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Posted: April 18th, 2024
The aim of this experiment is to observe the different stages of the garlic tissues during mitosis which are prophase, metaphase, anaphase and telophase in living tissue. Students are also required to consider the duration of the stages of mitosis in relation to the whole cell cycle. Other objectives are to calculate the percentage of cells in the area visible under the microscopes for each stages of mitosis.
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There are 5 different stages of mitosis that can be observed namely; interphase, prophase, metaphase, anaphase and telophase. By using certain chemicals to stain the cells, most of the cells can be observed under different stages of mitosis. Thus, the percentage of cells in each phase and the duration for each phase can be calculated.
Many cell divisions transform a single fertilized egg into a multicellular organism. A series of events called the cell cycle describes the sequence of events as a cell prepares for division and the divides. The cell cycle is a continual process and can be divided into two major stages which are interphase and mitosis (also known as M phase).
During interphase, the cell continues the basic biochemical functions and also replicates its DNA and other organelles for distribution to daughter cells. Interphase can be further divided into three stages namely G1 (gap), S (synthesis) and G2 phase. During G1 phase, the cell resumes synthesis of proteins, lipids and carbohydrates. Next, in the S phase, the cell replicates its entire genome, so that each chromosome consists of two copies joined together at centromere. Then, more proteins are synthesized when the cell enters G2 phase.
As mitosis begins, the replicated chromosomes are condensed and become visible under light microscope when stained. The two long strands of identical chromosomal material in a replicated chromosome are called chromatids. During prophase, the first stage of mitosis, DNA coils tightly, shortening and thickening the chromosome enabling them to separate easily. Toward the end of prophase, the nuclear membrane breaks down and nucleolus is no longer visible. The next phase in mitosis is metaphase, where chromosomes attach to the spindle at their centromeres and align themselves along the center of the cell, which is called the equator.
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Anaphase followed after metaphase. In the beginning of anaphase, the centromere regions of each pair of chromatids separate and are moved by the shortened spindle fibers toward opposite poles of the cell, dragging the rest of the chromatid behind them. These daughter chromosomes continue their movement until they form two compact clumps, one at each spindle pole. Telophase, the last stage of division, is marked by a pronounced condensation of the chromosomes, followed by the formation of a new nuclear envelope around each group of chromosomes. It start when the chromosome arrive at the pole of the cell. The chromosomes gradually uncoil to form the fine chromatin network seen in interphase, and the nucleoli and nuclear envelope reappear.
In this experiment, student needed to know whether which type of cell is suitable to be observed. Theoretically, massive mitosis process can be observed at the early development of embryo. However, observing this mass of cells in laboratory is inappropriate and could raised ethical issue. Therefore, observing mitosis in plant cell is much better and easier. In higher plants the process of forming new cells is restricted to special growth regions called meristems. These regions usually occur at the tips of stems (shoot) or roots. There are two parts in plant which actively dividing cells occur — the root tips and the shoot tips. Scientifically, these are called shoot apical meristem and root apical meristem respectively. The root is one of the best places in plant to see mitosis because the dividing cells are in more compact zone than elsewhere. Thus, observing different stage of mitosis would be easier here.
At the root tip there are few different zones or region of root development. This region can be divided into three. The zone of cell division is the region that contains the highest percentage of cells undergoes mitosis. Here, different stages of mitosis can be view under the microscope. The presence of root cap at the lower part serves as protective structure for the root. The region of cell elongation is area where growths of the cells occur. The cells enlarge and elongate to increase its length, forces the root tip down through the soil. The region of maturation or cell differentiation is where root hairs develop and where the cells differentiate to become phloem (sieve tube), xylem vessel and other tissues.
FIGURE 1 — Root tip of plant
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Apparatus: Scalpel, watch glasses, pipette fillers, microscope slides and coverslips, pair of fine forceps, mounted needle, soft tissue paper, microscope with magnification of x100 and x400 and eye protection.
Materials: Garlic roots, 1 M hydrochloric acid, toluidine blue stain, and distilled water.
1) A root tip at about 5mm is cut off from some growing garlic roots. The white tips with a firm rounded end are chosen and the brown tip is being removed by using a scalpel.
2) The root tip then is immersed in 2 cm3 1 M hydrochloric acid at about 5 minutes.
3) The root tip is being transferred to a watch glass containing approximately 5 cm3 of distilled water for 5 minutes. Then, the root tip is blotted gently by using soft tissue paper to prevent any damage to the garlic tissue.
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4) The root tip then is transferred to a clean microscope slide. Then, the root tip is being macerated by using mounted needles.
5) A few drops of toluidine blue is being added to stain the root tip and left for 2 minutes. The coverslip is used to cover the root tip and the coverslips is gently pressed by using the thumb to flatten it. Avoid lateral movement of the coverslip.
6) The nuclei (stained blue) of the garlic tissue are being located by using the lowest magnification (x40) first. Then, the lens is adjusted to a higher magnification of x100 and x400 to get a better image of the nuclei.
7) The different stages of mitosis are being observed and the cell is drawn to illustrate each stage.
