ALBG10TITLE :

Observing Mitosis

NAME : NUR SHUHADA BINTI MOHD FIRDAUS WONG

I/C NUMBER : 930409125468

GROUP : ALM12M16

STUDENT ID NUMBER: 2011889202

DATE OF PRACTICAL : 4 SEPTEMBER 2011

LECTURER’S NAME : SIR WILLIAM NGU

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Title

Observing Mitosis

Objective

This experiment investigates the process of mitosis by looking at the slides of the plant tissue

that has cells undergoing cell division. The objectives of the experiment are:

To prepare some slides of actively dividing plant tissue.

To observe the stages of mitosis in plant cells.

To use a microscope to observe cells

To make comparisons between cells at different stages of division.

Introduction

During the process of cell division the chromosomes are duplicated.Then they,along

with the remaining contents of the cell,are divided up in such a way that two identical daughter

cells are formed.The events of mitosis are continuous,but as in the case so many biological

processes it is easier to describe what is happening by breaking events down into phases,which

are interphase,prophase,metaphase,anaphase,and telophase.1

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Meristem cells

Mitosis in the higher plant cells takes place in regions called meristems (regions of the plant

that are actively growing) that are found in the tips of the roots and stems. In animals mitosis

can take part are over the organism, where ever new cells are required. However, some tissues

undergo division more than others for example the skin tissue: while cells in other tissues rarely

divide once they have reached maturity such as brain and heart tissue.1

To see a mitosis easily, observation must be done on a living cell. The best part to

observe mitosis is in the cells that are growing at rapid pace such as onion root cell tips. Onion

bulbs grow roots that have actively dividing cells in their tips. The root tips contain a special

growth region called the apical meristem where highest percentages of cells are undergoing

mitosis. Each cell has only 16 chromosomes so it is relatively easy to see the chromosomes once

they have condensed.

1 http://micro.magnet.fsu.edu/micro/gallery/mitosis/mitosis.html2

Stages of cell divisions

Onion root tips

Toluidine solution

Images showing cells in spleen,vessel wall and liver stained by toluidine solution

Toluidine blue is a polychromatic dye. Toluidine blue solution is used in testing for

lignin, a complex organic molecule that bonds to cellulose fibres and strengthens and hardens the

cell walls in plants. A positive toluidine blue test causes the solution to turn from blue to blue-

green. It absorbs different colours depending on the nature of its chemical binding with the

components of the tissue. The colour will depend upon the particular chemical structure of the

compound. At a lower acidic pH, toluidine blue gives only a blue or green colour. It is quite

useful to stain fixed tissue. TB provides only minimal information about the chemical makeup

of a tissue or organ. TB is also used clinically. It has a strong affinity for the granules in mast

cells, one of the wandering cells of connective tissue. TB is often requested as a specific stain for

mast cell tumours.3 In this experiment, by using the toluidine blue solution function as to make

the chromosomes being shown up so as easier to be observed.

33http://www.bio.net/bionet/mm/plant-ed/1997-October/002467.html

APPARATUS

Watch glass

Small sample tube

Small measuring cylinder

Microscope slides and coverslips

Pair of fine forceps

Mounted needle

Microscope with magnifications of x100 and x400

Eye protection

Stopwatch

MATERIALS

Garlic roots

1M Hydrochloric acid

Toluidine blue stain

Carnoy fixative

Soft tissue paper

Holding solution (70% ethanol)

PROCEDURE

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1. Two watch glasses labelled with “HCl” and “carnoy” were prepared.

2. 1 M hydrochloric acid and carnoy fixative were poured into their respective watch glass enough to cover the bottom of the watch glasses.

3. The root tips were put into a small beaker containing 2 cm3 of 1 M hydrochloric acid for

exactly 4 minutes.

4. The root tips were transferred to a watch glass containing carnoy fixative using forceps.

The root tips were left for 4 minutes, and then the root tips were dried on filter paper. The

root tips were handled gently as they will be very fragile.

