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TITLE: OBSERVING MITOSIS NAME: NUR AMALINA BT ZOLKEFLEE SID NUMBER: 2010843164 CLASS: 11M2 DATE OF EXPERIMENT: 24TH JANUARY 2011 LECTURER·S NAME: MISS FATHIAH ABDULLAH PARTNER·S NAME: FATIN NABILAH BT MOHD NASIR NUR AIN SOFIA BINTI HARITH SITI NASHUHA BINTI OSMAN

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All cells must replicate their DNA in order to pass it to the next generation of cells in the whole body. After DNA replication.OBJECTIVES Basically. we can develop certain experimental skills. The other purpose of this experiment is to observe the stages of the cell cycle in living tissue and to consider the duration of the stages of mitosis in relation to the whole cell cycle. Through this experiment also. new complementary DNA is produced thus yielding two identical DNA molecules. During mitosis. Not just that. we are also taught to calibrate an eyepiece graticule and use it to measure the size of the cells. For example. and producing valid results and recording results. metaphase. the process followed is mitosis which is important to ensure that each daughter cell receives one copy of each replicated chromosome. our experiment is done to prepare some slides of actively dividing plant tissues. INTRODUCTION y BACKGROUND INFORMATION Genetic information of human. The division is considered as complete once the cell undergo cytokinesis. the use of microscopes. During DNA replication. each human cell posses 46 chromosomes while onion cell posses 8 chromosomes. two strands of DNA separate and for each strand. anaphase and telophase. namely working safely. the chromosomes pass through several stages like prophase. plants and animals reside in unique structure called chromosomes. 2| ¢¡   e .

mitosis and cytokinesis. The centrioles move around nuclear envelope and locate themselves at opposite sides of the cell.cherokee.sister chromatids are joined at one region called centromere. The two strands of non.Figure 1 shows the cell cycle that includes interphase. The microtubules from cytoplasm form three dimensional structure called spindle fibre which are formed between two poles. Source: http://mysite.pdf The first stage in mitosis is prophase. chromatin condense to form highly condensed chromosomes. Nuclear envelope and nucleolus break down. 3| ¥¤ £ e . Chromosomes are visible at high magnification through light microscope.us/personal/gregg_schumaker/site/Important%20Class%20 Documents/1/Growth%20and%20Heredity/Observing%20Mitosis%20Lab.ga. During prophase.k12.

Figure 3 shows how the chromosomes arrange themselves at metaphase plate during metaphase.Figure 2 shows some events that take place during prophase. the centromeres of chromosomes line along the metaphase plate or equatorial plane.rhtml 4| ¨§ ¦ e . the cell has reached the end of metaphase. When this arrangement has been completed.com/2009/04/30/phases-of-mitoses/prophaselate__civyrosejpg/ During metaphase.sparknotes. Source : http://www.com/biology/cellreproduction/mitosis/section2. Source: http://chuck16. an imaginary line that is equidistant from centrosome poles.wordpress.

so that two sets of genetic information become enclosed in separate nuclei. During this stage. the centromeres split. Figure 5 shows the events take place during telophase.rhtml 5|  © e . One chromatid of aech chromosome is pulled to each of the poles. Figure 4 shows the events occur during anaphase. Chromosomes unravel and nuclear envelope reforms.com/biology/cellreproduction/mitosis/section3.sparknotes. Source: http://www.The third stage in mitotic division is anaphase. Anaphase ends when the separated chromatids reach the poles and the spindle breaks down. The spindle shorten to pull the two halves of each centromere in opposite directions. The events taking place during telophase is a reverse process of prophase.sparknotes.rhtml The last stage in mitotic division is telophase.com/biology/cellreproduction/mitosis/section2. Source: http://www.

bms.htm Overall. Cytokinesis is also known as cytoplasmic division. Can you imagine to have different colours of skin? This probably happen if mitosis do not occur as our genetic information is not restored in producing new cells. This is because the daughter cells are made to be ganetically identical to each other and parent cell. Figure 6 shows the sequence taking place during cytokinesis.ed. Mitosis is also important to animals and plants that practice asexual reproduction. Furthermore. plant cells divide due to formation of walls between two daughter cells. mitosis and cytokinesis.uk/research/others/smaciver/Cytotopics/Plant%20Cytokinesis. Source: http://www. For example starfish can regrow a completely new body from a fragment of its body. mitosis is also significant in replacing our dead cells sepecially 6|   e . During cytokinesis.ac.Cell cycle consists of three main stages which are interphase. New cell wall material is brought by microtubule system forming the phragmoplast. Genetic consistency is achieved by DNA replication prior to nuclear division and during the arrangement of the chromosomes on the spindle fibre and the separation of chromatids to the poles. a complex organelle consisting microtubules and actin filaments. mitosis is very crucial in order to maintain genetic consistency.

