International Education College

Title: Observing Mitosis

Name: Siti Juliana binti Jahidin

Lab partner: Nur Shafira Mohd. Noh
Noor Dayana Azmi
I/C No: 920514-01-5034
SID No: 2010671292
Group: 11M16
Lecturer’s Name: Madam Khaniza Hasliza Abdul Khalil

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Title
Observing mitosis

Objective
To prepare some slides of actively dividing plant tissue and to observe the stages of the cell
cycle in living tissue.

Introduction
The cell cycle

Cell cycle shows the sequence of events in cell division which involves several
different phases. The time taken for a cell cycle to complete can be varied either it will take a
short or a long time. It is controlled by a number of chemical signals made in response to
different genes. 1The major event in cell cycle is interphase and mitosis. Interphase
contributes the longest time in a cell cycle in which the cells prepare themselves for the cell
division. In this phase itself, interphase is divided into 3 stages which are G1 stage, S stage
and G2 stage. During this period of non-division, the cells undergo DNA replication and
continue their normal metabolic reaction. The cells are increase in mass and size along with
the increase in production of ATP. The end of interphase indicates the beginning of mitosis
which can only be proceeding after all that is needed is present and the parent cell is large
enough.

The next event following after interphase in a cell cycle is mitosis. This process is
further divided into a sequence of 4 significance phases which are prophase, metaphase,
anaphase and telophase. During prophase, each chromosome which represents by 2
daughter chromatids becomes shorter, denser and visible under the light microscope. The
nucleolus disappears and the centrioles begin to move to the opposite pole to form the
spindle fibre. Next, is metaphase in which the nuclear membrane has totally broken down
and the spindle fibre is fully formed. The chromosomes aligned themselves on the
metaphase plate, with each centromere attached to the spindle fibre. Then, anaphase comes

1 Edexcel AS Biology , Ann Fullick, Pearson Company, 2008

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in view. During this period, each pair of chromatid in a chromosome is separated from each
other at the centromere and is pulled towards the opposite poles at the centromere. The last
stage in mitosis is telophase. All chromatids already reach each pole and the membrane is
reforms. The chromosomes unwind themselves and the nucleoli are fully reformed. The
cytoplasm starts to divide leading the cell to the next stage, cytokinesis.

Cytokinesis is the continuation of the late telophase of the mitosis. At this stage, the
cytoplasm of the cell keeps on dividing until two new identical cells are formed. In animal
cells, the cytoplasm separates by the contraction of the ring of the contractile fibres around
the centre of the cell whereas in plant, the cell division occurs by the building up of the
cellulose cell wall. After that, the cell cycle is said to be complete and each of the new cell
enters interphase again. In multicellular organism, this cell cycle is repeated again and again
for the development of the cell and becomes slower or completely stop after the cell
becomes mature.

Cell cycle

Picture from: http://www.google.com.my

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The Toluidine blue

Toluidine blue is a dye. It is used to stain specimens for microbiologists and

other scientists. It is also used to stain living tissue for several reasons. One reason is to
screen for cancer, since precancerous cells and cancerous cells take up more dye that
normal cells. In the context of a sexual assault forensic examination, Toluidine blue is used
to locate and document injuries. Because the dye is selectively taken up by injured tissue
.Micro abrasions and lacerations can be visualized after the genital and perianal area are
stained with Toluidine blue and then destained. Any remaining blue after destaining is
indicative of cellular damage and may be due to injury from a sexual assault.2

Toluidine blue

Picture from: http://www.google.com.my

The carnoy fixative

Carnoy’s fixative is a chloroform-containing fixative. It penetrates tissues extremely
rapidly and can fix small tissue pieces in minutes rather than the hours required for other
fixatives (Chamberlain, 1932; Sass, 1958). Delicate tissues can be damaged when
transferred from aqueous solutions to this fixative, due to the extreme hydrophobicity of
chloroform and resultant rapid tissue dehydration. This fixative has traditionally been used for
cytological structures.3

2 http://everything2.com/title/Toluidine+blue

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 Carnoy fixative

Picture from: http://www.google.com.my

Cutting Back

If you're taking in too much caffeine, you may want to cut back. The best way is to cut back
slowly. Otherwise you could get headaches and feel tired, irritable, or just plain lousy.

Try cutting your intake by replacing caffeinated sodas and coffee with noncaffeinated drinks.
Examples include water, caffeine-free sodas, and caffeine-free teas. Keep track of how many
caffeinated drinks you have each day, and substitute one drink per week with a caffeine-free
alternative until you've gotten below the 100-milligram mark.

