MITOSIS AND DEVELOPMENT IN MULTICELLULAR ORGANISMS

Exercise 11
MITOSIS AND DEVELOPMENT IN MULTICELLULAR ORGANISMS
Student Learning Outcomes
(1) Be able to list and identify the phases of a cell’s life cycle known as Prophase, Metaphase,
Anaphase, Telophase and Interphase.
(2) Be able to draw a pie diagram indicating the relative proportions of time a plant, like Allium
(onion), spends in the different mitotic phases.
(3) Be able to diagram and describe the stages of embryonic development common to all
animals (zygote, morula, blastula, and gastrula).
(4) Be able to diagram and describe the function of a fertilization membrane surrounding a
fertilized Sand dollar egg.
(5) Be able to contrast the end results of the 2 kinds of cell division observed in Eukaryotic cells:
Mitosis and Meiosis.
Introduction
The tremendous diversity of structure and function that eukaryotic cells assume is remarkable
when you consider that most multicellular organisms begin life as a single fertilized egg, the
zygote. Through repeated divisions, this cell gives rise to all the cells that make up the organism.
The series of events experienced during cell division by actively reproducing cells is termed
mitosis. Mitosis also serves as the basic mechanism of reproduction in unicellular organisms. In
this lab, you will examine several essential aspects of mitosis, and of the role of mitosis in the
early development of multicellular organisms.
Mitotic processes are rather easily observed in situations where rapid increases in cell numbers
are occurring. Two cell types that will be used in this lab are 1) the cells produced as a
consequence of fertilization in the sand dollar, Dendraster, and 2) cells in the growing root tips
of the onion, Allium.
I. Mitosis in Plants
At no time other than cell division can a cell’s chromosomes be observed. Although it is also
possible to see chromosomes during meiosis, today we will examine mitosis in a rapidly growing
tissue, the root tip of the onion Allium.
Although animal and plant cells differ somewhat in structure as well as in some of the fine points
of mitosis, the mitotic phases are essentially the same in all eukaryotic cells. While the process is
continuous and there is some gradation among the various steps, four general phases can be
identified: Prophase, Metaphase, Anaphase, and Telophase. Mitosis is followed by
cytokinesis, the division of the cell’s cytoplasm, which also takes the two new nuclei into
separate daughter cells. When a cell in a growing tissue phase is not in mitosis, we say it is in
Interphase (this phase is not regarded as one of the phases of mitosis). During this relatively
long period, the DNA is replicated in the nucleus and extra amounts of other cellular components
are synthesized, in preparation for distribution to future daughter cells. No evidence of this
activity can be seen until the next mitosis, when the nuclear membrane dissolves and the
chromosomes condense into the short, thick, stainable bodies which are visible through the light
microscope.
For any given species, there is usually one characteristic number of chromosomes (n) found in
the gametes and double that number (2n) found in cells of the sexually reproducing adult. There
is wide variation in the number of chromosomes characteristic of species, and the n does not
necessarily indicate how much hereditary material is present. In some species, there is a large n
but the chromosomes are very small, while other species have fewer, larger chromosomes. The
chromosomes of onion cells are large and relatively few (2n=14), making them easier to study
than the cells of many other organisms.
A. Observation of Mitotic Phases in Onion (Allium) Root Tip Cells
The growing onion root tip is one of the most widely used materials for the study of mitosis,
since it is easily cultivated in quantity and preparations of the dividing cells are easily made.
There are regions of rapid cell division in root tips; therefore, the chances are good that within
such tissues one can identify every stage in mitosis. The following study will involve an
examination of preserved and stained thin sections of onion root tips, with the stains making the
chromosomes of the cells quite visible.
You will need a microscope for this portion. Be sure to use two hands. Make sure that the cord
is not dangling to prevent a tripping hazard. Before using the microscope, check for the
condition the microscope was left in from the prior class.
