BIOLOGY LAB REPORT
TITLE
: OBSERVING MITOSIS
PREPARED BY
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OBJECTIVE
To prepare some slides of actively dividing plant ti ssue
To observe the stages of the cell cycle in living tissue
To consider the duration of the stages of mitosis in relation t o the whole cell cycle
To develop certain experimental skills, namely working safely, the use of microscopes, producing producing valid results and recording results.
INTRODUCTION (2)
MITOSIS
Cell is known as unit of life. These cell divides either mitotically or meiotically , which is based on its location. Mitotic cell division take place in somatic cells which means all the cells in the body except the germ cells as they divide through another process called meiosis. Basically, mitosis is the process where an eukaryotic cell separates the chromosome chromosome in its cell nucleus into two identical sets in two separate nuclei which is known as two identical daughter cells. Mitosis doubles the number of cells without changing the genetic content. content. Mitosis which is known as M phase occurs after interphase which has three stages namely G1 phase , S phase and G2 phase. M phase is generally followed by cytokinesis which bring means of dividing the cytoplasm into two cells containing roughly equal shares the cellular components.
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Figure 1 : The roles of stages in Interphase
Figure 2 : Interphase
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Mitotic phase or M phase generally have four stages namely prophase, metaphase, anaphase and telophase. Some of stages can be even divided as early phase and late phase. Cytokinesis generally come together at the end of telophase stage. Cytokinesis in plant cell occur as the cell plate forms, thus dividing the daughter cell. Cell cycle is control by control chemical( cyclins). These cyclin build up and attach to enzymes forming cyclin-dependent kinases(CDKs), which means they add phosphate group to phosphorylates, changing shape and bring next stage of cell cycle. Mitosis is significant as they provide cell replacement, regeneration, and involve in asexual reproduction. During Prophase
Figure 3 : General prophase stage
Figure 4 : Early and late prophase
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During Metaphase
Figure 5 : Metaphase
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During Anaphase
Figure 6 : Anaphase
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Figure 7 : Early and late anaphase
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During Telophase
Figure 8 : Telophase
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Cytokinesis
Figure 9 : Cytokinesis in animal cell
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Figure 10 : Cytokinesis in plant cell
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Figure 11 : Mitosis is completed
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Figure 12 : In summary of Mitosis
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ONION CELL TIP
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Figure 13 : Onion cell tip
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Onion roots are used to view mitosis as the roots are easy to grow in large number, the cells at the root tip are actively dividing resulting in many cells that will be in mitosis stage, the tips can be squashed during preparation on microscopes slide so that individual chromosome can be observed and the presence of choromosomes that can be stained to make them more easily observable. There are three cellular regions near root tip of an onion. The root cap contains cells that cover and protect the underlying growth region as the root pushed through the soil. Second region of cell division known as meristem is where cells are actively dividing but not increasing significantly in size. And the third region is cell elongation, where the cell are increasing in size, but not dividing. Since each cell has only eight chromosomes so it is relatively easy to see them once they condensed.
HYDROCHLORIC HYDROCHLORIC ACID
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In order to see the chromosome chromosome inside the cells, the cells must be separated and spread out into a layer that is ideally just one cell thick. Plant cells such as onion cells are glued together by a middle lamella of pectins. Hydrochloric acid will break down the pectins that hold the cell together.
TOLUIDINE BLUE SOLUTION
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Toluidine blue is a polychromatic dye which is widely widely used in testing for lignin, a complex organic molecule that bond to cellulose fibres which strengthens and hardens the cell wall in the plants. TB is quite useful to stain fixed tissue but it only provides minimal information about the chemical make up of a tissue or organ. It has a strong affinity for the granules in mast cells, one of the wandering cells of connective tissues and also often requested as a specific stain for mast cell tumours. In this experiment, toluidine blue was used to stain the cells to make the chromosomes being shown up so they will be easier to be observed under the microscope later.
AIM To investigate mitosis in the cells of onion root tip
PROBLEM STATEMENT How do the cells in onion root tip divide ?
HYPOTESIS
Mitotically dividing cells are larger than normal cells.
The higher the percentage of dividing cells in one phase, the longer in time the cells spend on the phase the phases.
