LAB 4

Cell Division: Mitosis and Meiosis

Big Idea 3: Genetics and
Information Transfer

How do eukaryotic cells divide to produce genetically identical cells to

produce gametes with half the normal DNA?

Please be sure you have read the

student intro packet before you do this lab.

(If needed, the student intro packet is available at www.qualitysciencelabs.com/AdvancedBioIntro.pdf)

Lab Investigations Summary

Pre-lab and Questions: Loss of Cell Cycle Control in

Cancer
Lab Investigation 4.1: Environment Effects on Mitosis
Part 1 - How does the environment affect mitosis?
Null hypothesis and Chi-squared data analysis for significance
Part 2 - Student Guided Inquiry

Lab Investigation 4.2: Meiosis and Crossing Over in

Sordaria

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LAB 4 - Cell Division: Mitosis and Meiosis

Big Idea 3: Genetics and Information Transfer
How do eukaryotic cells divide to produce genetically identical cells to produce
gametes with half the normal DNA?

BACKGROUND
What are the differences between mitosis and meiosis?

Mitosis and meiosis are two of the most fantastically amazing processes in
life. Mitosis is a process in the cell cycle that allows the cell to duplicate without
fertilization. Prokaryotes (like bacteria) basically separate the cell contents into
two parts. This is called binary fusion. In eukaryotes, the process is more complex
and occurs in two main stages: mitosis and cytokinesis. Mitosis is the division of
the cell nucleus which is followed by the division of the cytoplasm or cytokinesis.
A common misconception is that interphase is the first stage of mitosis. However,
since mitosis is the division of the nucleus, prophase is actually the first stage.
Reproduction by mitosis is classified as asexual since the new daughter cells are
identical to the parent cell. Mitosis occurs as organisms grow and develop, like
The Cell Cycle

Cyclin Checkpoints
in red

c Phase
Mitoti
M Checkpoint

M
G2
Int

G1

Growth and
normal
metabolic
roles

S
erp hase

t Growth Phase
Firs

nd Growth Phase
o
c
Se

se

ha

Te
lop

Anapha
se

Growth and
preparation
for mitosis

hase

se

ha

p
Pro

Metap

G2 Checkpoint

DNA replication

G1 Checkpoint
(Restriction)

hesis Phase
Synt
2

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human skin cells that are being constantly replaced.

Meiosis involves two nuclear divisions: Meiosis I is the reduction
division. It reduces the chromosome number from diploid to haploid
and separates the homologous pairs. While different, meiosis I
looks similar to mitosis. There is crossing over of alleles from the
chromatids or recombinants, so the chromosomes are not identical to
the parent as in mitosis. Meiosis II, the second division, separates the
sister chromatids and results in four haploid gametes. Meiosis is how
sexually reproducing organisms produce gametes.

Cell Cycle, Cell Division, and Mitosis

The cell cycle is a series of events that cells go through as they

grow and divide. During the cell cycle, a cell grows, prepares for
division, and divides to form two daughter cells identical to the
mother cell. The purpose of mitosis is tissue growth, regeneration, or
asexual (vegetative) reproduction. There are three main stages during
the cell cycle: interphase, karyokinesis (mitosis), and cytokinesis.
Interphase is the longest lasting and includes three phases. G1
phase is a period of activity in which cells do most of their growing.
Cells increase in size and synthesize new proteins and organelles. S
phase is when DNA is replicated and key proteins associated with
the chromosomes are synthesized. During the G2 phase, many of
the organelles and molecules required for cell division are produced.
The M phase or mitosis (karyokinesis) now begins the process of cell
division.
Mitosis is divided into four main phases: prophase, prometaphase
and metaphase, anaphase, and telophase. Finally, cytokinesis occurs
which divides the cytoplasm and results in the production of the two
identical daughter cells to the parent cell.

Prophase

Metaphase

How is the Cell Cycle Regulated?