As you cut back on the amount of caffeine you consume, you may find yourself feeling tired. Your
best bet is to hit the sack, not the sodas: It's just your body's way of telling you it needs more
rest. Your energy levels will return to normal in a few days.

Reviewed by: Mary L. Gavin, MD

Date reviewed: January 2008
Originally reviewed by: Jessica Donze Black, RD, CDE, MPH

BackWhy the experiment is conducted?

3 http://microscopy.berkeley.edu/Resources/instruction/acid_fixatives.htm

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 This experiment is conducted to develop further understanding of the cell cycle that
we had learnt in class. Instead of learning this matter theoretically through various sources,
this subject is put into practical so that we can fully master the cell cycle by observing the
cells of the garlic root tip. During this experiment, we will be able to observe and identify
which stage is most of the cells are going through. We also can differentiate all phases
involve in mitosis and learn the characteristics of each phase by referring direct to the living
tissue. In order for this experiment to be a success, we need to apply the knowledge on the
cell cycle to determine which stage of the cell cycle the cell is going through, thus providing
us with better view and understanding.

Problem Statement

1. What is the most prominent stage in cell cycle of a cell?

2. What are the stages involve in mitosis?

Hypothesis
1. Interphase stage contributes to the largest number of cells compared to other stages.
2. Mitosis is a continuous process which involves four stages: prophase, metaphase,
anaphase and telophase.

Variables
• Manipulated variable: -

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 • Responding variable: -

• Fixed variables : Type and region of root tip used, volume and concentration of
hydrochloric acid and carnoy’s fixative

Apparatus

Scalpel, watch glasses, small sample tubes, small measuring cylinders, microscope slides,
cover slips, pair of fine forceps, mounted needle, microscope with magnification of x100 and
x 400.

Materials
Garlic roots, 1M hydrochloric acid, toluidine blue stain, carnoy fixative, cold distilled water,
and filter paper.

Procedure

1) 1-2 cm from several root tips of some growing garlic roots were cut off. White tips
with firm rounded end were chosen instead of brown tips which give poor results.
2) Next, the root tips were put into a small sample tube containing 2 cm³ of 1M
hydrochloric acid for exactly 4 minutes.
3) The root tips were transferred to another small sample tube containing 2 cm³ of
carnoy’s fixative. They were left there for another 4 minutes.
4) Then, the root tips were transferred to a watch glass and allowed to soak with
approximately 5 cm³ cold water for 4 minutes. After that, they were dried on filter
paper. The root tips were handled gently as they were already fragile.
5) One of the root tips was transferred to a clean microscope slide. The growing tip
was cut about 4-5 mm leaving the rounded tip while the rest was discarded.

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 6) The root tip was broke up gently with a mounted needle. This step is called as
maceration. One small drop of toluidine blue was added and was left to stain for 2
minutes.
7) The stained root tip was covered with a cover slip and it was blotted firmly with
several layers of filter paper. The cover slip was pressed gently to spread the root
tip.
8) After that, the root tip was viewed under microscope starting from the lowest
magnification and the cells with chromosomes were observed. The slide was
squashed again between two filter papers if the cells were overlapping.
9) Actively dividing cells that regularly shaped were searched under the microscope.
The number of cells that undergo cell division were detected and counted to find
the mitotic index. The phases of the dividing cells were recognized and the
observations made were recorded in a table.
10) The microscope slide was prepared again using another root tips from step 3 and
the following steps were repeated if the preparation was not very successful.

Results

Stage of cell cycle Number of cells Percentage of Size of the cell

visible cells (%)
(µm)
Interphase 134 87.58 25.0

Prophase 3 1.96 25.0

Metaphase 6 5.23 25.0

Anaphase 5 3.27 32.5

Telophase 5 3.27 35.0

Table 1: Percentage of cells at different stages

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Calculation of the mitotic index

Mitotic index = number of cells containing visible chromosomes total number of

cells in the field of view

Mitotic index = 19 153

= 0.124

Graph 1: The percentage of cell at each phase

Graph 2: The size of cell at each phase

Illustration based on the observation of the cell of each stage in the cell cycle

1) Interphase

Nucleus appears to be in dark stain.

Chromosomes are not visible
because they are not yet condensed
at this stage

2) Prophase

Chromosomes are condensed and

visible under the microscope. They
appear to be spread out around the
cytoplasm

3) Metaphase

The chromosomes lined up on the

metaphase plate, the equator of the
cell
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 4) Anaphase

The centomeres separate. The

spindle fibre contracts and shortens,
pulling each chromatid towards the
poles with the centromere heading
the way.

