Title: Observing mitosis in garlic roots Objectives:

1. To develop knowledge and understandings of mitosis by observing the

stages of cell cycle and estimating the duration of each phase in mitosis in cell cycle.
2. To develop problem solving and experimental skills; for example,

information is accurately processed, using calculations where appropriate, experimental procedures are planned, designed and evaluated properly, the use of microscopes, producing valid results and recording results. 3. To develop the techniques of preparing some slides for observing mitosis of actively dividing plant tissue.

Hypothesis:
Mitosis consists of prophase, metaphase, anaphase, and telophase. Mitosis occurs most frequently in apical meristems of plant cells. Anaphase is the shortest phase in mitosis while metaphase made up the longest phase in mitosis. Mitosis only accounts for a small duration in cell cycle.

Variables:
Manipulated variable: Invalid for this experiment Responding variable: Invalid for this experiment Fixed variable: The type of garlic roots, the region where the root tip has taken from, amount of stain used, temperature of the water bath, time taken for the for the root tip to stay in water bath and distilled water

Introduction:
Mitosis Mitosis consists of prophase, metaphase, anaphase, telophase, and cytokinesis. Plants cells do not involve spindle fibres in mitosis. All the phases have to work correctly in order for cell divisions to take place effectively in an error free condition.

Source: http://www.uhh.hawaii.edu/~ronald/392/Mitosis.JPG

Importance of mitosis Mitosis is important in growth and replacing old and worn out cells in an organism. Single celled bacteria can reproduce by asexual reproduction through binary fission, which is a form of mitosis. Mitosis is important for the growth of zygote after fertilization. Furthermore, in unicellular organisms, mitosis can make daughter cells inherit all the genetic content of the parent nuclei. This method of reproduction is suitable for an organism living in a stable environment. If mitosis mechanism does not functioning properly, mutation can occur which may be harmful to the organism. Garlic Allium sativum L., is known as garlic, is a species in the onion family Alliaceae. Garlic has been for both culinary and medicinal purposes. It is widely used as seasoning in cooking due to its pungent flavour. Garlic is easy to grow and can be grown all year round in mild climates. Meristem A meristem is an undifferentiated tissue that is actively dividing and found in zones of plant that growth is rapidly taken place. Cell divisions in meristems provide new cells for expansion and differentiation of new tissues and organs, which is vital for the functioning of the plant. Meristematic cells are packed closely together without intercellular cavities and they have thin primary cell wall. Stains and other chemicals Orcein ethanoic stain (aceto ethanoic stain) is a stain used for chromosomes in air-dried or squashed cell materials. According to MedicineWord, (2009), Toluidine blue solution is a stain in borax solution, used with heat on semi thick sections of epoxy embedded tissues. Hydrochloric acid is an acidic, aqueous solution of hydrogen chloride which is highly corrosive when concentrated and normally used as a laboratory agent. It is also found in gastric juices.

Apparatus and materials:

Garlic roots, scalpel or sharp knife, 1 M hydrochloric acid, toluidine blue solution, Cold distilled water, 2 watch glasses or small sample tubes, Hollow glass block or small sample tube, pipetters(and pipette fillers) or small measuring cylinders, microscope slides and coverslips, pair of fine forceps, mounted needle, filter paper or soft tissue paper, microscope with magnifications of X100 and X400, eye protection, immersion oil, gloves, safety goggles, hot plate, water bath at 60 C

Procedure:
Method 1: Using toluidine blue stain 1. 1-2 cm form several root tips of some growing garlic roots are cut off. Tips which are white and have a firm rounded end should be chosen. Brown tips will give poor results.
2. The root tips are put into a hollow glass block or small sample tube

containing 2 cm3 of 1M hydrochloric acid for exactly 5 minutes.

