ABSTRACT The chromosomes are the DNA molecule that packaged into thread-like structures.

The Feulgen reaction method and squash preparation was used to staining the chromosomes. This experiment was conducted to observe the morphology of the chromosome in the root tip and to study the pattern of chromosomes in different stages of the cell cycle. This experiment is also to carry out the Feulgen reaction of root tips of onion in order to study chromosomes morphology. The experiment was performed by using the root tips of onion. The root tips in the ethanol were transferred to the distilled water. Then, one of the root tips was transferred into the test tube that contains the 5% TCA (trichloroacetic acid), in the fume hood. After several minutes, the TCA treated root and the remaining non treated roots was transferred to the hot HCL in the separated beaker for several minutes at 60C and then all the roots were cooled by slowly adding distilled water. The liquid was being decanted. The TCA treated and untreated roots were transferred to the separated test tubes that have been cover with aluminium foil and then the Schiffs reagent was added. After half an hour, the Schiffs reagent was decanted and the bisulfate bleaching solution was added and then being decanted after 2 minutes. The bleaching procedure was repeated for two times and then then distilled water was added. Then, the squash preparation of the root tips was started by placing a drop of acetic acid on the glass slide and then the broad end of TCA treated root tips was cut and placed on the acetic acid. After it was covered with the cover slip, the root was squashed by using the application of the thumb pressure. The squashing step was repeated for untreated root tips. All the prepared slides were observed under light microscope using 1000x magnification. The results obtained are the root tip that is treated with TCA has no visible chromosome structure while the untreated root tips have visible chromosome structure at different stages of mitosis. As the conclusion, different stages of mitosis can be observed in the root tips of onion that are untreated through the Feulgen reaction.

INTRODUCTION In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called chromosomes. The chromosomes are one of the largest structures in the cell so they are the first component to be studied with the light microscope. Chromosomes were so named by W.Waldeyer, 1890 because of their tendency to stain intensely with many dyes (chromo = color; soma = body). They can also be identified with cytochemical reactions that are specific for DNA. Each chromosome is made up of DNA tightly coiled many times around proteins called histones that support its structure. Chromosomes are not visible in the cells nucleus not even under a microscope when the cell is not dividing. However, the DNA that makes up chromosomes becomes more tightly packed during cell division and is then visible under a microscope. Each chromosome has a constriction point called the centromere, which divides the chromosome into two sections. The location of the centromere on each chromosome gives the chromosome its characteristic shape, and can be used to help describe the location of specific genes. The cell cycle is the series of events that take place in a cell leading to its division and duplication or replication. The cell cycle divided into three major phase which are interphase, mitosis, and cytokinesis. It is during interphase that cell growth and DNA synthesis occur, the later during the S period. There are two period of interphase which are G1, where precedes DNA synthesis and G2 which follow G1. In Vicia faba, the total duration of cell cycle is 19.3 hours, which mitosis occupying 2.0 hours (Evans and Schott, 1964). Since mitosis last approximately 10% of the duration of the cell cycle, approximately 10% of the meristemic cells are in mitosis. The chromosomes of V.faba are very large and the diploid number are relatively low (2n=12), thus the details of the chromosome morphology can be readily observed. In the interphase nucleus, the chromosomal material, or chromatin, is dispersed as a rather homogenous mass of fibers. There are usually several small clumps of condensed, deeply stained heterochromatin, and there is at least one nucleolus. Nucleoli are rich in RNA and protein, as they are the structures in which the ribosomal subunits are assembled. Each nucleolus appears as large, round or oval structure within the nucleus. As interphase cells proceed into mitosis, the nucleolar material disperses and the chromatin condenses into compact structures, the chromosomes. The most useful stages for the study of chromosome morphology are late prophase and early metaphase.

The Feulgen reaction proposed by R. Feulgen and H. Rossenbeck in 1924 is one of the cytohistochemical reactions most widely used in biology and medicine. It is highly specific for DNA and also permit the DNA content of individual cells to be quantified my microspectrophotometry. This technique is used to measure the intensity of the dye that binds stoichiometrically to some class of molecule in the cell. With the Feulgen reaction, the DNA content of the cell can be quantified. Such measurement permits the relative amounts of DNA to be determined at different stages of mitosis and meiosis. Besides that, Feulgen reaction allows DNA in situ to be specifically stained based on the reaction of Schiff or Schiff-like reagents with aldehyde groups engendered in the deoxyribose molecules by HCl hydrolysis. The staining intensity is proportional to the DNA concentration. After the Feulgen reaction, the cell needs to be squash. The squash preparation is invaluable for the study of both plant and animal chromosomes. Unlike sectioned material, in which only a part of cell may be present, each cell in squash preparation contains all the chromosomes. The squashing also flattens the cells so that the chromosomes are equally dispersed and are often in the same focal plane. In this preparation, the cells are placed in the drop of the 45% acetic acid on a slide, and after that the tissue is broken up by tapping it with the end of the glass rod. The acetic acid provides a medium viscosity for dispersing cell. After the cover slip is placed on the preparation, the cells are squashed by application of thumb pressure on the cover slip.

OBJECTIVES 1. To observe the morphology of the chromosomes in the root tips of onion. 2. To study the pattern of chromosomes in the different stages of cell cycle. 3. To carry out the Feulgen reaction on the root tips of onion.