Procedure:
1. Plug in the cord and turn up the light intensity.to its maximum value and adjust the iris
diaphragm to its most closed setting. As you proceed you can increase the light passing
through the specimen by gradually opening the iris diaphragm.
2. Adjust the distance between the oculars: Without placing the prepared slide on the stage yet,
look through the oculars. You are likely going to see two circles of white light. Do not try to
focus your eyes on any one thing, as nothing is in focus yet. Slowly, move the two oculars
together and/or further apart until you see the single Field of View.
3. Lower the stage using the coarse focus knob, and make sure the (shortest) scanning objective
is facing the stage, so that there is no chance of the slide scratching any objective lenses.
4. Obtain a slide of Allium root tips. Hold the slide above a sheet of white paper, and note the
series of dark streaks on the slide. Each streak is a very thin longitudinal section through an
onion root tip.
5. Before looking through the eyepiece (ocular), open the stage clip, and place the slide on the
stage of the microscope beneath the objective, with the coverslip visible on the upper side.
The stage clip should be holding the slide in place, not pressing the slide under it. Center the
object below the objective without looking through the oculars. Nothing is in focus yet.
6. Coarse adjustment with scanning lens: With the scanning lens in place, move the stage up
to its highest point without looking through the oculars. Nothing is in focus yet.
Looking through the ocular with your right eye only (squint or cover your left eye), bring the
specimen into focus by turning the coarse focus adjustment knob slowly until the specimen is
generally in focus. Then turning the fine adjustment knob will bring the specimen into
sharper focus.
7. Focus your left eye: Viewing the specimen with both eyes through both oculars, turn the left
ocular diopter until the specimen is clear in both eyes.
8. Adjusting on low and high power objectives – use fine adjustment knobs only: If you wish
to view the specimen using higher magnification, center the specimen in the field, and
carefully rotate the revolving nosepiece to bring the next higher power objective into place
beneath the body tube. The specimen will no longer be in focus. In order to sharpen the
image of the specimen, adjust the focus using only the fine focus adjustment knob.
(Again, the light may have to be adjusted with the iris diaphragm.) Each time you move to
the next higher power objective, be sure you center the specimen beforehand.
9. Locate one of the root longitudinal sections under scanning power and then move to low
power, to determine whether it shows clear mitotic stages. Since each section is very thin, not
all will be equally good for study. A good section will have many cells showing the dark,
strap-like, chromosomes. Notice that the “best” area for mitosis is not at the very tip end of
the root or high up the root where it was cut, but above the root cap (See Figure 1).
10. After preliminary examination under low power, change to high-dry power, being very
careful not to break the slide. Keep in mind the sequence in which the different stages occur,
but do not try to find them in sequence. Thus, if you happen to find an Anaphase first, study
it before proceeding to another stage. Chances are that most of the cells will be in interphase.
(You may even see cells which appear to have no nucleus. Remember that this is a very thin
section and the slice you see captured the edge of some cells, missing the nucleus.) Keep this
slide for the rest of the lab exercise.
11. Obtain one of the folders entitled Plant Mitosis (set #55), view it through the mini-scope,
and use it to become familiar with the stages of mitosis. Figure 4 shows these stages also.
Refer to the plant mitosis model sequence on the side lab bench to help you visualize the
process.
12. Scan the prepared slide of the Allium root tip until you can reliably identify the four mitotic
phases. Be able to describe the chromosomal changes which occur during each phase. (It is
not necessary to be able to count every chromosome; just describe their positions, shapes,
and orientations.) For your information, descriptions of these phases follow.
Mitotic Phases
Prophase
During Prophase the chromosomes become distinguishable in the nucleus. The nuclear membrane breaks
down and the chromosomes become distributed haphazardly through the cytoplasm. At this stage in the
onion root tip the chromosomes often appear as a partially coiled mass. These elongated
chromosomes later become condensed into shorter chromosomes and the nuclear membrane disappears.