3. The root tips are transferred to a watch glass containing approximately

5 cm3 of cold water. The root tips are left for 4-5 minutes. They are then dried on filter paper. The root tips should be handled gently as they will be very fragile.
4. One of the root tips is transferred to a clean microscope slide. About 4-

5mm of the growing root tip is cut. The rounded tip should be kept and the rest should be discarded. The meristem tip is usually a denser white and more rounded at the cut end. Maceration will be more difficult if you take the wrong end. 5. The root tip is broken up gently with a mounted needle. In other words, this is maceration. One small drop of toluidine blue is added and left for 2 minutes.
6. The root tip is covered with a cover slip. It is then blotted firmly with

several layers of tissue or filter paper. The root tip is pressed gently to spread out. Alternatively, the coverslip can be tapped gently with the end of a pencil. 7. The root tip is viewed under the microscope by increasing the magnification gradually from 40X to 400X magnification. The cells are located first before the nucleus and chromosomes are located. If cells are overlapping, the slide should be squash again between two wads of filter paper. 8. Regularly shaped, actively divising cells are the cells should be looked for. DNA stains dark blue with toluidine blue stain so you should be able to see blue groups of chromosomes against a paler background.
9. If your preparation is not very successful, other root tips should be

repeated from step 3. The procedure should be adjusted to remedy the problem; for example, the time for the cells left in the stain should be adjusted if your cells are over- or under stained.

10. The number of cells in each phase is counted. Mitotic index is calculated using the formula below:

Method 2: Using orcein ethanoic stain

1. A test tube containing 2 cm3 of 1M hydrochloric acid into is inserted into

a water bath at 60 C. 2. 5mm of root tips are cut off from some growing garlic roots. Root tips which are white and have a firm rounded end are chosen; tips that are brown will give poor results. 3. 20-30 drops of orcein ethanoic stain and 3 drops of preheated 1M HCI is added to each cut root tip. 4. The stained root tips are heated gently for 3-5 minutes using a hot plate. 5. One of the root tips is transferred to a clean microscope slide. About 45mm of the growing root tip is cut. The rounded tip should be kept and the rest should be discarded. The meristem tip is usually a denser white and more rounded at the cut end. Maceration will be more difficult if you take the wrong end.
6. The root tip is broken up gently with a mounted needle. In other words,

this is maceration. Additional orcein ethanoic stain is added and left for 2 minutes.
7. The root tip is covered with a cover slip. It is then blotted firmly with

several layers of tissue or filter paper. The root tip is pressed gently to spread out. Alternatively, the coverslip can be tapped gently with the end of a pencil. 8. The root tip is viewed under the microscope by increasing the magnification gradually from 40X to 400X magnification. The cells are located first before the nucleus and chromosomes are located. If cells are overlapping, the slide should be squash again between two wads of filter paper.
9. Regularly shaped, actively divising cells are the cells should be looked

for. DNA stains dark red with orcein ethanoic stain so you should be

able to see red groups of chromosomes against a paler pink background. 10. If your preparation is not very successful, other root tips should be repeated from step 3. The procedure should be adjusted to remedy the problem; for example, the time for the cells left in the stain should be adjusted if your cells are over- or under stained.
11. Number of cells in each phase is counted. Mitotic index is calculated

using the formula below:

Risk assessment:
1. Avoid the lateral movement of coverslip during the squashing of the cells to prevent the cell contents from damaged or the cells from stacking together. Wear gloves and eye protection to avoid contact with skin as orcein ethanoic is corrosive, causes severe burns, has an irritating vapour and stains. If contact of orcein ethanoic stain does occur, wash the area thoroughly with water for 10 minutes. Mop up the spillage immediately. Wear gloves and eye protection. Toluidine blue will stain skin and clothes. If you are using a microscope with separate illumination, make sure that direct sunlight is not reflected through the optics to prevent dazzling when viewing the specimen. Handle the root tips gently as they are very fragile.

2.

3.
4. 5.

6.

Results:
Phase Prophase Picture with labels Description Chromatins are condensed and visible. Chromosomes are spread throughout the cytoplasm so we can infer that nuclear membrane has broken down.

Metaphas e

Chromomosomes line up on the equator.

Anaphase

Chromatids are sperated from each other and moved towards opposite poles.