8) The number of cells in the area visible under the microscope for each stage of mitosis is counted. The results are recorded in a table.
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9) The percentage of cells in each stage of mitosis is calculated. These values are ranked from highest to lowest. The percentage of cell can be calculated by using this formula:
Apparatus: Scalpel, water bath at 60 oC, test tubes, watch glasses, pipette fillers, microscope slides and coverslips, pair of fine forceps, mounted needle, soft tissue paper, microscope with magnification of x100 and x400 and safety goggles.
Materials: Garlic roots, 1 M hydrochloric acid, ethanoic alcohol (acetic alcohol), orcein ethanoic stain (acetic orcein), and distilled water.
1) 2 cm3 of 1 M hydrochloric acid is being preheated in 60 oC water bath.
2) A root tip at about 5mm is cut off from some growing garlic roots. The white tips with a firm rounded end are chosen and the brown tip is being removed by using a scalpel. The root tip then is transferred to a watch glass.
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3) 20-30 drops of acetic orcein are added together with 3 drops of the preheated 1 M of hydrochloric acid.
4) The root tip then is gently heated by using a hot plate for about 3-5 minutes. Additional drops of acetic orcein and hydrochloric acid then are added with the ratio of 5:1 respectively when the solution started to boil.
5) The heated root tip then is transferred to a clean microscope slide. Then, the root tip is being macerated by using mounted needles.
6) Additional stain is added on the root tip and then covered with coverslip. The coverslip is gently pressed by using the thumb to flatten it. Avoid lateral movement of the coverslip.
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7) The nuclei (stained red) of the garlic tissue are being located by using the lowest magnification (x40) first. Then, the lens is adjusted to a higher magnification of x100 and x400 to get a better image of the nuclei.
8) The different stages of mitosis are being observed and the cell is drawn to illustrate each stage.
9) The number of cells in the area visible under the microscope for each stage of mitosis is counted. The results are recorded in a table.
10) The percentage of cells in each stage of mitosis is calculated. These values are ranked from highest to lowest. The percentage of cell can be calculated by using this formula:
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In this experiment, my group was assigned to carry out the second method, by using orcein ethanoic in order to stain the chromosome of the garlic root cells. However, we did not manage to obtain the expected result. Although we manage to see the shape of the cell and its nucleus, we failed to observe the shape of the chromosome with further magnification. Due to this unexpected result, I had taken the results of the cells from the other group. They are using the first method staining the root tip cells by using toluidine blue stain. The chromosome of the cell can be seen clearly and the different phases of the mitosis can be detected. The results are shown in the table below.
This is the cell during interphase. The distinctive chromosomes become dispersed in interphase, in the form of chromatin (not visible).
This is the cell during prophase. Chromosomes become denser and visible under microscope. Nucleolus and nuclear envelope start to disappear.
This is the cell during metaphase. The chromosomes migrate and line up themselves on the equator (metaphase plate).
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This is the cell during anaphase. Chromatids separates at the centromere and spindle fiber pull them to opposite poles.
The last stage of mitosis is telophase. Chromatids decondense and form chromatin again. Nucleolus and nuclear membrane reappear.
The percentage of cells in each phase of the cell cycle using this equation is calculated:
Percentage of cell in interphase 34/52 100
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65.4%
Percentage of cells in prophase 14/52 100
26.9%
Percentage of cells in metaphase 1/52 100
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1.9%
Percentage of cells in anaphase 1/52 100
1.9%
Percentage of cells in telophase 3/52 100
5.8%
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From the percentage obtained, we can calculate the duration for each phase. In order to do that, we must assume that it takes an average of 1440 minutes (24 hours/ 1 day) for garlic root tip cell to complete the cell cycle. The time taken for each phase can be calculated by using the formula below:
Time for interphase 65.4 1440 / 100
941.76 min (15.646 hour)
Time for prophase 26.9 1440 / 100
387.36 min (6.456 hour)
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Time for metaphase 1.9 1440 / 100
27.36 min (0.456 hour)
Time for anaphase 1.9 1440 / 100
27.36 min (0.456 hour)
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Time for telophase 5.8 1440 / 100
83.52 min (1.329 hour)
In this experiment, there is a huge limitation as the method of experiment that my team had used does not work. The result obtained cannot even been analyze. Although we manage to see the shape of the cell and its nucleus, we failed to observe the shape of the chromosome with further magnification. After doing some research and careful post- mortem, I found out that the procedure may be wrong. Firstly, I do not placed the root tips in a watch glass containing acetic alcohol for a minimum of 12 hours. Secondly, the root tip is not being placed in ice cold water. Thirdly, during the experiment, hydrochloric acid is added together with acetic orcein at the same time. Due to these steps, the cells fail to stain the acetic orcein. Other problem arises when our specimen was overheated causing the watch glass to break.
