divisions that are
required to produce an individual organism from a fertilized egg, the telomeric sequences
become shorter with each cell division because of the nature of DNA polymerase, the
enzyme that mediates DNA replication prior to cell division and because of the lack of
telomerase activity in these later stage cells. Thus telomeres may act as replication
clocks that are set at fertilization. As the telomeres shorten, the chromosomal DNA may
become increasingly vulnerable to damage and in some way that is still unclear the
chromosomes may lose their ability to be replicated. When chromosome replication does
not take place, cell division is blocked and the cells become senescent. Aging on the
level of entire organisms may be the result in part of this cellular senescence. Recent
research has found that cells taken directly from malignant tumors and from permanent
(transformed) cell lines frequently contain telomerase activity, these findings lend support
to the hypothesis that telomere length is related to cell division capability as both of these
latter types of cells are able to divide an apparently unlimited number of times (Kim, et
al. 1994, Science. 266:2011-2015).
BANDING: Chromosome pairs can be individually identified by using specific staining
treatments. Several of the treatments produce characteristic bands on the arms or at the
centromeric constrictions. Some recently developed techniques result in painting entire
chromosomes of a pair by using fluorescent dyes bonded to DNA sequences that localize
to those chromosomes in a process called DNA hybridization. In lab we will analyze Gbanded chromosomes from normal human cells as well as preparing our own
chromosome spreads from cultured cells. G-banding involves treating the chromosomes
lightly with pancreatic protein-digesting enzymes (primarily trypsin and chymotrypsin)
and then staining them with Giemsa, a mixture of dyes that stain nucleic acids and
proteins. The results resemble the banding patterns in Figure 9.6. Some regions will
stain intensely (G-bands or G-dark bands) and other regions will stain much less
intensely (G-light bands). Interestingly, the G-dark bands, which are A - T rich, tend to
contain the genes that code for cell type specific proteins (such as hemoglobin), while the
G-light bands, which are G - C rich, contain the housekeeping genes which are
expressed in all cell types.
The localized affinity of the stain for the chromatin is presumably the result of
differential distribution of proteins that are associated with the DNA. Partial digestion

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with pancreatic proteases selectively alters the structure of some chromosomal proteins
more than others so that either the DNA in different regions of the chromosomes is
exposed by the enzyme treatment and binds the Giemsa stain more or less efficiently, or
the proteins themselves are staining differentially. The method is reproducible, indicating
that as the chromatin fiber of each chromatid loops and condenses to form a sister
chromatid, it does so in a regular orderly fashion. The fact that the banding patterns are
consistent seems quite remarkable given the structure of metaphase chromosomes
(review the condensed, looped structure of the human metaphase chromosome in Figure
9.7).

CELLS USED FOR CHROMOSOMAL ANALYSIS

Chromosomal analysis requires a population of cells in which a reasonably large
proportion is dividing. A variety of different cell types can be obtained from individuals
grown in culture (as described below) and used for such an analysis. For fetal diagnosis,
cells are obtained by withdrawing a sample of amniotic fluid that contains cells derived
from the fetus or by sampling chorionic villi (small finger-like processes on the placenta
which are fetal in origin). Chromosomal analysis that is done for children and adults
generally involves culturing lymphocytes that are isolated from a blood sample. Cells
from tumors and cultured cell lines are also analyzed for chromosomal abnormalities.
The HeLa cells we will analyze are human cervical carcinoma cells that have been grown
in culture since 1951. These seemingly immortal cells are used in research world-wide
and much of what we know about the molecular biology/biochemistry of human cells has
been derived from HeLa cell studies. Interestingly, the HeLa cell line was found to be
one of the cell lines that have telomerase activity in the research described above. Over
the years the HeLa chromosomes, like those of other permanent cell lines, have
undergone duplication, deletions and rearrangements and are now quite different from the
normal human karyotype. In the lab we will analyze the differences. Why do you think a
cell line could survive with grossly abnormal chromosomes whereas an organism can
not?