Method 1: Using toluidine blue stain Phases in cell cycle Number of cells in viewing field 1 40 12 10 1 0 Number of cells in viewing field 2 51 19 9 1 0 Number of cells in viewing field 3 46 15 10 1 0 Average number of cells 46 15 10 1 0

Interphase Prophase Metaphase Anaphase Telophase

Method 2: Using orcein ethanoic stain

Phases in cell cycle

Number of cells in viewing field 1 53 14 9 1 0

Number of cells in viewing field 2 48 16 10 1 0

Number of cells in viewing field 3 50 15 10 1 0

Average number of cells 50 15 10 1 0

Interphase Prophase Metaphase Anaphase Telophase

Discussion:
Using of garlic root tips Garlic root tips are used in this experiment because it is easy to obtain. In addition to that, the chromosomes are large and easy to stain. The colour of the chromosome after staining is easily distinguishable from its surroundings. We use root tips in our investigation because root tips contain apical meristemetic cells which are not differentiated yet and are rapidly dividing. Therefore, probability of finding a cell dividing in the zone of cell division is high. Thus, it is suitable for observing mitosis. Besides, each garlic root cells has only 8 chromosomes so it is relatively easy to see the chromosomes once they have condensed. Controlling of variables We counted the number of cells because the number of cells undergoes a certain phase in a random observation field is roughly proportional to the duration. This is because the longer the cell spends its time in a certain stage, the higher the chance of finding the cell at a specific time. The amount of stains used can be controlled by using a dropper. The

same garlic roots are used for the whole experiment to eliminate this factor from affecting the rate of mitosis. White, rounded region of the root is cut to give the best result. The stages of mitosis are drawn to show the distribution of chromosomes in each phase of the cell cycle. This also shows what happens to cells in each stage of mitosis. Temperature of the water bath can be controlled by using a heater. The time taken for a root tip to immerse in stains can be monitored by using a stop watch. Explanation of experimental methods Using toluidine blue stain is simpler, cheaper, safe, reliable and quicker compared to acetic ethanoic stain. When cutting root tips from the garlic roots, white, firm, rounded root tips are chosen because the root tips are newly grown which are actively dividing. The root tips are immersed in 1M hydrochloric acid to break down the pectins and the matrix of calcium pectate that hold the cells together while leaving the cell wall unaffected. This is because plant cells are hold together by middle lamella of pectins. This will make the squashing of the cells and maceration in the later of the experiment easier. Moreover, hydrochloric acid can fix the cell contents in position. The root tips are immersed in cold water to clean excess hydrochloric acid from the root tips. 4-5 mm of root tips are cut for the easy handling of the root tip and maceration. The root tip is macerated to ensure a single layer of root tip is obtained. Toluidine blue solution is added to stain the chromosomes. The root tip is blotted with a few layers of tissue papers to absorb excess toluidine blue solution. The root tips are squashed gently to spread out the cells. This step is important to give a single layer of cells for observations to be done under the microscope. The slide is viewed under low power first to locate the group of cells with visible nucleus stained blue before focusing specifically on the dividing nucleus. For experimental method 2, the root tips are heated after adding the stains to maximize the absorption of the stains by the chromosomes. Improvement of experimental methods Maceration can be improved by the help of a magnification glass. This will make further maceration of the root tip easier. A pen or pencil is preferably used for squashing the cells because your thumb may be shaking when you tried to squash the cells gently. This will indirectly causes lateral movement of cells. Immersion oil is needed if the objective lens of your microscope has to touch the slide in order to gain a clear view of the chromosomes. This may happen when you opt for 100 X and 400 X magnifications. The immersion enable the objective lens to slide across the slide without scratching it while do not alter the viewing of the chromosomes when using the microscope. Fine adjustment knob should be used to prevent the cracking of the microscope slide when using high powered lenses. If you want to prevent the slides from drying out, mount them in 50% glycerol.

Squashing of garlic cells can be delayed for several hours. This allows the cells to harden, reducing the danger of bursting when the slide is squashed. Avoid placing too much stains or materials on the slide to enable undisturbed view of the chromosomes. A photograph may be taken to make the counting of the cells easier and more accurate. For experimental method 2, the acetic ethanoic stain may dry up during heating. More stains should be added to the root tip to prevent the root tips from overheating and damage of cell contents. Explanation of process of mitosis Prophase is where the chromatin in the nucleus begins to condense and becomes visible in the light microscope as chromosomes. The nucleolus disappears. Golgi apparatus and endoplasmic reticulum disintegrates. Some spindle fibres cross the cell to form the mitotic spindle. Metaphase is a short period during which the chromosomes are lined up along the metaphase plate of the cell. Anaphase is the centromeres divide. Sister chromatids separate and move toward the corresponding poles. Spindle fibres contract to pull the chromatids towards the opposite ends of the cell. Telophase is the final stage of mitosis, chromatids arrive at opposite poles of cell, and new nuclear membrane forms around the daughter nuclei. Golgi apparatus and endoplasmic reticulum reforms. The chromosomes disperse and are no longer visible under the light microscope. The spindle fibers disperse, and cytokinesis or the division of the cell may also begin during this stage. Interphase is not considered a part of mitosis because genetic movement does not occur at this stage. Organelles such as nucleolus, spindle fibres, golgi apparatus, centrioles, chromatin, endoplasmic reticulum are only visible if an electron microscope is used.

