mitosis report

INTERNATIONAL EDUCATION CENTRE (INTEC) UiTM Section 17 Campus 40200 Shah Alam Selangor Darul Ehsan. TEL: 603-55227000

TITLE: OBSERVING MITOSIS

NAME: NUR AMALINA BT ZOLKEFLEE SID NUMBER: 2010843164 CLASS: 11M2 DATE OF EXPERIMENT: 24

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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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OBJECTIVES Basically, our experiment is done to prepare some slides of actively dividing plant tissues. 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, we can develop certain experimental skills, namely working safely, the use of microscopes, and producing valid results and recording results. Not just that, we are also taught to calibrate an eyepiece graticule and use it to measure the size of the cells.

INTRODUCTION y

BACKGROUND INFORMATION Genetic information of human, plants and ani mals reside in unique structure called chromosomes. For example, each human cell posses 46 chromosomes while onion cell posses 8 chromosomes. All cells must replicate their DNA in order to pass it to the next generation of cells in the whole body. During DNA replication, two strands of DNA separate and for each strand, new complementary DNA is produced thus yielding two identical DNA molecules. After DNA replication, the process followed is mitosis which is important to ensure that each daughter cell receives one copy of each r eplicated chromosome. During mitosis, the chromosomes pass through several stages like prophase, metaphase, anaphase a nd telophase. The division is considered as complete once the cell undergo cytokinesis.

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

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Figure 2 shows some events that take place during prophase. Source: http://chuck16.wordpress.com/2009/04/30/phases-of-mitoses/prophaselate__civyrosejpg/ During metaphase, the centromeres of chromosomes line along the metaphase plate or  equatorial plane, an imaginary line t hat is equidistant from centrosome poles. When this arrangement has been completed, the cell has reached the end of metaphase.

Figure 3 shows how the chromosomes arrange themselves at metaphase plate during metaphase. Source : http://www.sparknotes.com/biology/cellreproduction/mitosis/section2.rhtml

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The third stage in mitotic division is anaphase. During this stage, the centromeres split. The spindle shorten to pull the two halves of each centromere in opposite directions. One chromatid of aech chromosome is pulled to each of the poles. Anaphase ends when the separated chromatids reach the poles and the spindle breaks down.

Figure 4 shows the events occur during anaphase. Source: http://www.sparknotes.com/biology/cellreproduction/mitosis/section2.rhtml The last stage in mitotic division is t elophase. The events taking place during telophase is a reverse process of prophase. Chromosomes unravel and nuclear envelope reforms, so that two sets of genetic information become enclosed in separate nuclei.

Figure 5 shows the events take place during telophase. Source: http://www.sparknotes.com/biology/cellreproduction/mitosis/section3.rhtml 5|

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Cell cycle consists of three main stages which are interphase, mitosis and cytokinesis. Cytokinesis is also known as cytoplasmic division. During cytokinesis, plant cells divide due to formation of walls between two daughter cells. New cell wall material is brought by microtubule system forming the phragmoplast, a complex organelle consisting microtubules and actin fila ments.

Figure 6 shows the sequence taking place during cytokinesis. Source: http://www.bms.ed.ac.uk/research/others/smaciver/Cytotopics/Plant%20Cytokinesis.htm

Overall, mitosis is very crucial in order to maintain genetic consistency. This is because the daughter cells are made to be ganetically identical to each other and parent cell. 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. Genetic consistency is achieved by DNA replication prior to nuclear division and during the arrangement of the chromosomes on the spindle fibre a nd the separation of chromatids to the poles. Mitosis is also important to animals and plants that practice asexual reproduction. For example starfish can regrow a completely new body from a fragment of  its body. Furthermore, mitosis is also significant in replacing our dead cells sepecially

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our skin cells which are easily rubbed by the friction. So, in order to maintain in good condition, mitosis will occur to replace those ol d cells. In this experiment, we are using onion roots due to several specialities that it owns. Firstly, the onion roots are easily grewn in large numbers. Therefore, 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. Secondly, cells at the tip of roots are actively dividing and thus many cells will be in stages of mitosis. 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. 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, therefore the onion root cells is the most suitable to be used in this experiment. There are three cellular regions at the tip of onion root. The first one is root cap. 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). Here, the cells ar e 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.