Even at this early stage each chromosome has doubled, although this will be difficult to see on the slide.
Metaphase
During Metaphase the chromosomes become arranged near the center of the cell, with the ends of the
chromosomes generally pointing away from the midline. This stage is apparently a preparation for the
equal division of chromosomes between the daughter cells, a process that begins in the next phase. During
or somewhat before Metaphase, small, threadlike structures (spindle fibers), form in the dividing cell.
Some of them appear to be attached to the chromosomes and seem to provide the machinery for the
movement of the chromosomes, although the way in which this is accomplished is not yet understood.
These fibers usually appear most clearly in Anaphase. The composition of the spindle fibers is not known.
It has been suggested that they form by the aggregation of protein molecules. Under the electron
microscope these fibers appear as fine, straight, hollow tubules. Although they lengthen and shorten
during Mitosis, they do not appear to get thicker or thinner. This suggests that they do not stretch or
contract but that new material is added to the fiber or removed from it as the spindle changes shape.
Anaphase
At the beginning of Anaphase, the two members of each of the previously doubled chromosomes separate
and move toward opposite ends of the cell. This stage can be recognized in the onion by the two groups of
V-shaped chromosomes on opposite sides of the cell. The sharp end of the V is pointed away from the
center toward the cell wall. An onion cell has 14 chromosomes; hence it is seldom possible to see all of
them at one time. Reduce the light coming through the objective of the microscope and see if you can find
any spindle fibers near the center of the cell. They will appear as very fine lines between the two groups
of chromosomes, but they are not often visible in a study of this kind.
Telophase
Cell division is completed during Telophase, and reorganization of the cell contents of the two daughter
cells begins. It is often difficult to distinguish between late Anaphase and early Telophase in the cells of
the onion root tip. During Telophase, however, a cell plate starts to form as a fine line across the center of
the cell. When complete, the cell plate will divide the original cell into two daughter cells. In some cells it
will be indistinct. As Telophase progresses, the nuclei begin to reorganize, and the chromosomes
become indistinct. In both plant and animal cells, the daughter cells resulting from mitotic division have
the same number and kinds of chromosomes as the original cell from which they came. Thus, in the onion
each daughter cell has 14 chromosomes, just as the original cell had.
Figure 1. A
simplified drawing of a longitudinal section through the root tip of an onion (Allium).
Refer to the laboratory models and charts, your slides, or your textbook’s diagrams of the mitotic
phases in plants. In the spaces below, make realistic rough sketches (not cartoons) of each stage
of the cell cycle (including interphase) as one onion root tip cell divides into two daughter cells.
Make diagrams here: List the main actions that
happen during this phase here.
Use arrows and words to label
important parts:
Prophase:
Metaphase:
Anaphase:
Telophase:
Interphase:
Figure 2. Diagrams Showing the Different Phases an Onion Cell
Moves Through during a Mitotic Cell Division.
B. Durations of Phases in the Cell Cycle
The cell cycle is the name for the entire sequence of events from one division to the next,
including all the steps of mitosis, plus cytokinesis and interphase. The time it takes for the cell
cycle varies widely, with cells of developing embryos dividing the fastest. (You may have
observed the first cleavage of the sand dollar less than an hour after fertilization.)
As you will observe, some of the mitotic phases take longer than others. Now that you can
identify the various stages of mitosis, you are ready to determine the relative percent of time
spent in each phase during the life of a cell.
We do not have enough lab time to sit at the microscope and watch one live root tip cell go
through its entire cycle, since that might take many hours. Therefore, we will examine a group of
cells in a root tip at one point in time and deduce the relative time spent on the different phases
of the cell cycle. In order to make this determination, some assumptions are necessary:
a. In the growing region of a root tip, all of the cells are assumed to have the same overall
cell cycle time (including interphase).
b. The phase of the cycle which takes the most time (i.e., Interphase) will be that phase
which is seen in the greatest number of cells.
c. Since cells quickly pass through phases which require less time, the phase which takes
the least time will be seen in the fewest cells.
d. If we census a large block of cells in the growing area of a root tip, identifying the phase
of each one, the relative numbers will tell us which phases require more time and which
require less time.