APPARATUS Watchglasses, glass slide, micrometer slide, beakers, coverslips, forceps, microsope, stopwatch, safety goggles, dropper
MATERIALS -3
Onion root tips, carnoy fixative, holding solution (70% ethanol), 6 moldm hydrochloric acid (18 %), toluidine blue stain , carbon fuchsin, distilled water, paper towels
PROCEDURE 1. Two watchglasses labeled “ HCl “ and “ Carnoy” were prepared. -3
2. 6 moldm hydrochloric acid and carnoy fixative were added in each watchglasses by using dropper enough to cover the bottom of the watchglasses. solution (70% 3. An onion root tip was transferred into the “ HCl “ watchglass from holding solution
ethanol) using a forceps. It was then left for 4 minutes (time taken using using stopwatch). Carnoy “ watchglass watchglass for another another 4 4. The root tip then transfer using forceps into “ Carnoy
minutes ( time taken using stopwatch) 5. The root tip was then taken out using a pair of forceps and was placed on a glass slide. 6. The root tip was then stained with with a few drops off toluidine blue ( or carbol fuschin fuschin ) for 2 minutes ( time taken using stopwatch). 7. The excess stain surrounding the root tip was blotted way or soaked up gently and carefully using tissue paper. 8. One or two drops of distilled water was dropped on the root tip when the stain has been blotted away. 9. A cover slip was lowered gently to cover the root tip so that no bubble is trapped inside the cover slip. Lateral movement of the coverslip was avoided 10. The cover slip was then pressed firmly firmly using thumb carefully with putting putting paper towel on it to spread the root tip into a single layer cells making the cells more easily and clearly viewable. 11. The preparation slide was observed under the microscope ( X 100 magnification). The cells that undergoing different stages of mitosis was searched and identified. 12. The following were counted : i.
The total number of cells in microscopic field
ii.
The total number of cells containing visible chromosome
13. The mitotic index of the slide was calculated by using the fo llowing formula :
14. The size of cells cel ls that undergoing each stages of mitosis was calculated by comparing the scalee on the eyepiece graticule with the micrometer slide. 15. Tables were drawn to summarise the results.
RESULTS
Stage of mitosis
Number of cells
Percentage of cells (%) ( number of cells / total cells with chromosomes counted) X 100%
Interphase
95
95 /120 X 100% = 79.167
Prophase
17
17 /120 X 100% = 14.167
Metaphase
1
1 /120 X 100% = 0.833
Anaphase
6
6 /120 X 100% = 5.000
Telophase
1
1 /120 X 100% = 0.833
Table 1: Stage of mitosis corresponding to their percentage of cells
25 / 120 = 0.208
TELOPHASE
ANAPHASE
INTERPHASE
METAPHASE
Figure 14 : Onion cell under microscope(x 400 mag nification)
Table 2 : The illustration il lustration of the stages in cell cycle
CALCULATION
Calibration of the eye piece graticule per unit : Magnification (X 400) 100 eyepiece graticule = 25 stage microscale microscale units 1 eyepiece graticule
= 0.25 stage microscale units = 0.25 X 0.01 X 1000 = 2.5μm
Magnification (X 100) 100 eyepiece graticule = 100 stage microscale units 1 eyepiece graticule
= 1 stage microscale units = 1 X 0.01 X 1000 = 10 μm
Magnification (X 40) 40 eyepiece graticule = 100 stage microscale microscale units 1 eyepiece eyepiece graticule graticule = 2.5 stage microscale microscale units = 2.5 X 0.01 X 1000 = 25 μm
Formula : Size of cell = Number of units in eyepiece graticule x 0.025 μm
Stage of mitosis
Number of units in eyepiece
Size of cell in each stage
graticule
(µm)
Interphase
14
14 x 0.025 = 0.35
Prophase
0
0 x 0.025 = 0.00
Metaphase
1
1 x 0.025 = 0.025
Anaphase
1
1 x 0.025 = 0.025
Telophase
1
1 x 0.025 = 0.025
Table 3 : Size of cell in each stage of the cell cycle (x 400 magnification)
Stage of mitosis corresponding to their percentage of cells
INTERPHASE PROPHASE METAPHASE ANAPHASE TELOPHASE
Pie Chart 1 : Stage of mitosis m itosis corresponding to their percentage of cells
DISCUSSION
ANALYSIS OF DATA The experiment is started with onion root tip that is already prepared by laboratory assistant , dipped into HCL solution ( to break down the middle lamella), and then into carnoy fixative solution continued with a few drops of toluidine blue .The stain is blotted and the tip is covered with few drops of water. It is then covered with coverslip and pressed gently using thumb forming single layer of cell. From low power to high power of a microscope, the cell is observed and the mitotic rate is recorded. The preparation of onion root tip freezes the mitosis process at one point, thus, the higher the percentage of the cell in one stage , the higher the time spent on that stage. Based on table one, interphase