Figure 7 shows the three regions at the tip of onion root Source: http://www.excellup.com/InterBiology/morphologyplant.aspx

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In order to get an accurate result, we have to make sure that we know how to use the microscope correctly because our r esult is only depending on microscope. What we see through the microscope indicate how successful our experiment is. Firstly, you have to put your microscope on flat surface. T hen, 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. Use the lowest power objective (usually 4x for 40x magnification). Place the slide on the stage and adjust the large coarse focus knob until specimen is in focus. After that, adjust the small fine focus knob until specimen is cl early in focus. Then, adjust the diaphragm to get the best lighting. Rotate the objective lens to 10x objective for  100x magnification. Refocus and view the specimen carefully. Repeat this step by using 40x objective. It is an optional for you to use the 100x objective for 1000x magnification. You need to put 1-2 drops of immersion oil over the slide coverslip before viewing it at highest power.

Figure 9 shows a microscope Source : http://www.hometrainingtools.com/how-to-use-a-microscope/a/1120/

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In this experiment also, we are using toluidine blue instead of carbol fusc hin. Toluidine blue is a polychromatic dye that absorbs different colours depending on the nature of its chemical binding with components of the tissues. At pH 4.4, it will bind to pectins in cell walls and colour them pink. Compounds containing benzene rings such as lignin will be coloured green. At lower acidic pH, toluidine blue gives only blue or only green colour. At higher basic pH (11.1) 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. By observing onion meristem cells under microscope, we can see the interphase, the four stages of mitosis that is prophase, metaphase, anaphase and telophase and c ytokinesis clearly. The most time spent in a stage is in prophase and the least time spent in a stage is anaphase. All the stages can be differentiated by observing the chromosomes in nucleus that is the chromosomes¶ position within the nucleus. If only long strand can be seen, it indicates the cell is undergoing interphase. If the chromosomes scattered in t he nucleus, it indicates the prophase stage while if the chromosomes arrange themselves at the centre which is known as metaphase plate, it shows metaphase stage. On the other hand, if the spindle looked shorter and like pulling the chromosomes to the pole, it means that the cell is undergoing anaphase. The telophase stage can be observed when you can see two sets of  chromosomes in a cell. Cytokinesis is the easiest st age to be detected; t hat is when you can see cell plate formed in between t he two sets of chromosomes.

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MATERIALS AND APPARATUS MATERIALS: carnoy fixative, holding solution(70% ethanol), HCl acid( 18%), toluidine blue. APPARATUS : microscope slides, coverslips, small beakers, watch glasses, blades, Forceps, filter paper, compound microscope. METHOD Preparing the sample

1. Sample of onion root cells is obtained from the holding solution. 2. Two cups of solutions are prepared. The first cup contains HCl acid while the second one contains carnoy solution. 3. By using a forcep, an onion root tip is transferred into HCl a cid solution for four  minutes. 4. Then, the root tip is transferred into carnoy solution for four minutes. 5. By using a blade, 1 mm of the root tip is cut and is put on microscope slide. 6. A few drops of toluidine blue is dropped into the root tip and is left for two minutes. Then, a few drops of water is put onto the root tip to dilute the concentrated toluidine blue. 7. By using a filter paper, the stain surround the root tip is blotted away. 8. Then, a cover slip is put over the specimen. 9. The slide is covered with a paper tissue and the cover slip is pressed with the thumb. 10. Then, the slide is observed under microscope.

