The purpose of this experiment is to detect the different phases of the garlic tissues during mitosis which are prophase, metaphase, anaphase and telophase in life tissue. Students are besides required to see the continuance of the phases of mitosis in relation to the whole cell rhythm. Other aims are to cipher the per centum of cells in the country seeable under the microscopes for each phases of mitosis.
Hypothesis:
There are 5 different phases of mitosis that can be observed viz. ; interphase, prophase, metaphase, anaphase and telophase. By utilizing certain chemicals to stain the cells, most of the cells can be observed under different phases of mitosis. Therefore, the per centum of cells in each stage and the continuance for each stage can be calculated.
Introduction:
Many cell divisions transform a individual fertilized egg into a multicellular being. A series of events called the cell rhythm describes the sequence of events as a cell prepares for division and the divides. The cell rhythm is a continual procedure and can be divided into two major phases which are interphase and mitosis ( besides known as M stage ) .
During interphase, the cell continues the basic biochemical maps and besides replicates its Deoxyribonucleic acid and other cell organs for distribution to daughter cells. Interphase can be farther divided into three phases viz. G1 ( spread ) , S ( synthesis ) and G2 stage. During G1 stage, the cell resumes synthesis of proteins, lipoids and saccharides. Following, in the S stage, the cell replicates its full genome, so that each chromosome consists of two transcripts joined together at centromere. Then, more proteins are synthesized when the cell enters G2 stage.
As mitosis begins, the replicated chromosomes are condensed and become seeable under light microscope when stained. The two long strands of indistinguishable chromosomal stuff in a replicated chromosome are called chromatids. During prophase, the first phase of mitosis, DNA coils tightly, shortening and inspissating the chromosome enabling them to divide easy. Toward the terminal of prophase, the atomic membrane interruptions down and nucleolus is no longer seeable. The following stage in mitosis is metaphase, where chromosomes attach to the spindle at their kinetochores and align themselves along the centre of the cell, which is called the equator.
Anaphase followed after metaphase. In the beginning of anaphase, the centromere parts of each brace of chromatids separate and are moved by the sawed-off spindle fibres toward opposite poles of the cell, dragging the remainder of the chromatid behind them. These girl chromosomes continue their motion until they form two compact bunchs, one at each spindle pole. Telophase, the last phase of division, is marked by a marked condensation of the chromosomes, followed by the formation of a new atomic envelope around each group of chromosomes. It start when the chromosome arrive at the pole of the cell. The chromosomes bit by bit uncoil to organize the all right chromatin web seen in interphase, and the nucleole and atomic envelope reappear.
In this experiment, pupil needed to cognize whether which type of cell is suited to be observed. Theoretically, monolithic mitosis procedure can be observed at the early development of embryo. However, detecting this mass of cells in research lab is inappropriate and could raised ethical issue. Therefore, detecting mitosis in works cell is much better and easier. In higher workss the procedure of organizing new cells is restricted to particular growing parts called meristems.
These parts normally occur at the tips of roots ( shoot ) or roots. There are two parts in works which actively spliting cells occur aa‚¬ ” the root tips and the shoot tips. Scientifically, these are called shoot apical meristem and root apical meristem severally. The root is one of the best topographic points in works to see mitosis because the dividing cells are in more compact zone than elsewhere. Therefore, detecting different phase of mitosis would be easier here.
At the root tip there are few different zones or part of root development. This part can be divided into three. The zone of cell division is the part that contains the highest per centum of cells undergoes mitosis. Here, different phases of mitosis can be view under the microscope. The presence of root cap at the lower portion serves as protective construction for the root. The part of cell elongation is country where growings of the cells occur. The cells enlarge and elongate to increase its length, forces the root tip down through the dirt. The part of ripening or cell distinction is where root hairs develop and where the cells differentiate to go bast ( sieve tubing ) , xylem vas and other tissues.