Figure 7 shows the three regions at the tip of onion root Source: http://www. The chromosomes of onion cells can be stained to make them more easily observable and as the chromosoes are large and become very dark when stained. mitosis will occur to replace those old cells. Therefore. In this experiment. Secondly. the cells are actively dividing but not increasing in size while the third region is the region of cell elongation where the cells increase in size but not dividing. So. Firstly. in order to maintain in good condition.excellup.our skin cells which are easily rubbed by the friction. The first one is root cap. no ethical issues arise here as it is not an endangerd population and it will be much cheaper to be compared to other type of plants. cells at the tip of roots are actively dividing and thus many cells will be in stages of mitosis.com/InterBiology/morphologyplant. Here. we are using onion roots due to several specialities that it owns. the onion roots are easily grewn in large numbers. The tips can be prepared in a way that allows them to be flattened or microscopes slide(µsquashed¶) so that chromosomes of individual cells can be observed. It contains cells that cover and protect the underlying growth region as the root pushed through the soil. The second region is the region of cell division(meristem).aspx 7|   e . There are three cellular regions at the tip of onion root. therefore the onion root cells is the most suitable to be used in this experiment.

you have to put your microscope on flat surface. we have to make sure that we know how to use the microscope correctly because our result is only depending on microscope. After that. switch on the microscope¶s light source and then adjust the diaphragm to the largest hole diameter to allow the greatest amount of light passing through. Place the slide on the stage and adjust the large coarse focus knob until specimen is in focus. Refocus and view the specimen carefully. Use the lowest power objective (usually 4x for 40x magnification). Repeat this step by using 40x objective. Then. Rotate the objective lens to 10x objective for 100x magnification. What we see through the microscope indicate how successful our experiment is.hometrainingtools. adjust the diaphragm to get the best lighting. It is an optional for you to use the 100x objective for 1000x magnification. adjust the small fine focus knob until specimen is clearly in focus. Figure 9 shows a microscope Source : http://www.In order to get an accurate result. You need to put 1-2 drops of immersion oil over the slide coverslip before viewing it at highest power. Firstly. Then.com/how-to-use-a-microscope/a/1120/ 8|Page .

Toluidine blue is a polychromatic dye that absorbs different colours depending on the nature of its chemical binding with components of the tissues. Cytokinesis is the easiest stage to be detected. 9|Page . The most time spent in a stage is in prophase and the least time spent in a stage is anaphase. At lower acidic pH. All the stages can be differentiated by observing the chromosomes in nucleus that is the chromosomes¶ position within the nucleus.4. At higher basic pH (11. The telophase stage can be observed when you can see two sets of chromosomes in a cell. anaphase and telophase and cytokinesis clearly. it shows metaphase stage. it means that the cell is undergoing anaphase. toluidine blue gives only blue or only green colour. metaphase. Compounds containing benzene rings such as lignin will be coloured green. we can see the interphase. If only long strand can be seen. it indicates the prophase stage while if the chromosomes arrange themselves at the centre which is known as metaphase plate. By observing onion meristem cells under microscope.In this experiment also. If the chromosomes scattered in the nucleus. the four stages of mitosis that is prophase. if the spindle looked shorter and like pulling the chromosomes to the pole. we are using toluidine blue instead of carbol fuschin. it will bind to pectins in cell walls and colour them pink. it indicates the cell is undergoing interphase. At pH 4. On the other hand.1) the stained tissue will be coloured dark pink since most pectins will be charged and hinder the green colour of lignin. EXPERIMENTAL HYPOTHESIS Mitosis occurs in onion root tip and it is easily observable. that is when you can see cell plate formed in between the two sets of chromosomes.