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Preparing the microscope

1. A stage micrometer is slide on the stage of microscope. The smallest division on the stage micrometer is measured. 2. By using low power objective, the microscope is focused on stage micrometer. The eyepiece is rotated and the slide is moved. 3. 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. 4. Step 3 is repeated for medium and high power objectives. The cell size can be measured. RESULTS AND DATA

Figure 10 shows the drawing of overlapping cell walls. What we can see from the microscope is only overlapping cell walls.

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Figure 11 shows Nucleus is hardly seen due to compact cell wall. Source: http://mrswolfgang.wikispaces.com/Animal+and+Plant+Cells++Bizousky,+Byerly

Stage

of cell cycle

Number of cells in each stage

Interphase

Cannot be observed

Prophase

Cannot be observed

Metaphase

Cannot be observed

Anaphase

Cannot be observed

Telophase

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. After discussing with other groups that manage to conduct their experiment successfully, we found that ea ch stage in cell cycle can been observed clearly under microscope. Here is some pictures showing how chromosomes behave in each stage of mitotic division and interphase.

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Figure 12 shows all the cells that can be observed under microscope using low power  objective.

Figure 13 shows the chromosomes behaviour using higher power objective.

Source: http://www.practicalbiology.org/areas/advanced/cells-to-systems/celldivision/investigating-mitosis-in-allium-root-tip-squash,121,EXP.html Through our discussion too, we found that our hypothesis made is acc epted. That is most of the time in mitotic division is spent in prophase while the least time spent is in anaphase.

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Table 2 shows number of cells in each stage.

Source: http://www.practicalbiology.org/areas/advanced/cells-to-systems/celldivision/investigating-mitosis-in-allium-root-tip-squash,121,EXP.html

In calibrating the eyepiece graticule, 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. Number of division on eyepiece graticule is found to be three divisions equal to 1 smallest division of the stage micrometer. Therefore, each division on eyepiece graticule is equal to 33.33 micrometer.

Figure 14 shows the stage micrometer and eyepiece graticule scale.

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

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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. First, we must wear  the lab coat to prevent any stain onto our cloth. For example, toluidine blue can stain your  cloth if you do not wear lab coat. We have to make sure the concentration of HCl acid used is not too concentrated because it might be dangerous to be handled. Do not eat and drink throughout the experiment because the biology lab still keep the hazardous chemical substances even we are not using it. 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. The microscope is fragile and light bulbs can get so hot, so we have to be careful. Ethanoic ethanol is corrosive, so wear eye protection (goggles). Not just that, take care with scalpels and always carr y them on a white tile to prevent any injury occur during lab session. And always keep the laboratory in clean state. LIMITATIONS There are a few limitations that prevent us from getting a very ac urate and precise result. This is due to lack in time. Each step takes at least 2-3 minutes to be done. As there are many steps of them; overall, it takes about 30 minutes to prepare each specimen. Furthermore, it is not easy to observe the specimen under microscope. It takes about 10 minutes for us to actually see the specimen. Basically, this experiment consume a lot of  time. Next, the microscopes provided are not enough to accommodate all of us. F or  example, as our microscope is not in good condition, we have to use other group¶s microscope as there is no extra microscope provided. We have to wait to use a particular  microscope. This consume more and more time. Furthermore, this is our first experience 16 | P a g e

in conducting mitosis experiment. We are lacking in skill especially the skill to cut the root tip. MEDICATION AND FURTHER WORKS In order to get a more accurate result, a few suggestions had came across. 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. 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 have also to make sure that t he root tip is fully immersed in HCl acid and carnoy fixative so that the structure of t he cell can be clearly observed. Moreover, 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. From what I have read some tips from example reports, they are suggesting us to squash the root tip using blunt pencils. This could be a good alternative. CONCLUSION After we have done this 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. Therefore hypothesis is rejected and in order to verify this statement, we must undergo this experiment one more time by using different microscope, slides and other apparatus and material t o overcome any lacking during conducting this experiment. However through long discussion with other group members, 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.

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REFERENCES AND BIBLIOGRAPHY

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

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