OBSERVATION OF MITOSIS
In this inquiry using prepared slides of onion root tip, you will examine onion root cells in various stages of
mitosis, count the number of cells in the various stages, and approximate the time needed for cells to complete each
phase. You also will develop a hypothesis about the role of mitosis in cancer, and use comparisons of mitosis
phases for normal cells and cancerous cells to evaluate your hypothesis.
Materials
Compound Microscope
Prepared slides of onion (Allium) root tip, longitudinal section
Background
The onion (Allium) is one of the most widely used materials for the study of mitosis because it is readily available,
preparation of the dividing cells is easy, and the chromosomes are large and few in number. Root tips of plants
contain meristems, which are localized areas of rapid cell division, therefore, chances are good that in a specimen
of such tissues, one can find every stage of mitosis.
Obtain a slide of onion root tips, and note a series of dark streaks on it. Each tip is a very thin longitudinal section
through an onion root tip.
Place the slide on the stage of your compound microscope, and locate 1 of the sections using the scanning (4x)
lens. It is often possible to determine whether a given section shows good examples of mitotic stages (region of
most mitotic activity is above the tip). Because each section is very thin, not all will be equally good for study.
After your preliminary examination, change to the higher power objectives. Remember – use only fine focus knob
when using any objective other than 4x. You will need to visualize the cells under high power (40x objective) to
determine the appropriate stage.
Interphase
The interphase cell was named as such because it was thought to be a resting phase. It is now known that the cell is
actively undergoing cellular metabolic activities and synthesis of nucleic acids and proteins. Interphase cells are
characterized by a distinct nucleus bounded by a nuclear envelope (membrane). One or more nucleoli may be
visible.
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Prophase
During prophase, chromatin has condensed enough for chromosomes to be visible in the nucleus. The nuclear
envelope begins to break down, and the chromosomes are now free to be distributed throughout the cytoplasm. The
nucleolus disappears. Each chromosome consists of two identical sister chromatids, which are connected at the
centromere. Within this region, each chromatid contains a protein disc-shaped kinetochore. A bridge of
microtubules called the spindle apparatus begins to form between the two poles of the cell. The spindle fibers
insert into the kinetochore and run from them outward to the two poles of the cell.
Metaphase
Near the central region of the spindle apparatus is a plane referred to as the metaphase plate, at right angles to the
long axis of the spindle fibers. During metaphase the chromosomes are maneuvered into position by the spindle
fibers that are attached to the kinetochore of each chromosome. One sister chromatid of each chromosome is
connective to spindle fibers running to one pole, and the other sister chromatid is connective to spindle fibers
running to the other pole.
Anaphase
Anaphase begins when sister chromatids are pulled apart by the spindle fibers; the centromere splits, and the sister
chromatids become daughter chromosomes as they are pulled to opposite poles of the cell. This stage can be
recognized in the onion cell by the 2 groups of V-shaped chromosomes; the sharp end of the V is oriented toward
the pole. The onion has a diploid number of 16 chromosomes, so it is seldom possible to see all of them at one
given time.
Reducing the light from your microscope may allow you to visualize spindle fivers near the center of the cell. They
appear as very fine lines between the two groups of chromosomes.
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Telophase
As telophase begins, a nuclear envelope reforms by the fusion of its parts of the endoplasmic reticulum, and the
nucleoli reappear. Chromosomes de-condense into more diffuse chromatin. Mitosis ends with the assembly of 2
interphase nuclei, each with 1 complete set of single-stranded chromosomes.
Cytokinesis
The first indication that cytokinesis is beginning is the formation of a cell plate, which forms a fine line across the
center of the cell. Vesicles containing new cell wall material fuse together, elongating the plate, until ultimately the
cell divides into two daughter cells.
Procedure
Part A: Locating and counting cells in stages of mitosis
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2.
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4.
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6.
7.
Locate under the microscope on an onion root tip slide an area with cells in the process of mitosis. After
locating the cells under low power, switch to high power.
Count and record the number of cells in each phase and interphase. Count at least one hundred cells in the
field of view.
HELPFUL TIP: Remember that not all regions of the root tip will have cells that undergo mitosis.
Regions of the root tip that you will want to avoid include the zone of maturation (farthest from the tip)
and the root cap. See the figure below.
Move the slide so you are looking at a new area of cells (or another onion root tip).
Count and record the number of cells in each phase and interphase for this second area.
Repeat for a third new area (or another onion root tip).
Total the number of cells counted in each phase and interphase for the three areas. Record this figure
under "Total number of cells" in the table.
Add the total number of cells viewed in each phase and interphase to get the total number of cells
counted. Record this number in the table in Part B.
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Figure: Root tip of onion (Allium), showing regions of development.
Zone of maturation
Zone of elongation
Zone of initiation
Root cap
Part B. Determine the time required for each phase
Assume that the number of cells in a phase indicates the time spent during mitosis of that phase. Time spent in a
mitotic phase or in interphase can be calculated if the total time for the complete cell cycle is known. If onion cells
require 24 hours (1440 minutes) to complete the cell cycle (from interphase to interphase), then:
hours spent per phase = (# of cells in that phase ÷ total # of cells counted) x 24 hours
1. Calculate the time required for each phase of mitosis using your data. Use the total of the three areas counted.
Assume that the total time for the cell cycle is 24 hours.
2. Record the times in the last column of the table.
First area
Phase
Interphase
Second area
Third
area
Prophase
Metaphase
Anaphase
Telophase
Total # of
cells counted
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Total #
of cells
Time in hours
Questions
Please type out your answers to these questions. Multiple pages must be stapled together (no paper clips or unattached
pages). Don’t forget to include the above table!
1. Which cell cycle phase requires the longest time for completion?
2. Which mitotic phase requires the longest time for completion?
3. What important changes are occurring in the nucleus during the longest phase of mitosis? Does this justify the
amount of time spent in the phase?
4. Which mitotic phase requires the next longest time for completion?
5. Which mitotic phase requires the shortest time for completion?
6. Are there any assumptions made in using this method to estimate the timing of phases within the cell cycle?
What are the most important assumptions?
PART C. Testing hypotheses about cancer using observations of mitosis.
In the scientific method (hypothetico-deductive method), complementary hypotheses are evaluated to determine the
best explanation for a natural phenomenon. The scientific researcher develops both a testable hypothesis
(sometimes called an alternative hypothesis) and a complementary null hypothesis. Then the researcher collects
data to evaluate both hypotheses, and if the data is sufficient, determines that one of the hypotheses is the better
explanation.
Cancer is a disease that involves irregular accumulation of cells within the body tissues.
Develop a pair of hypotheses about cancer that includes both the cell cycle and mitosis.
Use the following table as evidence to test your hypothesis, and to answer questions 7 through 12:
Time (in minutes) of cell cycle phases for normal and cancerous chicken stomach cells
Normal
Cancerous stomach cells
stomach cells
Interphase
120
16
Prophase
60
15
Metaphase
10
2
Anaphase
3
1
Telophase
12
3
7. Which mitotic phase for normal cells requires the longest time for completion?
8. How do cancer cells differ from normal cells in time spent for each cell cycle phase?
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9. How do cancer cells differ from normal cells in total time required for mitosis?
10. What are your hypotheses?
11. Given the data presented here, what conclusion can you make about your hypotheses?
12. Imagine you decide you want to publish the results of your (this) research. What are some important
assumptions made in this study that you might need to defend in your written work?
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