A few drops of toluidine blue is dropped into the root tip and is left for two minutes. 9. the slide is observed under microscope. Forceps. 10. Sample of onion root cells is obtained from the holding solution. compound microscope. the stain surround the root tip is blotted away. filter paper. Then. a cover slip is put over the specimen. The slide is covered with a paper tissue and the cover slip is pressed with the thumb. APPARATUS : microscope slides. holding solution(70% ethanol). By using a blade. 5. 7. 1 mm of the root tip is cut and is put on microscope slide. watch glasses. METHOD Preparing the sample 1. toluidine blue. 2. By using a filter paper. 3. 6. Two cups of solutions are prepared. the root tip is transferred into carnoy solution for four minutes. Then. 4. 8.MATERIALS AND APPARATUS MATERIALS: carnoy fixative. an onion root tip is transferred into HCl acid solution for four minutes. Then. 10 | P a g e . HCl acid( 18%). small beakers. By using a forcep. a few drops of water is put onto the root tip to dilute the concentrated toluidine blue. Then. The first cup contains HCl acid while the second one contains carnoy solution. coverslips. blades.

Preparing the microscope 1. What we can see from the microscope is only overlapping cell walls. A stage micrometer is slide on the stage of microscope. The smallest division on the stage micrometer is measured. The eyepiece is rotated and the slide is moved. 11 | P a g e . The cell size can be measured. RESULTS AND DATA Figure 10 shows the drawing of overlapping cell walls. 3. 4. the microscope is focused on stage micrometer. By using low power objective. 2. Step 3 is repeated for medium and high power objectives. The number of divisions on eyepiece graticule is counted and is made equivalent to the smallest division on stage micrometer and hence the length that one eyepiece division is calculated.

we found that each stage in cell cycle can been observed clearly under microscope.+Byerly Stage of cell cycle Number of cells in each stage Interphase Prophase Metaphase Anaphase Telophase Cannot be observed Cannot be observed Cannot be observed Cannot be observed Cannot be observed Table 1 shows number of cells in each stage DISCUSSION Based on our result. we found that our result is not valid at all due to some errors that we encounter that we will discuss in detail in sources of error. Source: http://mrswolfgang.com/Animal+and+Plant+Cells++Bizousky.wikispaces.Figure 11 shows Nucleus is hardly seen due to compact cell wall. After discussing with other groups that manage to conduct their experiment successfully. 12 | P a g e . Here is some pictures showing how chromosomes behave in each stage of mitotic division and interphase.

html Through our discussion too.121. we found that our hypothesis made is accepted.practicalbiology.EXP. That is most of the time in mitotic division is spent in prophase while the least time spent is in anaphase.Figure 12 shows all the cells that can be observed under microscope using low power objective.org/areas/advanced/cells-to-systems/celldivision/investigating-mitosis-in-allium-root-tip-squash. 13 | P a g e . Source: http://www. Figure 13 shows the chromosomes behaviour using higher power objective.

each division on eyepiece graticule is equal to 33. Figure 14 shows the stage micrometer and eyepiece graticule scale.org/areas/advanced/cells-to-systems/celldivision/investigating-mitosis-in-allium-root-tip-squash.EXP. Number of division on eyepiece graticule is found to be three divisions equal to 1 smallest division of the stage micrometer. Therefore.33 micrometer. Source: http://www.html In calibrating the eyepiece graticule.practicalbiology.Table 2 shows number of cells in each stage. 14 | P a g e . we found that the smallest division on the stage micrometer equals to 100 micrometer. The eyepiece is rotated and the slide is moved to superimpose the scales of eyepiece graticule and stage micrometer.121.

VALIDITY AND RELIABILITY It is obvious that our experimental result is not valid. we assumed that the root tip has not been fully immersed in HCl and carnoy solution which later interrupt the image observed under microscope. the intensity of toluidine solution used is too low until the nucleus of the specimen cannot be observed under microscope. The lens of the microscope are not being wiped out first before we are using it. our result obtained can be considered as quite reliable but not valid. we come into conclusion that there might be errors due to apparatus because our experimental result is constantly the same. The emulsion oil can damage the structure of the cell. this experiment is a time critical experiment. Secondly. Next. it cause the specimen to overlap each other. we obtained the same results. the duration for the root tip to be immersed in HCl acid and carnoy solution is not exactly four minutes. the specimen might be damaged due to high pressure exerted by the thumb on it. The first one comes from microscope. SOURCE OF ERROR There are lots of errors that we have analysed. This is because we could not see any stages in the cells. So. Thirdly. Our group only manage to see the overlapping cell walls. But then. our experimental result is quite reliable because we have been repeated this experiment for three times but unfortunately. As we have been repeated this experiment three times. This is because a few steps in this experiment is constraint with time. It makes us difficult to trace the stage easily. 15 | P a g e . Not just that. As the root tip has been cut in a large size and not exactly 1 mm. Our group¶s microscope has been contaminated by the emulsion oil.Not just that.