Method I: Exploitation TOLUIDINE BLUE STAIN
APPARATUS AND MATERIALS:
Apparatus: Scalpel, ticker spectacless, pipette fillers, microscope slides and coverslips, brace of all right forceps, mounted needle, soft tissue paper, microscope with magnification of x100 and x400 and oculus protection.
Materials: Garlic roots, 1 M hydrochloric acid, toluidine blue discoloration, and distilled H2O.
Procedure:
- A root tip at about 5mm is cut off from some turning garlic roots. The white tips with a house rounded terminal are chosen and the brown tip is being removed by utilizing a scalpel.
- The root tip so is immersed in 2 cm3 1 M hydrochloric acid at about 5 proceedingss.
- The root tip is being transferred to a ticker glass incorporating about 5 cm3 of distilled H2O for 5 proceedingss. Then, the root tip is blotted gently by utilizing soft tissue paper to forestall any harm to the garlic tissue.
- The root tip so is transferred to a clean microscope slide. Then, the root tip is being macerated by utilizing mounted acerate leafs.
- A few beads of toluidine blue is being added to stain the root tip and left for 2 proceedingss. The coverslip is used to cover the root tip and the coverslips is gently pressed by utilizing the pollex to flatten it. Avoid sidelong motion of the coverslip.
- The karyon ( stained blue ) of the garlic tissue are being located by utilizing the lowest magnification ( x40 ) foremost. Then, the lens is adjusted to a higher magnification of x100 and x400 to acquire a better image of the karyon.
- The different phases of mitosis are being observed and the cell is drawn to exemplify each phase.
- The figure of cells in the country seeable under the microscope for each phase of mitosis is counted. The consequences are recorded in a tabular array.
- The per centum of cells in each phase of mitosis is calculated. These values are ranked from highest to lowest. The per centum of cell can be calculated by utilizing this expression:
Percentage of cell = Total figure of cell in stage ten 100
Entire figure of cells counted
METHOD II: Exploitation ORCEIN ETHANOIC STAIN
APPARATUS AND MATERIALS:
Apparatus: Scalpel, H2O bath at 60 oC, trial tubings, ticker spectacless, pipette fillers, microscope slides and coverslips, brace of all right forceps, mounted needle, soft tissue paper, microscope with magnification of x100 and x400 and safety goggles.
Materials: Garlic roots, 1 M hydrochloric acid, ethanoic intoxicant ( acetic intoxicant ) , orcein ethanoic discoloration ( acetic orcein ) , and distilled H2O.
Procedure:
- 2 cm3 of 1 M hydrochloric acid is being preheated in 60 oC H2O bath.
- A root tip at about 5mm is cut off from some turning garlic roots. The white tips with a house rounded terminal are chosen and the brown tip is being removed by utilizing a scalpel. The root tip so is transferred to a ticker glass.
- 20-30 beads of acetic orcein are added together with 3 beads of the preheated 1 M of hydrochloric acid.
- The root tip so is gently heated by utilizing a hot home base for about 3-5 proceedingss. Additional bead of acetic orcein and hydrochloric acid so are added with the ratio of 5:1 severally when the solution started to boil.
- The het root tip so is transferred to a clean microscope slide. Then, the root tip is being macerated by utilizing mounted acerate leafs.
- Additional discoloration is added on the root tip and so covered with coverslip. The coverslip is gently pressed by utilizing the pollex to flatten it. Avoid sidelong motion of the coverslip.
- The karyon ( stained red ) of the garlic tissue are being located by utilizing the lowest magnification ( x40 ) foremost. Then, the lens is adjusted to a higher magnification of x100 and x400 to acquire a better image of the karyon.
- The different phases of mitosis are being observed and the cell is drawn to exemplify each phase.
- The figure of cells in the country seeable under the microscope for each phase of mitosis is counted. The consequences are recorded in a tabular array.