APPLICATIONS FOR KARYOTYPING

The ability to identify individual chromosomes in the karyotype of organisms has
been very useful as a medical diagnostic procedure and in basic research in genetics and
evolution. One can detect variations from the normal karyotype with respect to
chromosome number (a condition referred to as aneuploidy) or with respect to
rearrangement of chromosomal regions (deletion, duplication, translocation from one
chromosome to another non-homologous chromosome, or inversion within a
chromosome).
For most animal species, increasing or decreasing the number of chromosomes or
number of copies of genes (as in duplicating or deleting regions of chromosomes) almost
always causes lethality early in embryonic development. Exceptions to this
generalization for the human species are trisomies (having three copies of a given
chromosome) for chromosome 8, 13, 18, or 21. Individuals carrying these trisomies are
initially viable but generally are mentally retarded, physically abnormal, and have a

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shorter life expectancy. Aneuploidy of sex chromosomes in many cases results in
viability. Although people with such karyotypes are frequently mentally retarded,
physically abnormal and/or infertile, XYY individuals are an exception in that they are
generally normal males.
Fetal diagnosis is an important application of karyotyping. It is estimated that
more than 50% of human pregnancies end in spontaneous abortion; very frequently
before the woman is even aware she is pregnant. More than half of the spontaneously
aborted embryos that have been studied have been found to have chromosomal
abnormalities. Karyotype analysis is routinely done in situations in which there is
increased risk of a fetus with abnormal chromosomes (e.g. a pregnant woman over 40
years of age).
There is also medical interest in more subtle chromosomal rearrangements such as
translocations and inversions. Many different types of leukemia and lymphoma (disease
of white blood cells and the organs in which they are formed) and several types of solid
tumors have been shown to be correlated with specific translocation, inversions and/or
deletions of chromosomal regions in the affected cells. There is considerable on-going
research seeking to identify and characterize the genes that are in the regions of such
rearrangements (especially at the breakpoints) as they are likely to be involved in
transforming the cells to their malignant growth pattern.
The ability to identify specific chromosomes has been useful in studies that
localize genes to chromosomes (e.g. somatic cell genetics and in situ hybridization
methods beyond the scope of our discussion here). This technology is being used in the
current international effort to map the human genome.

PREPARATION METHODS: CELLS

CULTURING AND BLOCKING CELLS: Cells from sources such as those described
above are grown in culture under carefully controlled conditions that maximize the rate
of cell division. Some cell types (e.g. lymphocytes from blood) require chemical
stimulation to divide in culture. At a stage when many cells in the culture are dividing, a
chemical agent (e.g. colchicines or vinblastine) is added to block cell division at
metaphase. Briefly review mitosis in Figure 9.8. What types of cells would accumulate
in the culture when metaphase is blocked? Why not block mitosis in early prophase or
after anaphase?
SWELLING THE CELLS: Cultured cells, which have been arrested in metaphase, are
swollen by treatment with a hypotonic solution. Water from the solution diffuses into the
cells, increasing their volume and making them fragile.
FIXING THE CELLS: The swollen cells are treated with an acetic acid/methanol
solution that interacts with the proteins of the cells to stabilize cellular structures. Cells
are preserved by the fixative and can be stored in a freezer for months with relatively
little degradation. Note also that the acetic acid/methanol treatment inactivates any
infectious agents that might be in the cells.

PREPARATION METHODS: CHROMOSOME SPREADS

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PREPARING THE SLIDES: Our starting material in the lab will be HeLa cells that
have been cultured, blocked in metaphase with colchicines, swollen by treatment with a
hypotonic solution, and then fixed. We will spread the chromosomes on slides by
dropping the cell suspension from a height of approximately 12 inches above the slide.
When the fragile swollen cells hit the slide, those that were arrested in late prophase and
metaphase will generally burst, releasing their chromosomes. What cellular structure is
lacking in these cells? How does this greatly facilitate making chromosome spreads? To
increase the likelihood that the chromosomes spread out and do not overlap, we will heat
them gently by exhaling warm air on them immediately after dropping them on the slide.
STAINING AND PHOTOGRAPHING THE SLIDES: We will stain the slides with a
very simple, rapid method that will produce a purple, uniform staining on the
chromosomes. After staining the slides, let them air dry and then examine them with a
microscope. Scan your slides at low power to locate areas with good metaphase spreads
(chromosomes close together, but with little or no overlap). After finding an area of
metaphase spread, proceed to viewing the spread under oil immersion.