5) Telophase

The chromatids reach the poles of the

cell and the spindle fibre disappears

Discussion

The purpose of this experiment is to prepare some slides of actively dividing plant
tissue and to observe the stages of the cell cycle in living tissue. Thus, garlic satisfies the
requirement for this experiment as it is easy to be obtained, can be handled easily and is not

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really hard to be observed under microscope. The apical meristem of the root tip was
selected during the preparation of slide as that part has actively dividing cells which helps us
to observe mitosis and other stages in cell cycle clearly.

In this experiment, there was no manipulating variable and responding variable

because the data was collected after the garlic root tip cells were observed. There is no need
to control those two variables as the main objective was to observe the stages of cell cycle in
living tissue. However, certain factors were kept constant throughout this experiment to make
the results obtained more reliable. The same type of plant was used which is garlic and the
same part was taken to prepare more than one slide. Same volume and concentration of
hydrochloric acid and carnoy’s fixative were used to immerse the garlic root tip with the same
period of time. The purpose of using hydrochloric acid in this experiment is to break down the
middle lamella of pectin. This middle lamella of pectin that holds the garlic cells together
needed to be broken down so that the cells can be spread out to one layer of cells only. The
hydrochloric acid also breaks the DNA of the chromosome to deoxyribose aldehyde which
reacts with the toluidine blue and gives the chromosome the blue colour. This will then help
us to study and observe the cell better. Meanwhile, the root tip was immersed in the carnoy’s
fixative as this fixative is good for preserving chromatin, nucleoli, and spindles. The
cytoplasm is preserved as a stringy, coagulated mass, but some organelles are dissolved
such as the mitochondria. Again, the root tip cells were needed to be spread out to one layer
by breaking up the root tip with a mounted needle. Then, the root tip was stain with toluidine
blue in order to stain the chromosomes. As the chromosomes were hardly observe, this blue
staining was a great helper to provide us with a better view.

Referring to Table 1, it shows the percentage of cell and size of cell at different
phases. For interphase, prophase, metaphase, anaphase and telophase, the percentage of
cell is 87.58%, 1.96%, 5.23%, 3.27% and 3.27% whereas the size of the cell is 25 µm, 25
µm, 25 µm, 32.5 µm and 35 µm. From this data, we can make early assumption, that
different phases have different number of cells going through that particular phases and
different size of cell except for certain phases that have the same number of cells and have
the same size of cell.

The results obtained were further analyzed by plotting a graph so that the graph was
focused on the subject that needs to be discussed. Thus graph 1 was produced consisting of
percentage of cells according to their phases. From that graph, we can see that interphase
stage contributes to the largest percentage of cells while prophase has the lowest
percentage of cells. This data was obtained due to the fact that interphase takes the longest

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time compared to other stages in cell cycle of a cell. In order for the cell to undergo a mitotic
division, a lot of preparations are needed so that the final two daughter cells from each
parent cell are genetically identical to each other. Thus, this stage needs the most time of the
cell cycle for fully preparation and chances for the cell to undergo it own metabolic reactions
which is important for the whole function of a living system. That is why the interphase stage
is the most prominent stage in a cell cycle. The graph also shows that during M phase,
metaphase takes the largest number of cells which indicates longest duration of phase
compared to other phases in the mitotic phase. This can be explained with the fact that the
chromosomes take time to reach the equator of the cell and to arrange themselves on the
metaphase plate.

Another graph was plotted with the same purpose but this time the stages in cell
cycle were compared with the size of the cell. Interphase, prophase and metaphase have the
same size of cell and the size is slightly increased during anaphase and telophase. During
interphase, the cell already increased in size with the replication of DNA and the synthesis of
new organelles for the formation of two daughter cells. That is why for the prophase and
metaphase there is no significant increase in size. However, the size of the cell starts to
obviously increase during anaphase and telophase. For this case, the chromatids are
separated during anaphase and start moving to the opposite poles during anaphase while
during telophase which occurs continuously with cytokinesis, the separated chromatids
already reached the poles and the cytoplasm start to divide itself. Thus, major increase can
be seen in this two phases in order for the parent cell to form two identical daughter cells.