recorded the highest number of cell followed by anaphase, metaphase and telophase. Cytokinesis is not identified as it is not a mitosis part, but incidence that hppen after mitotic phase. This shows that the time spent on interphase is the longest . Basically, when a group of cell is dividing rapidly, high proportion of the cell undergoes mitosis while nondividing group of cell stays in interphase stage of the cell cycle. Mitotic index is used to calculate amount of cell division occurring in a tissue . Figure 14 in my result section shows the image of onion root tip cell under microscope (x 400 magnification). In this field of view, as stated in table 3 , 3 cells can be viewed ( have visible chromosomes) chromosomes)
out of 17 cells. The non-visible chromosome chromosome cell either can be in
interphase or cytokinesis. From these observation, mitotic index can be calculated giving result of 0.43. The number of cells which undergo different stages of mitosis and their corresponding percentages are tabulated in Table 1. The table shows that the interphase stage has the highest number of cell in the field view which is 95 cells and thus gives 79.167 % of the total cells. This is followed by prophase stage which gives 17 cells thus record 14.167 % of the total cells. Then both metaphase metaphase and telophase stage have 1 counted cells and thus make up 1.667 % of the the total cells respectively. Another 5% is the anaphase. These data can be illustrated in pie chart as in the Pie Chart 1.
Stage of mitosis corresponding to their percentage of cells is shown in another form , pie chart. The Pie Chart 1 shows that the interphase has taken the largest portions. Hence, it shows that cells spend the longest time in interphase during the cell cycle compared to the other phases in mitosis. During interphase, interphase, the cell undergo three subphases namely G1 phase. S phase and G2 phase. During all three subphases, the cells growth by producing proteins and cytoplasmic organelles. Hence, it is clear that interphase spend the longest period of time in the cell cycle as the cells increase in mass and size, replicate their DNA and carry out normal activities. Meanwhile, among the other four phases in the M phase, the prophase is the longest periods spend by the cells followed by anaphase. Metaphase and telophase are the least timeconsuming by the cells. This is because, during the prophase, the chromosomes coil and condense , the centrioles begin to separate and start to form the mitotic spindle. Hence, these processes take a longer time compared to the other stages in the mitotic (M) phase.
Table 3 shows sizes of cell in each different cell stage. The sizes of the cells are increasing from the interphase to the telophase. The sizes of the cells that are undergoing interphase are the smallest compare to the other stages which is 0..025 μm. This is because during this stage, the chromosomes in the cells are uncoiled and are not divided. Since there is no prophase stage cell in viewed field, thus the size of the cell is cannot be justified. But, the overall study suggest that the prophase prophase stage has the second largest size of cell and followed by metaphase , anaphase anaphase and telophase. telophase. The activities that happen happen inside of the cells during different stages results in the size of the onion root tip cell. The sizes of the cells are calculated by comparing the scale on the eyepiece graticule and the scale on the micrometer slide.
FURTHER STUDY Other than using the onion root tips, the same experiment can also be carried out by using different type of cells such as garlic cells o r cheek cells. Beside using toluidine blue stain, orcein ethanoic stain can also be used as the other choice of stain t o conduct the experiment. In some preparation, only few dividing cell is observed. This is because maybe a wrong section is cut from the root which is not the root tip thus actively dividing apical meristem of the root is not being viewed. There is also the possibility that the root used no longer have dividing cell due to X- ray or heavy metal emanation. As stated earlier in the introduction, cellulose walls of the plant cells are held together by cement called middle lamella. Treatment with hydrochloric acid breaks this wall and this is essential as it allows cells to separate and produce one cell thick preparation, making viewing of individual cells possible. The result is reliable if repeated measurement which almost have similar values. Besides, the length of the cell is measured three times to obtain a mean value. Next, the eyepiece graticule is used with full care and it has been correctly calibrated using stage micrometer and under supervision of lecturer..