The microscope is fragile and light bulbs can get so hot. so wear eye protection (goggles). toluidine blue can stain your cloth if you do not wear lab coat. It takes about 10 minutes for us to actually see the specimen. Do not eat and drink throughout the experiment because the biology lab still keep the hazardous chemical substances even we are not using it. Basically. We must also be very careful when handling the lab apparatus like beakers and cavity slide which tend to break easily and will harm our safety. We have to make sure the concentration of HCl acid used is not too concentrated because it might be dangerous to be handled. First. so we have to be careful. This is due to lack in time. as our microscope is not in good condition. As there are many steps of them. it takes about 30 minutes to prepare each specimen. Each step takes at least 2-3 minutes to be done. We have to wait to use a particular microscope. Furthermore. we must wear the lab coat to prevent any stain onto our cloth. this is our first experience 16 | P a g e . the microscopes provided are not enough to accommodate all of us. And always keep the laboratory in clean state. Next. This consume more and more time. LIMITATIONS There are a few limitations that prevent us from getting a very acurate and precise result. Furthermore. Not just that. For example. it is not easy to observe the specimen under microscope. take care with scalpels and always carry them on a white tile to prevent any injury occur during lab session. overall. this experiment consume a lot of time. Ethanoic ethanol is corrosive. we have to use other group¶s microscope as there is no extra microscope provided. For example.SAFETY MEASURES AND PRECAUTION There are a few safety measures that must be taken into consideration in order to make sure that the experiment is going on smoothly without any accidents.

This could be a good alternative. MEDICATION AND FURTHER WORKS In order to get a more accurate result. We have also to make sure that the root tip is fully immersed in HCl acid and carnoy fixative so that the structure of the cell can be clearly observed. Therefore hypothesis is rejected and in order to verify this statement. a few suggestions had came across. 17 | P a g e . Moreover. they are suggesting us to squash the root tip using blunt pencils. From what I have read some tips from example reports.in conducting mitosis experiment. finally we came into conclusion that our experiment is not success to prove what had been stated in hypothesis that is the most time spent is in prophase and the least time spent is in anaphase. We have to make sure that the concentration of toluidine blue is just fine( neither too dilute nor too concentrated) so that we can see the nucleus clearly. we can conclude that mitotic stages of onion root tip can be observed with a light microscope and the most time spent is in prophase while the least time spent is in anaphase. we must undergo this experiment one more time by using different microscope. slides and other apparatus and material to overcome any lacking during conducting this experiment. CONCLUSION After we have done this experiment. we have to increase our alertness in recording time so that the duration taken for the root tip to be immersed is actually as required. However through long discussion with other group members. We are lacking in skill especially the skill to cut the root tip. The first one is we have to wipe the lens before we are using it to prevent any immersion oil that might be used by the previous group.

Accessed on 1st February 2011  FROM BOOK  Angela. et al . Edexcel Pearson. 2008. Accessed on 1st February 2011  2011.com/preview.asp?id=156102.net/pdecell/celldivision/oniontip. Available from : http://www.EXP.html.jccc.experiment-resources. Accessed on 1st February 2011  2011. Available from: http://staff. In Salters-Nuffield Advanced Biology or Edexcel AS Biolog. Available from : http://en.121.REFERENCES AND BIBLIOGRAPHY  FROM INTERNET  2011.H. London. Available from: http://www.org/wiki/Mitosis.wikipedia.org/areas/advanced/cells-tosystems/cell-division/investigating-mitosis-in-allium-root-tipsquash. Accessed on 1st February 2011  2011. 18 | P a g e .com/validity-andreliability. Accessed on 1st February 2011  2011.html.123helpme. Voice of Genome.practicalbiology.html. p. Available from : http://www.116-117.

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