In the calculation, I am trying to find which the duration for each phase. Firstly, the percentage of cells in each stage is calculated. Here it can see that most of the cells about 65% seem to be at interphase. The least number of cells is at metaphase and anaphase with only one cell at each phase. From here, cells are predicted to spend most of their time in interphase and only a short while in metaphase and anaphase. Then, the time for each phase is calculated. For this, a few assumptions is taken into account. Firstly the cells at the root tip take about 24 hours to complete one cell cycle. This means that in minutes, this process will take 1440 minutes. Thus, the percentage of cells in each stage calculated earlier is multiplied with 1440 and divides by 100.
From the result obtained, it is obviously seen that interphase takes the longest time in the cell cycle of the cells at the root tip. This is because interphase consists of another 3 sub stages and cell gone through complex series of metabolism process to prepare itself to divide. Anaphase and metaphase take the shortest time to get through this phases. This is due to the size of the cell which is small and the shortening of spindle fiber is relatively fast. This is a reason for a low percentage of cells at this stage. Prophase on the other hand is longer than telophase, anaphase and metaphase even though shorter than time taken in interphase. This is may be due to time taken for the chromosomes to condense, formation of spindle fiber, and disintegration of nucleolus and nuclear membrane.
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In this experiment, students are required to observe the different phases occur during mitosis. They are prophase, metaphase, anaphase and telophase. Garlic’s root tips are chosen to be observed in his experiment for some reasons. Firstly, in terms of number of chromosome, garlic cell only has six chromosomes. When the number of chromosome is less, each stages of mitosis can be observed clearly. Secondly, the garlic’s root tip is chosen as the root tip is the best part of a plant other than the shoot tip in order to observe mitosis. There are different zones in garlic root tip and one of them is the zone of cell division, where the cell is dividing very actively. Therefore, students should be aware when they are preparing the specimen as they need to know which part of the root tip is the zone of division. If they cut out the cell from the zone of elongation and differentiation, it is possible to observe the stages of mitosis.
In order to stain the chromosome of the cell, different chemicals which are hydrochloric acid together with acetic orcein or toluidine blue is used. In plant cell, they are being hold together by a middle lamella of pectin. Hydrochloric acid will breakdown the pectin and allows the cell to be separated into single units. This will allow us to observe and count the percentage of different stages of mitosis. When the root tips is being immersed in hydrochloric acid, the DNA of the chromosome will be hydrolyzed to form deoxyribose aldehyde which then react with the stain. Hydrochloric acid can also be used to remove the root cap, other than using scalpel. Besides, the root tip can be macerated easier when they are immersed inside the hydrochloric acid, but the concentration of the acid must be suitable as higher concentration will ruptured the cell of the root tips.
There are some limitations that can affect the outcome of this experiment. Firstly, due to time constraint, the root tips is not immerse in acetic alcohol for a minimum period of 12 hours. Only acetic orcein and hydrochloric acid is used in order to stain the chromosome. Secondly, the amount of stain used can also affect the result of the experiment. The used of excess stain to may cause the cells to become too dark in color. As the result, the chromosome become hard to be observed as the surrounding color is too dark. Meanwhile, if less amount of is used, the chromosome will not clearly visible. Therefore, for better results, the preparation steps should be repeated and the quantity of stain used must be adjusted. Besides, students should be extra aware when heating the specimen with a hot plate. The stain together with hydrochloric acid must be added continuously to prevent the stain and acid from being evaporated too much.
The use of light microscope is also considered as the limitation in this experiment. Under light microscope, the chromosome of the cell is not clearly visible but still can be observed. For better image of chromosome, the highest resolution can be used. However, the highest resolution can only be used with the aid of emulsion oil. Emulsion oil which has a high refractive index can overcome the problem of the highest objective lens which has short focal length. Lastly, the technique to macerate the cell can also affect the result of the experiment. The cell must be macerated nicely to avoid damage on the cell and try to macerate it as thin as possible because it is easier to observe the chromosome when the root tip is only one-cell thick.
During the experiment, safety precaution must be taken into account in order to avoid any possible accident that can happen due to carelessness. Firstly, the use of chemicals such as toluidine blue stain and acetic orcein can stain skin and cloth. Meanwhile, hydrochloric acid is corrosive, so avoid any physical contact with these chemicals. Students should remember to wear their lab coats and eye protection while handling these chemicals in order to reduce the risk of harmful injuries. If there is any contact with these chemicals, immediately wash the area of contact with tap water and clean up the spillage area.
During the observation process with microscope, make sure that there is no presence of air bubble between the cavity slide and the coverslip. If there is any, try to remove the air bubbles by using soft tissue paper to blot the slide firmly. Students also need to use the lowest magnification (x40) first in order to locate the chromosome. Once the chromosomes are located, use another magnification(x100 and 400x) by adjusting the rough adjustment knob and when the lens is getting closer to the glass slides, adjust the microscope by using fine adjustment knob. This is to ensure that the lens does not collide with the slide. In addition, students should be careful when they are handling the scalpel and forceps as both equipments are sharp and can cause serious cut.
As the conclusion, we can observe 5 different stages of mitosis namely; interphase, prophase, metaphase, anaphase and telophase. The result obtained from the observation is used to calculate the percentage of cells in each phase and the duration for each phase can be calculated. Hence, the percentage of cells and the duration can be used to differentiate each phase. The hypothesis is accepted.
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