- The per centum of cells in each phase of mitosis is calculated. These values are ranked from highest to lowest. The per centum of cell can be calculated by utilizing this expression:
Percentage of cell = Total figure of cell in stage ten 100
Entire figure of cells counted
Consequence:
In this experiment, my group was assigned to transport out the 2nd method, by utilizing orcein ethanoic in order to stain the chromosome of the garlic root cells. However, we did non pull off to obtain the expected consequence. Although we manage to see the form of the cell and its karyon, we failed to detect the form of the chromosome with farther magnification. Due to this unexpected consequence, I had taken the consequences of the cells from the other group. They are utilizing the first method staining the root tip cells by utilizing toluidine bluish discoloration. The chromosome of the cell can be seen clearly and the different stages of the mitosis can be detected. The consequences are shown in the tabular array below.
Appearance of cell under microscope
Description
- This is the cell during interphase. The typical chromosomes become dispersed in interphase, in the signifier of chromatin ( non seeable ) .
- This is the cell during prophase. Chromosomes become denser and seeable under microscope. Nucleolus and atomic envelope start to vanish.
- This is the cell during metaphase. The chromosomes migrate and line up themselves on the equator ( metaphase home base ) .
- This is the cell during anaphase. Chromatids separates at the kinetochore and spindle fibre draw them to opposite poles.
- The last phase of mitosis is telophase. Chromatids decondense and signifier chromatin once more. Nucleolus and atomic membrane reappear.
- The per centum of cells in each stage of the cell rhythm utilizing this equation is calculated:
Percentage of cell = Total figure of cell in stage ten 100
Discussion:
DATA ANALYSIS
In this experiment, there is a immense restriction as the method of experiment that my squad had used does non work. The consequence obtained can non even been analyze. Although we manage to see the form of the cell and its karyon, we failed to detect the form of the chromosome with farther magnification. After making some research and careful post- mortem, I found out that the process may be incorrect. First, I do non put the root tips in a ticker glass incorporating acetic intoxicant for a lower limit of 12 hours.
Second, the root tip is non being placed in ice cold H2O. Third, during the experiment, hydrochloric acid is added together with acetic orcein at the same clip. Due to these stairss, the cells fail to stain the acetic orcein. Other job arises when our specimen was overheated doing the ticker glass to interrupt.
In the computation, I am seeking to happen which the continuance for each stage. First, the per centum of cells in each phase is calculated. Here it can see that most of the cells about 65 % seem to be at interphase. The least figure of cells is at metaphase and anaphase with merely one cell at each stage.
From here, cells are predicted to pass most of their clip in interphase and merely a short piece in metaphase and anaphase. Then, the clip for each stage is calculated. For this, a few premises is taken into history. First the cells at the root tip take about 24 hours to finish one cell rhythm. This means that in proceedingss, this procedure will take 1440 proceedingss. Therefore, the per centum of cells in each phase calculated earlier is multiplied with 1440 and divides by 100.
From the consequence obtained, it is evidently seen that interphase takes the longest clip in the cell rhythm of the cells at the root tip. This is because interphase consists of another 3 bomber phases and cell gone through complex series of metamorphosis procedure to fix itself to split. Anaphase and metaphase take the shortest clip to acquire through this stages. This is due to the size of the cell which is little and the shortening of spindle fibre is comparatively fast. This is a ground for a low per centum of cells at this phase. Prophase on the other manus is longer than telophase, anaphase and metaphase even though shorter than clip taken in interphase. This is may be due to clip taken for the chromosomes to distill, formation of spindle fibre, and decomposition of nucleolus and atomic membrane.
Evaluation
In this experiment, pupils are required to detect the different stages occur during mitosis. They are prophase, metaphase, anaphase and telophase. Garlicas root tips are chosen to be observed in his experiment for some grounds. First, in footings of figure of chromosome, Allium sativum cell merely has six chromosomes. When the figure of chromosome is less, each phases of mitosis can be observed clearly.