Procedure:
1. Observe the Allium root tip longitudinal section provided above.
2. While looking at the root tip provided, one identify the mitotic phase (or Interphase) for each
cell observed, moving longitudinally up and down the root tip. Tally (Table 1) of the number
of cells in each phase. Skip those cells with no visible nucleus (what happened to these
cells?), but count all of the others, including those in Interphase. (Try to avoid counting the
same cell twice.)
3. The cell phases should be tallied until all cells with a nucleus visible have been observed and
counted. Record your tallies in Table 1 below.
Table 1. Number tally of onion root tip cells
in different phases of the cell cycle
Group Tally Other Group
Counts
Class
Total
Percent of
Time
Class
Avg.
Interphase X x x
Prophase X X X
Metaphase x x X
Anaphase X X X
Telophase x x X
Total Cells X X X
15. Calculate the percent of the cells’ total cycle time spent in each phase using the following
formula:
percent of time spent in phase = number of cells in phase
total number of cells counted
16. Record the values in Table 1 and in the class data table on the board.
Question 1. Based on your calculations which phase plant cells spend the most time. Be able to
explain why.
Question 2. Why is a class average more likely (than your group’s data alone) to reflect the true
percentage of time spent in each phase?
Question 3. Using your data for each phase (Table 1, previous page), draw a “pie chart” to
graphically demonstrate the relative amount of time an average onion root tip cell spends in each
phase of mitosis: Put the phases in the proper order going clockwise from the top center starting
with interphase.
Using the whole “pie” below to represent 100% of the cell cycle time, sketch in “wedges” which
reflect the portion of the pie (relative percent of time) spent in the different phases by the cells in
your onion root tips.
Figure 3. Pie Chart: Relative Amounts of Time
Spent in the Various Phases of Plant Mitosis
Question 4. Compare your chart with a general pie chart for plant mitosis found in your textbook
or some other reference. Describe the similarities and differences.
Question 5. Speculate on why our class’ pie chart percentages may not agree exactly with those
seen in your reference.
II. Fertilization and Early Embryonic Development in Sand Dollars
The development of the zygote into a complex of interdependent cells, tissues, and organs that
make up an adult animal is one of the more fascinating processes in biology. Using the following
procedure, you will examine the process of fertilization and the resulting mitotic divisions which
quickly follow. The complete developmental sequence from zygote to adult cannot be
accomplished within a one-lab time frame. However, several increasingly complex larval stages
could be observable over the next few days (depending, of course, on larval mortality rates in the
lab).
The most abundant sand dollar found along the San Diego coastline is a small organism (up to 3
1/2 inches in diameter) living in sandy areas below the surf zone. A member of the Phylum
Echinodermata, the sand dollar is covered with a very short blanket of movable spines (used for
locomotion), among which are short, moving hair-like projections called cilia. Sand dollars filter
small nutrient particles and plankton from the sea water. They have provided many generations
of biologists with a ready source of gametes for reproductive and developmental studies. Each
female will produce millions of eggs and each male will eject even more sperm.
A. Observation of Adult Sand Dollar
Procedure:
Take a few moments to study an adult sand dollar. The focus knobs are found on the arm of the
dissecting microscope. The magnification can be changed by rotating the knobs on the head of
the microscope. Then turn the Top light on. Place a few paper towels on the stage of a
dissecting microscope, and then place a sand dollar on the paper towels.

Question 6. Are the aboral (top side) and oral (under side) surfaces similar? If not, how do they
differ?