PREPARATION METHODS: KARYOTYPING

You will be provided with a photograph of a G-banded, normal human
lymphocyte chromosome spread (see worksheet 3). The chromosomes were prepared by
the basic method outline in the lab protocol and the chromosome preparation was
photographed with a 35 mm camera. (The photographs were obtained from the
Cytogenetics Lab at the Medical Center). Use this photo to construct your normal human
karyotype. Begin by covering the back of the print with double-sided tape, then cut out,
sort and identify the chromosomes using size, centromere position and banding as criteria
and arrange them as a karyotype.

Procedure:
1. Practice dropping 2-3 drops of solution (use water here) from a Pasteur pipette
onto a slide from a height of about 12 inches. The slide can be flat on the lab
bench or held at an angle of up to 60 degrees. If you are holding the slide on the
angle, have the drops land on the slide near the label and then run down the slide.
2. When you feel confident that you can hit the slide with reasonably accuracy,
remove a slide from the Coplin jar of ethanol (please cover the jar after removing
the slide).
3. Dry the slide thoroughly with a Kimwipe and let the instructor know you are
ready for the HeLa cells.
4. Draw up the cell suspension with a dry Pasteur pipette (fill only two-thirds of the
narrow part of the pipette).
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5. Hold your slide at the angle you prefer and from a height of 12 inches, drop the
cell suspension onto the slide.
6. After the fluid runs down the slide and before it dries, hold the slide in front of
your mouth and gently exhale warm air onto the surface carrying the cells. Do
this 3-4 times. What is the effect of warming the chromosome spreads at this
stage? What would be the effect of blowing forcefully?
7. Allow the slide to air-dry. Make sure you have labeled it with your initials.
8. Dip the slide in stain #1 for 1 second only, and then dip it again for 1 second
only.
9. Drain off the excess stain by holding the slide vertically and touching the end to
paper towels; carefully wipe off the back of the slide (do not contact the surface
onto which you dropped the cells).
10. Dip the slide in stain #2 for 1 second only, and then dip it again for 1 second
only.
11. Rinse off unbound stain with 5 dips in water. Wipe off the back of the slide with
a Kimwipe.
12. Allow the slide to air-dry after the staining, then examine it beginning with the
low power objective and find an area with a number of good chromosome
spreads. The most useful spreads are those in which the chromosomes are close
together but not overlapping.
13. Look for metaphase spread using the oil immersion objective in your
microscope.
Answer the following questions:
A. Examine the chromosome preparation you have made under oil objective
(100X objective). Is there a difference in overall length of the chromosomes
among the spreads? ____________ Why would you expect to be such a
difference?

B. What prominent cellular structure breaks down during prophase of mitosis?

How is the breakdown of this structure useful for making the chromosome
preparations?

C. (a) Construct a karyotype (page 30) of the chromosomes from the normal
human cell (worksheet 3) as directed in the protocol. Attach the
chromosomes to the underlying template provided. Label the groups on
your karyotype and indicate which are metacentric, submetacentric, and
acrocentric. Fill in the normal cell data in the table below.

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(b) Count the HeLa cell chromosomes on your print (Use worksheet 2 instead).
Determine how many are metacentric, submetacentric, and acrocentric.
Enter that data on the table below.
Normal Human
Somatic Cell

HeLa Cell Results

# of Chromosomes per cell

# of Metacentric Chromosomes per cell
# of Submetacentric Chromosomes per cell
# of Acrocentric Chromosomes per cell

(c) Briefly, discuss any differences you found between the normal and HeLa
cell karyotypes in terms of the criteria in the table.

(d) What other types of differences do you think you would probably detect if
you compared G-banded chromosomes of both types of cells?