Evaluation on procedures and results This experiment is fair because the experiment is repeated 3 times to minimize the errors in the experiment. This experiment also takes into account the different types of staining methods. Different stains are used for the experiment to ensure that the investigation is comprehensive. The usage of different stains may affect the results of the experiment because chromosome may react differently to each stain by absorbing different amount of stains. The amount of stains absorbed may affect the visibility of

the condensed chromosomes. Insufficient acid treatment results in easy handling of the root tips but difficult maceration; too severe acid treatment results in difficult handling but easy maceration. There are some anomalies in the number of cells observed because most of the cells are in prophase stage where chromosomes are spread throughout the cytoplasm. We have repeated 6 slides of root tips. From the results, we have prophase as the longest phase and telophase as the shortest phase but in fact metaphase is the longest phase and anaphase is the shortest phase in mitosis. After some discussion, our group think that the there may be some problems in staining the chromosomes. Over stained nucleus may results unclear patterns of chromosomes. We may need to improve our techniques of maceration so that a layer of root tips cells can be obtained for easy observations to be made. Stacking of nucleus can give wrong pictures on the stages of mitosis. Furthermore, we need to cut the zone of cell division of the root to get an accurate result. We unable to observe telophase in this experiment probably because daughter cells enter interphase immediately after the telophase at the instant we were preparing for the slides. There is a difference between two mitotic indices because toluidine blue stains are better absorbed by chromosomes compared to acetic orcein solution.

Conclusion:
From the results obtained, telophase is the shortest phase in mitosis whereas prophase is the longest phase in mitosis. Interphase accounts for a large part in cell cycle while mitosis only accounts for a small part in cell cycle. This is because we found more non-dividing cells than total number of cells undergoes mitosis. About 48 cells without visible chromosomes compared to 26 cells containing visible chromosomes in average. The result is in fact incorrect because anaphase is the shortest phase while metaphase is the longest phase. Mitotic index is the ratio of cells undergoes mitosis to total number of cells in a cell cycle. Mitotic index for toluidine blue solution is 0.3611 compared to 0.3421 for second method of staining. Overall, mitosis made up 35.11% of cell cycle. The hypothesis is partly accepted by this experiment that is mitosis accounts for a small part in cell cycle.

Reference:

Fullick, A. 2008. Edexcel AS Biology. 272p. Essex: Pearson Education Limited Medicine word. 2009. Alkaline Toluidine Blue O -Medicine Word Explain Alkaline Toluidine Blue O. Available from: http://www.medicineword.com/alkaline+toluidine+blue+O.shtml Accessed on: 6 February 2009 Morgan, W.R. 1997. Mitosis and Meiosis Lab Instructions: Observing Mitosis and
Cytokinesis in Plant Cells. Available form:

http://www.wooster.edu/biology/wmorgan/bio306/Observing_Plant_Mito sis.html Accessed on: 5 February 2009 Plant and Science for Schools. 2009. Staining a Root Tip and Calculating Its Mitotic Index. Available from: http://wwwsaps.plantsci.cam.ac.uk/worksheets/scotland/mitosis.htm Accessed on: 6 February 2009 Shannah.S. 2008. The Cell Cycle. Available from: http://www.uhh.hawaii.edu/~ronald/392/Mitosis.JPG Accessed on: 6 February 2009 Wikipedia. 2009. Mitosis. Available from: http://en.wikipedia.org/wiki/Mitosis Accessed on: 5 February 2009 Wikipedia. 2009. Mitosis. Available from: http://en.wikipedia.org/wiki/Garlic Accessed on: 6 February 2009

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