Question 7. Closely observe the flat oral surface. Locate the main opening in the center,
surrounded by the five-pointed star-like structure. The star-like structure is often difficult to see
in a sand dollar as it covered by many of the moveable spines. However, this structure is much
more easily seen in a relative of the sand dollar, an urchin. View the following video to observe
this structure. https://www.youtube.com/watch?v=hLg4EbxWqNo What is this structure’s
function?
B. The Gametes: Eggs and Sperm
The external anatomy of sand dollars will not reveal their sex, but by injecting them with a few
milliliters of an isotonic potassium chloride solution, they can be induced to shed their gametes.
Due to the decreasing numbers of echinoderms, such as sand dollars, in general, we will observe
the gametes using videos.
Question 8. Observe the following short video: Video”A Sea Biscuit’s Life) 3:36 minutes

Use the above Video to answer questions 8, 9, 10, and 12.
Above is a Sand Dollar Egg, notice the difference between it and the Sea Biscuit egg (35 seconds
into the video). What do you see as the major differences between the two species?
Question 9. Describe the sperm movement (31 to 33 seconds in video). Does it appear to be
directed or random?
Question 10. In the spaces below, briefly describe the form (shape) of an egg and a sperm.
Then describe the mobility (movement), if any, of the two kinds of cells. Now compare the
gametes’ sizes (make sure you are viewing them both at the same magnification).
Sperm Egg
Form
Mobility
Size
Suggest a possible explanation for the observed differences.
Question 11. Surrounding the egg cells, you will notice a protective gelatinous material
containing colored granules. This material is outside the cell membrane of the egg. (Label this
material in the image labeled Unfertilized Egg in Figure 4.)
C. Fertilization
Question 12. It is usually not possible to observe fertilization (nuclear union) but you can tell
when it has occurred, for a fertilization membrane (40seconds) will develop around each
fertilized egg, inside the egg’s gelatinous cover. (Label this on the image of the Fertilized Egg in
Figure 4.) This halo-appearing membrane emerges from the surface of the cell and signals the
formation of the zygote. Draw a fertilized egg showing the fertilization membrane. What might
be its function?
D. Mitosis and Cleavage
Cleavage is a form of mitotic cell division which is not accompanied by cellular growth. In fact,
each successive cleavage will increase the number of cells while their volume is halved. Thus,
the total mass of the embryo remains the same while the cells proliferate.
The cleavage sequence includes all the stages from zygote through blastula. However, even
though all the mitotic stages (Prophase, Metaphase, Anaphase, etc.) are occurring with each
cleavage stage, we would not be able to actually see the sand dollar chromosome. This is due to
their small size, the large quantity of opaque cytoplasmic material in the cells, and the need for a
specialized staining technique to make them visible. Chromosomes will be more easily observed
in the stained onion root tip cells found on prepared slides used in Part I of today’s experiment.
In mitosis the nucleus divides in a process known as karyokinesis, which apportions identical
chromosomal material to each of the two daughter nuclei. This is soon followed by the more
obvious cytokinesis, or division of the cell body. This first mitotic cell division requires only a
few minutes and should occur within an hour after fertilization. Subsequent divisions will
probably occur at longer intervals. For your reference, data are available for the time frame of
cleavage in the sea urchin, a relative of the sand dollar (see Table 2).
Question 13. Draw arrows between each of the stages in Figures 4 and 5. Then on the top of the
arrow, label what process is happening (formation of fertilization membrane, first cleavage, etc.)
based on what is listed in Table 2 below. On the bottom of the arrow, label how long the process
takes (e.g. 2-5 minutes, etc.). See the example on Figure 4.
Table 2. Approximate Time Sequence for the Development
of Fertilized Sea Urchin Eggs
(Note: cell division is temperature dependent)
formation of fertilization membrane
2-5 minutes
first cleavage 50-70 minutes
second cleavage 78-107 minutes
third cleavage 103-145 minutes
blastula 6 hours
hatching of blastula 7-10 hours
gastrula 12-20 hours
pluteus larva 24-48 hours
Question 14. On entering the egg, the sperm nucleus carrying the male chromosomes unites
with the nucleus of the egg to produce a zygote with the chromosomes of both egg and sperm.