D. Why do you think a cell line such as HeLa could survive with a grossly
abnormal karyotype whereas an embryo can not?

Exercise 9.4 Meiosis in Sordaria: A Study in Crossing Over

Sordaria fimicola is a fungus that spends most of its life as a haploid mycelium.
When conditions are favorable, cells of filaments of two different mating types fuse.
Ultimately the nuclei fuse and 2N (diploid) zygotes are protected within a structure called
the perithecium. Each 2N (diploid) zygote undergoes meiosis, and the resulting cells
(ascospores) remain aligned. The position of an ascospore within the ascus depends on the
orientation of separating chromosomes on the equatorial plate of meiosis I. After meiosis
I, each resulting ascospore divides once by mitosis, resulting in 8 ascospores per ascus.
This unique sequence of events means that it is easy to detect the occurrence of crossing
over involving chromatids carrying alleles that encode for color of spores and mycelia.

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Materials Needed:
Petri plate containing hyphae resulting from a cross between black and tan
spores
Slides and cover slips
Dropping bottle of methyl cellulose
Procedure:
1. Follow steps 1-19 on page 9-8 and 9-9. Answer questions and record results.
A)

How can you tell where a mating between the wild black strain (+) and the
mutant tan strain (-) took place?

B)

What is an ascus?

C)

What is a perithecium?

D)

In the table below, record the number of asci with spores all of one color
(indicating that the zygote was formed by fusion of cells of the same strain),
black and tan spores with crossover absent, and black and tan spores with
crossover present.

Table 9.4: Number of asci in each category

All spores of one color (all tan or all
black)
Crossover absent
Crossover present
E)

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What percentage of asci observed resulted from fusion of cells from different
strains?

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F)

What percentage of those resulting from the fusion of different strains

demonstrates crossovers?

References:
Abramoff, P. and Thomson, R. 1991. Laboratory Outlines in Biology V. Freeman.
Helms, D. 1994. Biology in the Laboratory. 2nd Ed., Worth Publishers.
Morgan, J. and Carter, M.E. 1993. Investigating Biology. Benjamin/Cummings.
Acknowledgements:
We wish to thank the late Professor B. Fritze of Broward Community College for
allowing us to use his Lab ideas and techniques that have been incorporated into this Lab
(pp. 9-6 to 9-9). We would also like to thank Professor Rosemond Potter of the University
of Chicago for sharing her ideas and techniques involving Human Chromosome Analysis
that appears in this Lab (pp. 9-11 to 9-21).

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Review Questions
Mitosis and Meiosis
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.

What are the stages of the cell cycle?

What is karyokinesis?
What is cytokinesis?
Which types of cells undergo: a) mitosis
b) meiosis?
What is the a) haploid number, b) diploid number of an organism?
What are the stages of Interphase?
What are the stages of mitosis?
What events occur at each stage of a) interphase, b) mitosis, c) meiosis?
Which process a) reduces the chromosome number; b) keeps it the same?
(mitosis or meiosis)
Which structure(s) is (are) found in plant cells but not in animal cells (or vice
versa) during mitosis, meiosis or cytokinesis?
Which organism has a polytene chromosome?
How many chromosomes are found in a somatic human cell?
What is the function of the fixative?
What is the function of the preservative?
What was used to stain the onion root tip?
Be able to identify the stages of mitosis/meiosis.
Be able to recognize the results of crossing over in Sordaria.
What is a perithecia?
What is an ascus?
What are autosomes and how many do humans have?
How many sex chromosomes are there?
What is a karyotype?
What are telomeres?
Which terms describe the position of the centromere?
What are the names given to the long and short arms of the chromosomes?
Which stain gives the chromosomes their banding pattern?
Which bands are rich in G-C / A-T?
What are HeLa cells? How are they different from normal cells?
Which chemical blocks the formation of the spindle fibers?
Which important structure disappears and allows the formation of a metaphase
spread?

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Worksheet 1

REPRESENTATIVE FIELD WITH METAPHASE SPREADS

(Enlarged Picture Taken Under 10X Objective)
a. metaphase
b. interphase nucleus

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Worksheet 2

METAPHASE SPREAD (HeLa Cells)

KARYOTYPE (46 chromosomes, XY)

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Worksheet 3.