Second, the garlicaa‚ root tip is chosen as the root tip is the best portion of a works other than the shoot tip in order to detect mitosis. There are different zones in garlic root tip and one of them is the zone of cell division, where the cell is spliting really actively.
Therefore, pupils should be cognizant when they are fixing the specimen as they need to cognize which portion of the root tip is the zone of division. If they cut out the cell from the zone of elongation and distinction, it is possible to detect the phases 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 works cell, they are being hold together by a in-between gill of pectin. Hydrochloric acid will breakdown the pectin and allows the cell to be separated into individual units. This will let us to detect and number the per centum of different phases of mitosis. When the root tips is being immersed in hydrochloric acid, the Deoxyribonucleic acid of the chromosome will be hydrolyzed to organize deoxyribose aldehyde which so react with the discoloration. Hydrochloric acid can besides be used to take the root cap, other than utilizing 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 suited as higher concentration will tear the cell of the root tips.
There are some restrictions that can impact the result of this experiment. First, due to clip restraint, the root tips is non immerse in acetic intoxicant for a minimal period of 12 hours. Merely acetic orcein and hydrochloric acid is used in order to stain the chromosome. Second, the sum of discoloration used can besides impact the consequence of the experiment.
The used of extra discoloration to may do the cells to go excessively dark in colour. As the consequence, the chromosome go difficult to be observed as the environing colour is excessively dark. Meanwhile, if less sum of is used, the chromosome will non clearly seeable. Therefore, for better consequences, the readying stairss should be repeated and the measure of discoloration used must be adjusted. Besides, pupils should be excess cognizant when heating the specimen with a hot home base. The discoloration together with hydrochloric acid must be added continuously to forestall the discoloration and acid from being evaporated excessively much.
The usage of light microscope is besides considered as the restriction in this experiment. Under light microscope, the chromosome of the cell is non clearly seeable but still can be observed. For better image of chromosome, the highest declaration can be used. However, the highest declaration can merely be used with the assistance of emulsion oil. Emulsion oil which has a high refractile index can get the better of the job of the highest aim lens which has short focal length. Last, the technique to macerate the cell can besides impact the consequence of the experiment. The cell must be macerated nicely to avoid harm on the cell and seek to macerate it every bit thin as possible because it is easier to detect the chromosome when the root tip is merely one-cell midst.
SAFETY MEASURES
During the experiment, safety safeguard must be taken into history in order to avoid any possible accident that can go on due to carelessness. First, the usage of chemicals such as toluidine blue discoloration and acetic orcein can stain skin and cloth. Meanwhile, hydrochloric acid is caustic, so avoid any physical contact with these chemicals. Students should retrieve to have on their lab coats and oculus protection while managing these chemicals in order to cut down the hazard of harmful hurts. If there is any contact with these chemicals, instantly wash the country of contact with tap H2O and clean up the spillage country.
During the observation procedure with microscope, do certain that there is no presence of air bubble between the pit slide and the coverslip. If there is any, attempt to take the air bubbles by utilizing soft tissue paper to blot the slide steadfastly. Students besides need to utilize the lowest magnification ( x40 ) foremost in order to turn up the chromosome. Once the chromosomes are located, utilize another magnification ( x100 and 400x ) by seting the unsmooth accommodation boss and when the lens is acquiring closer to the glass slides, adjust the microscope by utilizing all right accommodation boss. This is to guarantee that the lens does non clash with the slide. In add-on, pupils should be careful when they are managing the scalpel and forceps as both equipments are crisp and can do serious cut.
Decision:
As the decision, we can detect 5 different phases of mitosis viz. ; interphase, prophase, metaphase, anaphase and telophase. The consequence obtained from the observation is used to cipher the per centum of cells in each stage and the continuance for each stage can be calculated. Hence, the per centum of cells and the continuance can be used to distinguish each stage. The hypothesis is accepted.