In the early 1970's, a series of experiments confirmed the cell cycle

is controlled by specific signaling molecules in the cytoplasm that
triggers and coordinates key events. In the cells cycle control system,
there are three main checkpoints where stop and go-ahead signals
regulate the cycle. These signals are both internal and external. The
G1 checkpoint is the most important. If the cell receives the goahead signal, it will usually complete the G1, S G2,and M phases and
divide(exception include mature muscle and nerve cells which never
divide, but are in the G0 phase.
Cell division is strictly controlled by specific protein complexes
called cyclins containing enzymes called cyclin-dependent kinases
(CDKs). CDKs can turn on or off the different processes involved in
cell division. There are CDKs specific for each phase of the cell cycle
and their levels change during the cycle. CDK is activated when it is
bound to cyclin. As each cyclin is turning on or off, CDK causes the
cell to move through the stages in the cell cycle.
Looking back at the cell cycle diagram, there are three checkpoints
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Anaphase

Telophase

that cyclins and CDK control before allowing the cell to proceed to the next phase:
G1, G2, and Mitosis at metaphase and anaphase. For example, the G2 checkpoint
checks for damage after DNA is replicated, and if there is damage, it prevents the
cell from going into mitosis. Cancer cells often are found to have the cell cycle
genes that interfere with proper cell cycle control.

Fertilization, Meiosis, and Crossing Over of Chromosomes

Meiosis involves the reduction division of chromosomes in phase I (Meiosis I)

resulting in two diploid daughter cells, but pieces of the chromosomes have been
exchanged (crossing-over). During this phase, homologous chromosomes form
a tetrad. Crossing-over occurs where chromatids cross over on another and the
crossed sections of the chromatids are exchanged. The homologous chromosomes
separate and two new cells
1
2
3
4
5
are formed.
Although
they look like the result of
mitosis, they are different
with sets of chromosomes
and alleles that are different
from each other (and
from the original diploid
6
cell) after crossing over
occurred. Phase II (Meiosis
II) has the chromosomes
lining up in the center of
7
each cell and separating.
Each of the four daughter
8
cells produced receive
two chromatids and are
9
haploid. Each daughter
cell is genetically different
from one another and from
10
the original cell. These
haploid cells are referred
to as gametes. In animals,
the female gamete is an
egg and the male gamete is
sperm. After fertilization,
then mitosis begins as the
11
cells in the fertilized egg
undergo cell divisions. See
the figure to the left.
Meiosis

Meiosis Legend
1. Interphase G
2. Interphase S
3. Prophase I
4. Metaphase I
5. Anaphase I
6. Telophase I

7.
8.
9.
10.
11.

Prophase II
Metaphase II
Anaphase II
Telophase II
Cytokinesis

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PREPARATION
Materials and equipment are listed with each lab separately.
Timing and length of lab activities

Lab Investigation 4.1 needs about five days prep for growth of onion roots.
During the last three days, the onions will be exposed to the variable caffeine.
Two lab periods will be required for staining and preparing slides, collecting data,
and mathematical analysis. Additional two lab periods are needed for the guided
inquiry. The Pre-lab cancer cell analysis and the Lab Investigations 4.2 do not
require lab bench time, or if done in class require one class period each.

Learning objectives aligned to standards and science

practices (SP)
To describe the events that occur in the cell cycle (3A2 and SP 1.2).
To construct an explanation, using visual representations, as to how the DNA
in chromosomes is transmitted to the next generation via mitosis, or meiosis
followed by fertilization (3A2 and SP 6.2).
To represent the connection between meiosis and how crossing over leads to
increased genetic diversity (3A2 and SP 7.1).

General safety precautions

Students should wear gloves and safety goggles or glasses when handling the
chemicals and razor blades. To avoid injuries, students should use a pencil eraser
instead of their thumbs when pressing down on cover slips.

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Pre-lab and Questions:

Loss of Cell Cycle Control in Cancer
Perhaps you know someone who has battled cancer in some form. Cancer can
occur when cells lose control of their cell cycle and divide abnormally. This happens
when tumor-suppressant genes, such as p53 or Rb (retinoblastoma), are mutated.
In normal cells, mitosis usually is blocked if there is DNA damage. Sometimes,
however, DNA damage makes cells divide more often as with certain forms of
leukemia.
Consider the following questions before you start this investigation:
How is the cell cycle controlled in normal cells? (See background info at the
beginning of this lab.)
What are cyclins and CDKs? What do these proteins (enzymes) do in a cell?
How are normal and cancer cells different from each other?
What are the main causes of cancer?
What goes wrong during the cell cycle in cancer cells?
What makes some genes responsible for an increased risk of certain cancers?
Do you think the chromosomes
might be different between normal and
cancer cells?