METAPHASE SPREAD (Normal cell)

Note: Use this worksheet to do the karyotype.

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LAB REPORT #9
Date: ___________________
Name(s) :____________________________________________________

MITOSIS AND MEIOSIS

Exercise 9.1 The Cell Cycle and Mitosis
1.

In the circles below, draw onion cells you observed from your squashed root tips that illustrate the
important stages of the cell cycle. Name each stage. Draw one cell in each circle.

2.

Table 9.1: Percent of Cells in Each Phase of the Cell Cycle

Cell Stage
Interphase
Prophase
Metaphase
Anaphase
Telophase
Grand Total

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Field 1 Field 2 Field 3 Total

% Grand Total (total/grand x 100)

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3.

Using the information in page 9-4, calculate the following:

(a) Time spent in Prophase
Time spent in Metaphase
Time spent in Anaphase
Time spent in Telophase

_____hr ______min
_____hr ______min
_____hr ______min
_____hr ______min

(b) What was the total time spent in mitosis? ____________

What was the total time spent in interphase? __________
(c) How do your results compare with what is known about the Allium cepa
cycle?

4.

cell

Mitotic Index see page 9-5 to complete the table below:

Table 9.2: Mitotic Index for Onion Cells

Area 1 Area 2 Average

Total # of Cells
# Cells in Mitosis
Percentage of Cells in Mitosis
5.

Mitosis in Animal Cells List three (3) differences between mitosis in animal cells and mitosis in
plant cells.

(a) ___________________________________________________
(b) ___________________________________________________
(c) ___________________________________________________

Exercise 9.2 Giant Chromosomes of Drosophila

1.

Do you see any bulges along the length of the chromosome? __________ What do you think they
represent? __________________________

2.

How many chromosomes do you see? __________________________

(Note: Homologous chromosomes are paired along their entire length, so what appears as a
chromosome is actually two).

Giant Polytene Chromosome

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Exercise 9.3 Human Chromosome Analysis

1.

Examine the chromosome preparation you have made under oil (100X objective). Is there a
difference in overall length of the chromosomes among the spreads? ____________ Why would
you expect to be such a difference?

2.

What prominent cellular structure breaks down during prophase of mitosis?

How is the breakdown of this structure useful for making the chromosome preparations?

3.

(a) Construct a karyotype(see back of this page) of the chromosomes from the normal human cell
(worksheet 3) as directed in the protocol. Attach the chromosomes to the last page of the Lab Report
using the underlying template as a guideline. Label the groups on your karyotype and indicate
which are metacentric, submetacentric, and acrocentric. Fill in the normal cell data in the table
below.
(b) Count the HeLa cell chromosomes on your print. Determine how many are metacentric,
submetacentric, and acrocentric. Enter that data on the table below.

Normal Human
Somatic Cell

HeLa Cell Results

# of Chromosomes per cell

# of Metacentric Chromosomes per cell
# of Submetacentric Chromosomes per cell
# of Acrocentric Chromosomes per cell
(c) Briefly, discuss any differences you found between the normal and HeLa cell karyotypes in
terms of the criteria in the table.

(d) What other types of differences do you think you would probably detect if you compared Gbanded chromosomes of both types of cells?

4.

Why do you think a cell line such as HeLa could survive with a grossly abnormal karyotype
whereas an embryo can not?

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Use Worksheet #3 to do this karyotype.

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Exercise 9.4 Crossing Over in Sordaria

1.

How can you tell where a mating between the wild black strain (+) and the mutant tan strain (-) took
place?

2.

What is an ascus?

3.

What is a perithecium?

4.

In the table below, record the number of asci with spores all of one color (indicating that the zygote
was formed by fusion of cells of the same strain), black and tan spores with crossover absent, and
black and tan spores with crossover present.
Table 9.4: Number of asci in each category

All spores of one color (all tan or all black)

Crossover absent
Crossover present
5.

What percentage of asci observed resulted from fusion of cells from different strains?

6.

What percentage of those resulting from the fusion of different strains demonstrates crossovers?

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Worksheet 3.

METAPHASE SPREAD (Normal cell)

Note: Use this worksheet to do the karyotype.

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