5. One of the root tips were transferred to a clean glass slide. About 1-2 mm from the

growing root tip was cut. The rounded tip was kept and the rest was discarded.

6. One small drop of toluidine blue was added and it was leaved to stain for 2 minutes.

7. After 2 minutes, the excess stain surrounding the root tip was soaked up gently using the

paper towel.

8. 1 to 2 drops of water was dropped on the root tip when the stain has been blotted away.

9. The slide with the root tip was covered with a cover slip, and then it was blotted with sof

tissue papers. It was then pressed gently to spread the root tip.

10. The slide of the root tip was viewed under the microscope (x400 magnification).

Chromosomes of the cells were identified. The cells in various stage of cell division were

detected. Once the located cells in division, the power of microscope was changed to high

power.

11. The regularly shaped and actively dividing cells were viewed. DNA stains dark blue with

toluidine blue stain enable the blue groups of chromosomes against a paler background.

12. The views of the cell from the microscope were drawn in the boxes. Each cell was filled

the entire box, be as much detail as possible, and the cells were labelled with as many

structures that can be identified.

13. The following were counted;

(i) The total number of cells in the microscopic field

(ii) The number of cells containing visible chromosomes

14. Steps 9- 10 were repeated for various microscope slides.

15. The mitotic index of the slide was examined (mitotic index is the fraction or percentage

of cells containing condensed chromosomes). Mitotic index was calculated by the

following formula:

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Mitotic index = numberof cells containing visible chromosomes

total number of cells∈the f ield of view

16. By using a stage micrometer and eyepiece graticule, appropriate measurements were

made to allow to compare the size on interphase with those that are undergoing

cytoplasmic division. The eyepiece graticule had been first cablirated with stage

micrometer.

17. Tables were drawn to summarise the results.

Results

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Stages in cell cycle Number of cells Percentage of cells

Interphase 31 77.5 %

Prophase 3 7.5%

Metaphase 2 5.0%

Anaphase 2 5.0%

Telophase 1 2.5%

Cytokinesis 1 2.5%

Total 40 100

Table 1 : Number of cells that undergo different stages in mitosis and the percentages

The mitotic index of the slide was calculated by using the following formula:

Mitotic index = numberof cells containing visible chromosomes

totalnumber of cells∈the field of view

= 9/40

= 0.225

The illustration of the stages of mitosis

1) Interphase

The nucleus may have one or more dark stained nucleoli and is filled with a network of threads, the chromatin. During the S Phase of interphase DNA replication occurs.

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2) Prophase

The first sign of division occurs in prophase. Here the replicated chromosomes joined by a centromere begin to coil and thicken. In late prophase the nuclear membrane and nucleoli disappears. The spindle apparatus appears in the cytoplasm.

3) Metaphase

At metaphase, the chromosomes have moved to the center of the cell, moved by the spindle fibres. The centromeres are attached to the spindle fibres

4) Anaphase

At the beginning of anaphase, the chromosomes are separated and moved, by the spindle fibres to the poles of the cell.

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5) Telophase

During telophase the nuclear membrane reforms the spindle fibres disappear. Cytokinesis occurs dividing the cell in two daughter cells.

6) Cytokinesis

A ring of contractile fibre tightens around the centre of cell.Continue to attract until the two cells have been separated.

Discussion

The aim of this experiment is to prepare some slides of

actively dividing plant tissue. in this experiment, the

garlic root tips are used since they are easy to get and

easy to be handle and observing the cells. Thus, the

stages of the cell cycle in the living tissue of that garlic

root tips were observed by a light microscope. In order to

see the chromosomes inside the cells, the cells must be

separated and spread out into a layer that is ideally just

one cell thick. Plant cells are glued together by a middle

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lamella of pectins which then had been broken down with hydrochloric acid. The mitosis stages

had been determined by observing the cell one by one and recognize the stages had occurred.