Human karyotype (normal cells) from

www.daviddarling.info/encyclopedia/K/karyotype.html

Background on HeLa cells

HeLa cells are cervical cancer cells
first isolated from a woman named
Henrietta Lacks.
Her cells have
been cultured since 1951 and used
in numerous scientific experiments.
Henrietta Lacks died from her cancer
not long after her cells were isolated.
Lacks cancer cells contain remnants of
HPV (human papillomavirus), which
we now know increases the risk of
cervical cancer.

Materials:
Human karyotype pictures above and on the next page of
normal and cervical HeLa cancer cells.

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Human karyotype (HeLa cervical cancer cells) from Modal karyotype

of the HeLa Ohio cervical adenocarcinoma cell line htcl.cytspb.rssi.ru

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Procedures:
1. Write a hypothesis as to how the chromosomes of a cancer cell
might appear in comparison to a normal cell and how those
differences are related to the behavior of the cancer cell.

2. Compare the normal karyotype picture to the HeLa cell

karyotype. Count the number of chromosomes in each type of
cell and note any differences in their appearance.

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Questions
HeLa Cells:

1. Do your observations support your hypothesis?

2. If not, what type of information might you need to know in

order to understand your observations?

3. If yes, what type of information can you find that would validate
your conclusions?

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Lab investigation 4.1:

Part 1 - Environmental Effects on Mitosis

How do environmental factors affect the rate and quality of mitotic division?
Scientists are interested in this question from the perspective of disease, specifically,
the uncontrolled division of cells known as cancer. This investigation will allow
you to make a simplified study between the relationship between environment
and mitosis. You will test the rate of growth of onion roots when exposed to an
environmental chemical, caffeine in the form of coffee.

Hypothesis:

Make a hypothesis to describe the effect of caffeine on the stages of mitotic division.

Materials:
Onion bulbs (2)*

Paper towels*

Toothpicks (8)

Distilled water*

Glass canning jars (2)*

Microscope slides (4)

Concentration of caffeine
(coffee): 0.5% (0.5 g/100 mL)*

Microscope coverslips (4)

Microscope*

Metric rulers (2)

0.5% Toluidine Blue stain

Digital scale

0.1 M HCl (hydrochloric acid)

Razor blade, straight edge

Light bulb heat source*

Forceps

Stopwatch

*items not included

10

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Procedures:
Advance Preparation:

Grow onion bulb roots in water to 1 cm a few days before

experiment starts.

1. After onion roots have reached 1 cm, change one of

the jars from water to caffeine; monitor onion root
growth in the water and caffeine jars over a three day
period. Record daily measurements in centimeters
of the root lengths.

2. After the third day of growth in caffeine and water,

harvest and stain the onion root tips. If you wait
longer, the cells may not show much mitotic activity.
3. Using the scalpel, cut five 1-2 mm pieces of root
tips from the onion in the water and place them on
a slide. Add five drops of 0.1 M HCl to the root
tips on the slide. Place the slide about 8 cm from a
45 watt light bulb for 30 minutes. When the HCl
starts to dry up, add a few drops more. (The base of
a lava lamp works great. See photos to the right.)

4. Remove the slide from the heat source. Tear a

paper towel into pieces and using the ragged edges,
dab and soak up the excess HCl.
5. Next, add 3-5 drops of 0.5% Toluidine Blue stain to
the slide with the five root tips. Return the slide to
the heat source for five minutes. Again, if the stain
starts drying out, add one or two more drops.
6. Remove the slide from the heat source, dab the
excess stain and add another two to three drops

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11

of stain. Transfer two root tips from the slide to a new slide,
making sure there is a bubble of stain covering the root tips.
Add the cover slip (at a 45 degree angle to minimize air bubbles)
and proceed to the following squash technique:

7. Place the slide on a paper towel cushion and cover the slide and
coverslip with a piece of paper towel. Push down on the coverslip
with the eraser of a pencil. You can press the eraser of the pencil
rather hard as long as you do not twist or torque. Press straight
down over the section that has the root tip.

