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ALBG10

TITLE : 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

LECTURERS 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: y y y y 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

Meristem cells 1

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

Stages of cell divisions

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.

Onion root tips

http://micro.magnet.fsu.edu/micro/gallery/mitosis/mitosis.html

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 bluegreen. 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.

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

APPARATUS y y y y y y y y y 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 y y y y y y Garlic roots 1M Hydrochloric acid Toluidine blue stain Carnoy fixative Soft tissue paper Holding solution (70% ethanol)

PROCEDURE 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) (ii) The total number of cells in the microscopic field 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: Mitotic index =
            

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

Stages in cell cycle Interphase Prophase Metaphase Anaphase Telophase Cytokinesis Total

Number of cells 31 3 2 2 1 1 40

Percentage of cells 77.5 % 7.5% 5.0% 5.0% 2.5% 2.5% 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 = = 9/40 = 0.225
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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.

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.

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 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 G phase (first gap), the S phase (synthesis), and the G 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.

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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 mm 1 unit = 1 mm 100 = 0.01 mm = 10 m By using the scale on the stage micrometer and eyepiece of x400 magnification, the size of the cell have been measured
40 x magnification (40x 10) 100 division on graticule = 25 division on slide micrometer 1 division on graticule = 25 100 = 0.25 division = 0.25 x 10 m = 2.5 m

Stage phase Interphase Prophase Metaphase Anaphase Telophase Cytokinesis

Size of the cell (m) 8 2.5 = 20.0 7 2.5 = 17.5 7 2.5 = 17.5 10 2.5 = 25 6 2.5 = 15.0 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

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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
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.

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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 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.

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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.

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
y y Edexcel AS Biology , Ann Fullick, Pearson Company, 2008 International Edition BIOLOGY seventh edition, Pearson Education, Neil A. Campbell and Jane B. Reece, 2005 y Mitosis : http://micro.magnet.fsu.edu/micro/gallery/mitosis/mitosis.html Retrieved on 7/10/11 y Toluidine blue solution : http://www.bio.net/bionet/mm/plant-ed/1997October/002467.html Retrieved on 7/10/11

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Toluidine blue solution : http://education.vetmed.vt.edu/Curriculum/VM8054/Labs/Lab2/Examples/extolblu.ht m Retrieved on 7/10/11

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

y y

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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