There are four stages of mitosis of the root tips of the garlic, which are prophase, metaphase,

anaphase and telophase. From Table 1 , prophase stage gives the highest number of cells in the

field of view which is 3 cells thus give 7.5% of the total cells. It is followed by metaphase and

anaphase which gives 2 counted cells each and thus record 5.0% each of the total cells.

Then,telophase and cytokinesis which both give 1 cell thus each record 2.5% of the total cells

respectively. A group of cells that is not dividing is in interphase,which makes up 77.5% of the

cells. The result shows that the mitotic index of the garlic root tips is 0.500. The mitotic index is

used for studying tumour growth in cancer patients

In this experiment of mitosis, prophase record the longest duration in the mitosis as its show

the highest number of cells compared to the other stages of mitosis.The longest time in the cell

cycle is interphase which often accounts for about 50% of the cell cycle. Cells spend the longest

time in interphase during the cell cycle compared to the mitotic (M) phase. It is during the

interphase that the cells grows and copies its chromosomes in preparation for the cell division.

Interphase can be further divided into three subphases: the G₁ phase (“first gap”), the S phase

(“synthesis”), and the G₂ phase (“second gap”). During all three subphases, the cells growth by

producing proteins and cytoplasmic organelles such as mitochondria and endoplasmic reticulum.

Hence, it is clear that interphase spend the longest time in the cell cycle as the cells increase in

mass and size, replicate their DNA and carry out normal activities.

Length of cell during cytokinesis is longer than length of cell during interphase. This is

because during cytokinesis, the parent cell is dividing into two daughter cells whereas during

interphase, the cell only grows bigger to prepare for the cell division.It is shown by the

calculations below.

Calibrating eyepiece graticule:100 units = 1 mm1 unit = 1 mm

100 = 0.01 mm = 10 µmBy using the scale on the stage micrometer and eyepiece of x400 magnification, the size of the cell have been measured

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40 x magnification (40x 10)100 division on graticule = 25 division on slide micrometer1 division on graticule = 25

100 = 0.25 division = 0.25 x 10 µm = 2.5 µm

Stage phase Size of the cell (µm)

Interphase 8 × 2.5 = 20.0

Prophase 7 × 2.5 = 17.5

Metaphase 7 × 2.5 = 17.5

Anaphase 10 × 2.5 = 25

Telophase 6 × 2.5 = 15.0

Cytokinesis 16 × 2.5 = 40.0

Toludine blue is used rather than the orcein ethanoic stain because the procedure is much

simpler than the other one. Besides, orcean ethanoic stain is corrosive, irritant, causes burns and

has an irritating vapour.

There are only few dividing cells because the surrounding environment is not favourable for

the cell division. They have already been treated by hydrochloric acid and toluidine blue, there is

only a little nutrients present in the surrounding environment. Cells will only undergo division

when they are in optimum environment. In addition, we might have cut the wrong region of the

onion root. Some of the region of elongation has been cut. There is only a few apical meristem

cells present in that particular region. Hence, we see only a few dividing cells in the root tip.

The cellulose walls of plant cells are held together by compound in the middle lamella.

Treatment with hydrochloric acid only breaks up the middle lamella. This is very useful in the

preparation because hydrochloric acid will break down the pectins that hold the cell together.

Hydrochloric acid will dissolve the pectins but leave the cellulose cell walls unaffected. The cells

separate so that a preparation that is only one cell thick can be produced, making viewing of

individual cells possible.

Validity

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To get more accurate readings, the root tips must be macerated well to break down the cell

so it will be only one cell thick. Alternatively, another microscope slide can be placed at right

angles to the original slide to form a cross, and squash the tip between the two slides. This

method will provide two samples for staining and allow the root tip to be only one cell- thick.

Lateral movement of the cover slip must be prevented to avoid overlapping. Besides that,

validity can be ensured by carefully using an eyepiece graticule which has been calibrated

correctly using a stage micrometer. For more accurate reading somehow, graticule scale with

smallest divisions can be used.

Reliability

The experiment is unreliable as we only study the mitotic process of apical meristem cells by

preparing 1 slide. We should prepare more similar slides. Then, we compare the results so that

we can get a more reliable result.