8. Repeat steps 3 to 7 for your caffeine exposed root tips slide staining.
9. Look at your slides under a microscope at low and high powers
for cells undergoing mitosis. The cells will not be neatly arranged
with the squash technique. Examine the size, shape, and
position of the chromosomes in each treatment in order to help
you identify the different stages of mitosis. Count and record
the number of cells in each phase of mitosis on Table 4.1a.

10. Sketch the stages of mitosis observed from roots in each treatment.
11. Analyze the class data with Chi-squared data analysis IF you
have access to the data.
Collect the class data for each group and record it in
Table 4.1b. Calculate the mean and standard deviation
for each group.
Compare the number of cells from each group in
interphase and in mitosis using Table 4.1c.

Use a chi (pronounced ki) squared distribution test to

statistically analyze the data in Table 4.1d.

12. Enter the number of caffeine cells in interphase and mitosis as

observed (o) in Table 4.1d.
Calculate the percentage of cells in interphase and
mitosis in the control group from Table 4.1b.

Multiply the percentages by the total number of cells in

the caffeine group; this will give the expected numbers (e).
Calculate the chi-square (X2) value for the test.

Compare this value to the critical value in Table 4.1e.

13. Enter the number of caffeine cells in interphase and mitosis as

observed (o).
Calculate the percentage of cells in interphase and
mitosis in the control group from Table 4.1b.

Multiply the percentages by the total number of cells in

the caffeine group; this will give the expected numbers
(e) in Table 4.1d.

Calculate the chi-square (X2) value for the test (Table 4.1d).
Compare this value to the critical value in Table 4.1e.
12

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 14. See Table 4.1e. The degrees of freedom (df ) equals the number of
groups minus one. In this case, there are two groups, interphase
and mitotic; therefore, df = 2-1 or 1.
At a p value of 0.05, the critical value is 3.84. If the
calculated chi-square value is greater than or equal to
this critical value, then the null hypothesis is rejected and
caffeine DOES have a significant effect on the mitotic
growth of onion root tip cells.
If the calculated chi-square value is less than this critical
value, the null hypothesis is not rejected and caffeine does
NOT have an effect on the mitotic growth of onion root
tip cells. The NULL HYPOTHESIS says that there is
NO EFFECT OF CAFFEINE ON THE MITOTIC
GROWTH OF ONION ROOT TIP CELLS.

Conclusions and Post-lab Review:

1. Was there an increase in the number of cells in mitosis in the
caffeine treated roots? If you were able to analyze a large set of
data, was the difference significant (at p=0.05)?

2. Why is it important to collect class data when performing chisquare analysis?

3. Does an increase in the number of cells in mitosis mean that

these cells are dividing faster than the cells in the roots with the
lower number of cells in mitosis?

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Table 4.1a: The Number of Cells in Interphase, Mitosis, and Cytokinesis

Number of Mitotic Phases in Each Treatment
Interphase

Prophase

Metaphase

Anaphase

Telophase

Cytokinesis

Control
H 2O
Caffeine
(0.5%)
Table 4.1b: Class Data
Number of Cells

Group

Interphase

Mitotic

Total

Control
Caffeine
Table 4.1c: Comparison of Interphase and Mitotic Cells
Interphase Cells

Mitotic Cells

Total

Control
Caffeine
Table 4.1d: Calculation of Chi-Square X 2 = (o-e)2/e =

Interphase
Cells

# Observed
(o)

# Expected
(e)

(o-e)

(o-e)2

(o-e)2 / e

Mitotic Cells
Table 4.1e: Critical Values of the Chi-Squared Distribution
Probability

14

Degrees of Freedom (df)

1

0.05

3.84

5.99

7.82

9.49

11.1

0.01

6.64

9.21

11.3

13.2

15.1

0.001

10.8

13.8

16.3

18.5

20.5

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Part 2 - Student Guided Inquiry,

Environmental Effects on Mitosis
Going back to Lab Investigation 4.1 on the environmental effects on mitosis and
plants, what other factors might increase or decrease the rate of mitosis? Design
and conduct an investigation to determine the effects of such factors as: salinity,
temperature, pH (acid rain), humidity, fertilizers: N, K, P, Fe, sugar, CO2; or biotic
organisms like beetles, ants, roundworms, etc. which might alter root growth.
Step 1:

Chose your driving question to investigate.