Evaluation

The validity and the reliability of the results obtained in an experiment are important to
determine whether the experiment conducted is successful or not. For this experiment that
requires us to observe the mitosis, the results obtained are valid if all the stages in a cell
cycle can be observed. The results obtained are also valid if the percentage of cells for every
stage in cell cycle were correspond or almost similar to the theoretical value. The size of cells
measured for all stages must be logically matched the characteristics of every stage in cell
cycle. In order to gain this validity, the root tip used must be macerated well using the

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mounted needle. If cells are overlapping, the slide must be squashed again between two
wads of filter paper. Any lateral movements of the coverslip must be avoided. A stage
micrometer and an eyepiece graticule are used to measure the size of cells.

Meanwhile, the reliability of these results can be ensured when the same results are
obtained by other individuals that conduct this experiment with the respect to certain
variables that involved. Repeating any readings or measurements helps in making the results
more reliable. All variables that are counted for this experiment must be well controlled.
Therefore, it is advised to prepare more than one slide using the same part of root. The
number of cells must be counted for several times and the size of the cells must be
measured for at least 3 times that end up with the average of the 3 readings taken. These
steps confide us that the results obtained are not gained through chance.

However, in this experiment, there still several limitations and errors occur that make
the results less valid and less reliable. The root tip of the garlic is very fragile and easily
damage especially after it was immersed in the carnoy’s fixative. The root tip cells also had
damaged easily during the preparation of slide causing difficulties in observing the cells as
the nucleus was hardly visible or not visible at all. This problem was overcome by preparing
more than one slide and the root tip was carefully handled during the preparation of slide.
Moreover, the presence of air bubbles on the slide unable us to see clear image. This led us
to wrong observations and inaccurate readings. However, this limitation was prevented by
slowly lowering the cover slip while covering the root tips using a needle. Furthermore,
problem also had occurred while observing the cells when the root tip was over stained by
the toluidine blue. Some chromosomes might were unclear and did not take into account
during the process of counting. Thus, it was solved by using only a small drop of toluidine
blue and the excess toluidine blue was removed using a filter paper. It was really hard to
count the cells straight away from the microscope. Thus, there was high possibility for us to
miscount the cells. In order to get the accurate number and accurate measurement, the
image of the cells were first captured using a camera. This had helped us to count the cells
accurately and the process of counting can be repeated for several times.

.Safety Precautions

There are some safety precautions that need to be followed when conducting the

experiment. Before entering the lab, we must make sure that we wear closed shoes. It must

be worn all the time in the lab to protect our feet from any sharp or pointed apparatus or any

harmful chemicals. Lab coats need to be wore all the time you conducting the experiment

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especially to prevent the toluidine blue that can badly stain our clothes and skin. Use the

scalpel carefully when cutting the root tip of the garlic. Hydrochloric acid is corrosive and can

cause irritation when in contact with our skin. Therefore, extra care must be taken when

handling this solution. The process of transferring the root tip from the hydrochloric acid must

be done using forceps and not using our own hands. Carnoy fixative is flammable, can cause

severe burns, irritating to skin and dangerous to health by prolonged exposure through

inhalation and if swallowed. Any contact of this chemical with the skin should be avoided. If it

does occur, skin should be rinsed with plenty of water. The bottle containing carnoy’s fixative

should be covered tightly after used. The fragile apparatus such as microscope slide and

cover slips should be handled with care. If any of the apparatus broken, report immediately to

the people in charge for the lab.

Conclusion

The interphase stage has the highest number of cells compared to other stages which
indicates the stage as the most prominent stage in cell cycle. Mitosis is a continuous process
which consists of 4 phases, mainly prophase, metaphase, anaphase and telophase.

Therefore, the hypothesis is accepted.

Further work

Further investigation can be done to help us with better understanding about mitosis and it
relation with certain factors also to compare the mitotic index. The rate of mitosis is different
for different age of root tip taken. Roots of different ages are taken and the same method is
used in preparing the slide. This experiment will enable us to determine at what age the
mitosis occurs rapidly and at what age the mitosis occurs at slowest rate or completely stop
going through mitosis. The number of cells needs to be counted to calculate the mitotic
index.

References

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i. Edexcel AS Biology , Ann Fullick, Pearson Company, 2008

ii. http://www.associatedcontent.com/article/2924780/observe_mitosis_in_plants_using_

onions.html

iii. http://www.google.com.my

iv. http://microscopy.berkeley.edu/Resources/instruction/acid_fixatives.htm

v. http://www-saps.plantsci.cam.ac.uk/worksheets/scotland/mitosis.htm

vi. http://everything2.com/title/Toluidine+blue

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