What is the relative chromosome number of the zygote compared to the gametes? THINK!
E. Early Cleavage Stages (See also Figure 4)
Procedure:
Question 15. Were the photographs of all the stages up to gastrulation pictured in the video of
the Sea Biscuit seen earlier taken at the same power magnification?
a. Within individual stages in the early cleavage stages, how do the individual cell sizes
compare?
b. How does the size of the entire cell mass compare at these different stages?
F. Late Cleavage Stages (See also Figure 5)
Procedure:
1. Use figure 5 and the video of the Sea Biscuit.
Question 16. Find the mass of cells called a morula (1:22 in the Video) and on figure 5 Draw
the morula. Be sure to label it as a solid sphere of cells.
2. After the sixth or seventh division the cells undergo their first morphogenic migration,
meaning that cells move quite a distance from one area of the embryo to another. The cells in
the center begin to move to the outside, leaving a hollow center. The hollow ball of cells is
known as a blastula.
Question 17. Draw a blastula (only seen in Figure 5). Be sure to label it as a hollow, fluidfilled
sphere.
Question 18. Compare the size of a blastula to that of an unfertilized egg. Has the embryo
actually grown? Estimate the number of cells the blastula contains:
Question 19. Describe how you tell if an embryo is a simple mass of cells (“morula”) or
actually a blastula (fluid-filled sphere of cells).
G. Gastrula
In the late blastula phase, the cells undergo their second morphogenic migration. At one point
on the blastula surface the cells migrate inward toward the other side of the sphere and results in
the formation of an embryo composed of two primary cell layers. The outer cell layer is the
ectoderm and the inner one the endoderm. Within a short time the mesoderm will begin to
form between these layers and by midgastrulation these three germ layers will have developed.
All subsequent tissues and organs will develop from these three primary germ layers.
Procedure:
Refer the Sea Biscuit video (1:26 minutes) and Figure 5 Cleavage and also observe gastrulas
development.
Question 20. Draw a gastrula. Label the ectoderm and endoderm (using arrows to point to
parts).
Question 20. A gastrula will develop into free-swimming “pluteus” larvae (1:34 minutes) in a
day or two. In contrast a sea star forms a bipinnaria larva. What does gastro- (as in
gastrointestinal tract) mean?
Questions 22. At what point in development would you logically expect the larvae to be capable
of growth (growing in size)? Explain your answer.
When the sand dollar embryos reach the larval stage, they have a complete digestive system and
are capable of feeding. They consume phytoplankton and become significant members of the
zooplankton trophic level. The free-swimming larvae will, in time, metamorphose into tiny sand
dollars and settle to the bottom of their marine environment. Those which settle in an appropriate
sandy area will become the new generation and they will compete with their parents for the
available habitat.
Figure 4. Early Stages in the Developing Sand Dollar
Fertilization of fertilization membrane
2 – 5 min.
Figure 5. Later Stages in the Developing Sand Dollar
I. Review of Sand Dollar Development
Answer the following questions with reference to the letter designations in the chart below.
Processes Stages
a. fertilization f. larva
b. morphogenic migration g. gametes
c. hatching h. blastula
d. cleavage i. zygote
e. metamorphosis j. gastrula
Question 23. Which of the following represents the sequence of occurrence for the
developmental processes?
1. a, b, c, d, e
2. a, d, b, c, e
3. b, e, d, c, a
4. e, d, b, a, c
5. a, d, e, c, b
Question 24 . Which of the following represents the sequence of occurrence for the
developmental stages?
1. f, g, h, i, j
2. j, h, i, f, g
3. g, h, i, j, f
4. g, i, h, j, f
5. i, g, f, j, h