Step 2:

Fill in the ExD (Experimental Design) form on the next page

to plan your experiment. Remember to include a control and to
test only one variable at a time while keeping all other potential
variables constant.
Step 3:

Design data tables and collect data.

Step 4:

Analyze the data (graphs chi-square calculations).

Step 5:

Summarize your conclusions.

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15

Experimental Design (ExD) Form

Complete this ExD pre-lab planning form before beginning your lab
1.

Independent variable: (What is the cause agent? What are you changing?)

2.

Dependent variable: (What is being measured?)

3.

Lab set-up:
Experimental
Groups
Number of Trials

4.

Control: (What is the experimental group being compared to?)

5.
Hypothesis: (Use an if [Independent Variable], then [Dependent Variable] format.
State the cause and effect relationship between the I.V. and the D.V. It must be testable.)

16

6.

Lab title: (The effect of ____[I.V.] ____on ____[D.V.]____.)

7.

Experimental constants: (Name at least six variables NOT altered during the experiment.)

8.

Sketch of experimental set up with labels:

9.

Detailed procedure:

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LAB Investigation 4.2:

Meiosis and Crossing-Over in Sordaria
The fungus Sordaria fimicola is an
ascomycete fungus that can be used to
demonstrate the results of crossing over
during stage 1 of meiosis. Sordaria is a
haploid organism for most of its life cycle.
It becomes diploid only when the fusion
of the mycelia (filament-like groups of
cells) of two different strains results in
the fusion of the two difference types of
haploid nuclei to form a diploid nucleus.
The diploid nucleus must then undergo
meiosis to resume its haploid state.
Meiosis, followed by one mitotic
division, in Sordaria results in the
formation of eight haploid ascospores
contained within a sac called an ascus
(pleural, asci). A single gene determines
the spore color. In the illustration on the
following page (Figure 4.2d), a cross was
made between wild type (black) and tan
strains. The resulting zygote produces
either parental type asci, which have four
black and four tan spores in a row; or
recombinant asci, which do not have this
pattern (refer to Figures 4.2b and 4.2c).

Mature perithecium
containg many asci

Ascospores
8 haploid nuclei
Spore discharge

4 haploid nuclei
Diploid zygote
Meiosis
Fertilization

Ascospore

Mitosis

Mitosis

Mycelia fuse
Mitosis

Filament

Mycelium

The Life Cycle of Sordaria fimicola

Figure 4.2a

Meiosis without
crossing over
Figure 4.2b

or

Meiosis with
crossing over
Figure 4.2c

or

or

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or

17

Sordaria fimicola asci tetrad analysis

Figure 4.2d

Materials:
Figure 4.2d above, an illustration of Sordaria asci

Procedures:

1. For at least 50 asci in Figure 4.2d above, count the number

showing crossover and the number not showing crossover. Enter
your data in Table 4.2a.
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Number of 4:4 (non-crossovers)

Table 4.2a

Number of asci showing crossover

Total Asci
% Asci Showing Crossover Divided by 2
Gene to Centromere Distance(map units)

Data Analysis:
The frequency of crossing over appears to be governed by the distance between
the genes, or in this case, between the gene for spore coat color and the centromere.
The probability of a crossover occurring between two genes on the same chromosome
(linked genes) increases as the distance between those genes becomes larger. The
frequency of crossover, therefore, appears to be directly proportional to the distance
between genes.
A map unit is an arbitrary unit of measure used to describe relative distances
between linked genes. The number of map units between two genes and the
centromere is equal to the percentage of recombinants. Customary units cannot
be used because we cannot directly visualize genes with the light microscope.
However, due to the relationship between distance and crossover frequency, we can
use the map unit.
Using your data from Table 4.2a above, determine the distance between the
gene for spore color and the centromere:
1. Calculate the percentage of crossovers by dividing the
number of crossover asci by the total number of asci X 100.
2. To calculate the map distance, divide the percentage of
crossover asci by 2. Record your results in Table 4.2a.

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19

Evaluation and Conclusions:

1. Why did you divide the percentage of asci showing crossover
(recombinant) by 2?

2. The published map distance between the spore color gene and
the centromere is 26 map units. How did your data (or class
data) compare with this distance?

3. Illustrate what happened during meiosis to produce the results

you found.

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