EVALUATION
Limitation and improvement Several limitation are found in this experiment. Onion root tips are very fragile and easily damage. The presence of xylem in the onion root tip makes the maceration more difficult. Thus, to prevent misleading results, the end of root tips were not cut. The onion root tips should be handle with extra care. The onion root tips were prepared earlier by the laboratory assistant and placed in holding solution (70% ethanol). Cutting the wrong end of onion root tip will give arise to inaccurate results, thus it was not carried out . Since the prepared specimen are very thin ( one cell layer thick) , it enable us to be clearly observed under the microscope. But extra care have to be taken as the cover slip is pressed. It should be pressed gently co that the pressure will spread the cells into single layer. Lateral movement were avoided and only one direction is used to give pressure to make sure that the samples were not damaged and prevent them from giving overlapping result. Another limitation is the time constriction. In order to obtain percentage of cells in different stages, one have to calculate the number of cells in different region of onion root tip. However, it is not possible to be carried out as we only have limited time. Thus, eash student manage to get only one field of view only. To overcome this limitation, the observation is shared and observed to get better idea on mitotic process.
Validity and reability Since , looking at only one slide of onion root tip is not enough to get accurate result as different region of onion root tips have different rate of dividing or mitotic phase. Thus, every student prepare onion root tip each and the best result among those specimen were chose. This give rise to accurate reading as a lot of sample specimen is used. This ensures the validity of the result. Extra care was taken when lowering the glass slide co that no ai r bubbles will trap inside the slide. Presence of air bubble inside the slide will inhibit observation process under microscope. In order to prevent this phenomenon, excess toluidine blue stain solution was removed by using tissue paper. This guarantee the validity and reability of data.
SAFETY PRECAUTION In order to avoid any accident or injury during the experiment in laboratory, the precautionary steps should be taken and applied. Wearing lab coat and a pair of suitable shoes are compulsory when conducting an experiment in the lab at all times to protect the skin and clothing from chemical substance. This is to ensure that no chemical solutions such as toluidine blue solution is spilled to our skin and clothing as it will stain badly. Furthermore, the glassware such as beakers and boiling tubes should be handled with full care because they are fragile. Not only that, sharp objects such as forceps must be used properly to prevent any contamination and injuries. Avoid consuming consuming any solution that used in this experiment because because they might be contaminated. Clean and dry microscope slides , cover the microscope and place the apparatus back in the places after finishing the experiment to prevent any accident and to maintain the good state of the apparatus. CONCLUSION A dividing cell undergo mitosis which contain 4 phases namely prophase, metaphase, anaphase and telophase. The higher the percentage of dividing cells in one phase, the longer in time the cells spend on the phase. The hypothesis is accepted.
REFERENCES 1. http://www.google.com.my/search http://www.google.com.my/search?q=mitosis&hl=en& ?q=mitosis&hl=en&safe=off&biw=1366 safe=off&biw=1366&bih=681&pr &bih=681&pr md=imvns&tbm=isch&tbo=u&sourc md=imvns&tbm=isch&tbo=u&source=univ&sa=X&ei=jrSWTubiAs6 e=univ&sa=X&ei=jrSWTubiAs6mrAedmdjuAw& mrAedmdjuAw&ved= ved= 0CFEQsAQ 2. http://en.wikipedia.org/wiki/Mitosis 3. http://www.docstoc.com/ http://www.docstoc.com/docs/3665608/Mitosis-in-Oniondocs/3665608/Mitosis-in-Onion-Root-Tips Root-Tips 4. Edexcel AS Biology – Practical 3.1- Ann Fullick, Pearson Company, 2008 5. http://en.wikipedia.org/wiki/Tolonium_chloride