This experimental result is valid. The procedure is designed well to study the mitotic process of

apical meristem cells. Besides, the length of cells during interphase and cytokinesis is measured

using appropriate devices, which are stage micrometer and eyepiece. The method used is easy to

be carried out. We stain the chromosomes with toluidine blue and then we observe the result

under a microscope.

Limitations,Sources of Errors and Ways to overcome

The existence of some unavoidable limitations can cause the results to be less accurate than it

should be. Firstly it only had about two hours in which to conduct the experiment. Students may

not be able to get proper results. So, accurate way or method plus exact skill may help to reduce

the wasting time for repeating the experiment if the cell had been damaged and cannot be

observed. As a result, the time would be used wisely.

Toluidine blue also will affect the panoramic view of the cell. Too much toluidine blue for

staining the root tip will cause the cell become too dark and cannot be seen when it is viewed

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under microscope. Thus, excess stain must be removed by using filter papers and layers of tissue

or by blotting process. If the cells are over- or under stained, the quantity of the stain added. In

other way, the root tip can be immersed in some distilled water after being stained to prevent the

slide of the cell from being too dark.

Apart from that, when lowering the cover slip on the slide, it must be done at angle of 45°. This

is to ensure there is no air bubble that will produce the artefact in the observation.

Safety Precautions

Laboratory coats are worn during the investigation to avoid the toluidine blue stain spill on

skin or clothing as it will stain very badly. Besides that, the glassware is handled with extra care

such as glass slide since the apparatus can break easily and might cause injury.

The garlic root tip is very fragile and easily to damage which then affect the structure of the

cell. Thus, the garlic root tip should be handled very carefully. The coverslip should be tapped

gently to avoid the lateral movement which can cause the cells is overlapped.If the microscope

slide is blotted directly by using the thumb, it might leave the finger print on the coverslip and

make the observation of the slide under the microscope not clear and alter the results.

Be careful when handling the glassware such as beakers, glass slides and watch glasses since

the apparatus are fragile. The scalpel is extremely sharp. Make sure your fingers are clear when

you make the cut of the root tip. This will prevent any injuries and unwanted incident

Make sure direct sunlight is not reflected through the optics when using the microscope with

separate illumination as it may damage the eyes.

Further investigation

Instead of using toluidine blue solution, this experiment also can be done by using the orcein

ethanoic to stain the chromosomes of the cells. The chromosomes will appear pink rather than

blue. Hence, we can investigate which solution will give a clearer image of the cells under the

microscope.

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Conclusion

The number of cells in each of the stage of mitosis may vary. Mitosis of the root tip of the garlic

consists of 4 stages which are prophase, metaphase, anaphase and telophase. Prophase gives the

highest number of cells and thus occupies the longest time in mitosis.

References

Edexcel AS Biology , Ann Fullick, Pearson Company, 2008

International Edition BIOLOGY seventh edition, Pearson Education, Neil A. Campbell

and Jane B. Reece, 2005

Mitosis : http://micro.magnet.fsu.edu/micro/gallery/mitosis/mitosis.html Retrieved on

7/10/11

Toluidine blue solution :

http://www.bio.net/bionet/mm/plant-ed/1997-October/002467.html Retrieved on

7/10/11

Toluidine blue solution :

http://education.vetmed.vt.edu/Curriculum/VM8054/Labs/Lab2/Examples/

extolblu.htm Retrieved on 7/10/11

Image of mitosis stages :

http://micro.magnet.fsu.edu/micro/gallery/mitosis/mitosis.html Retrieved on 7/10/11

Interphase stage : http://www.agedlibrary.com/070020.pdf Retrieved on 7/10/11

Onion root tips :

http://www.biology.arizona.edu/Cell_BIO/activities/cell_cycle/cell_cycle.html

Retrieved on 8/10/11

Mitosis lab : www.nclark.net/MitosisLab.pdf Retrieved on 8/10/11

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