APPARATUS AND MATERIALS Forceps Scapels Test tubes Root tips 70% ethanol 5% TCA (trichloroacetic acid) Distilled water Hydrochloric acid Schiffs reagent Bisulfate bleaching solution Parafilm Aluminium foil Acetic acid Paper towel Glass slide Cover slips Microscope

PROCEDURES Feulgen reaction 1. One root tip was transferred from 70% ethanol to a test tube of distilled water for a few minutes by using the forceps. The root was grasp at the broad end. 2. The root tip was transferred from the distilled water to the test tube of 5% TCA at 90C in fume hood, and was treated for 15 minutes. 3. The TCA treated root tip was transfer by using the forceps to a test tube of distilled water, in which it should be remain until all root tips are ready for the HCL treatment. 4. The 70% alcohol was decanted from the test tube containing the remaining or the untreated of the root tips and the distilled water was added. After several minutes, the untreated root tips were transferred to test tube of 1N HCl at 60C. The TCA treated root tip also was transferred to a separated test tube of 1N HCL at 60C. 5. After the root tips have been in the hot HCL for 10 minutes, the acid in each test tube was diluted and cooled by slowly adding the distilled water. 6. The liquid was decanted and then about 10ml of Schiffs reagent was added to each test tube in fume hood. Each test tube was covered with parafilm and then with the aluminium foil to prevent light from entering. The root tips were treated with Schiffs reagent for 30 minutes. 7. In the fume hood, the Schiffs reagent was carefully decanted and 10 ml of freshly prepared bisulfate bleaching solution was added to each test tubes. After 2 minutes, it was decanted and these bleaching procedures were repeated for two more times. 8. The root tips were rinsed with several changes of distilled water. The root tips was kept in the distilled water until the squashing is made. Squashing preparation 1. An alcohol-cleaned slide was label with pencil and a drop of 45% acetic acid was placed at the center of glass slide. 2. By the broad end, the root tips was picked up by using the forceps and the root tip was gently rub by using the paper towel to removed root cap. The root tip was immediately placed in the drop of the acetic acid.

3. With a scapel, all parts of root tip was removed except or the terminal around 1.5 mm, which contain the dividing cells. The tissue was allowed to remain in the acetic acid for about 1 minute. 4. The root tissue was tapped with the flat end of a glass rod until tissue is thoroughly dispersed in the drop of acetic acid. Then, the cover slip was lowered on the preparation 5. Excess liquids were removed before the squashing. The paper towel was placed on the slide, at one end of the slide the towel was hold and then the index finger of the hands was moved across the slide, it was lightly pressed. 6. With a paper towel held in one place on the slide, the preparation was squashed with the thumb. A rolling motion was used to ensure the liquid is pressed to the edge of the cover slip where the towel can absorbs it. Thumb pressure being increased in successive round of squashing instead of being applied all at once. After applying the maximum pressure with the thumb, a final round of squashing with the eraser end of the pencil was carried out without moved the cover slip. 7. The steps were repeated for the TCA treated root tip. All of the prepared slides were observed under the light microscope by using the 1000x magnification power. Precautions Rinse the forceps with water after transfer of root tips from any caustic solution. Perform experiment in fume hood when using the TCA because it is a strong acid, and Schiffs reagent is dangerous that may causes burns. Always worn protective gloves when handling the entire chemical.

RESULT AND OBSERVATION Root tips TCA treated root tip Observation No visible chromosomes and mitosis stages in the cell. Untreated root tip There are visible chromosomes in the cell. The pattern of chromosomes in prophase, anaphase and telophase stages of mitosis can be observe.

PROPHASE

ANAPHASE

TELOPHASE

UNTREATED ROOT TIP

TCA TREATED ROOT TIP

DISCUSSION DNA is a nucleic acid that contains the genetic instructions used in development and functioning of all living organisms. While a chromosome is an organized structure of DNA and protein found in cells. Cell cycle on the other hand, is the series of events that take place in a cell leading to its division and duplication. Cell cycle can be divided in two stages. Interphase is the phase during which the cell grows, accumulating nutrients needed for mitosis and duplicating its DNA and the mitosis phase is the phase which the cell splits itself into two distinct cells, often called daughter cells and the final phase, cytokinesis, where the new cell is completely divided. Chromosomes are only visible when the cell is dividing. They can be made more visible using appropriate staining techniques. The chromosome pattern was found to be different in each phases of cell cycle. This experiment was done to observe the different pattern of chromosomes in each phase of cell cycle and focusing more on mitosis stage of plant cells that is onion cells. Onion root tips are used to be observed because the cells at the tip of the roots are actively dividing, and thus many cells will be in stages of mitosis. Furthermore, the tips can be prepared in a way that allows them to be flattened on microscopes slide that is to be squashed so that the chromosomes of individual cells can be observed. Not only that, the chromosomes of onion cell is also found out to be slightly larger compared to other plant and very dark when stained thus making them more easily observable. The root tip of onion was soaked into 70% ethanol to kill microbes and it is one of the aseptic techniques. 70% alcohol used instead of pure alcohol because pure alcohol coagulates protein in contact. 70% ethanol, a diluted alcohol, also coagulates the protein, but at slower rate, so it penetrates all the way through cell before coagulation can block it. The cell is then coagulated and then the organism or microbes dies. Some of the root tip is soaked in TCA, trichloroacetic acid, as a treated control slide. The TCA is used because it extracts all cellular nucleic acid, both DNA and RNA, thus induce chromosomal damage. The root tip is soaked in HCl so that HCl hydrolysis can occur to removes the purine from DNA, thus exposing aldehyde group at the 1 position of the purinated deoxyriboses. The

tissue is then treated with Schiffs reagent that reacts with the aldehydes on the depurinated DNA to form a reddish purple product. Each molecule of Schiffs reagent combined with two aldehyde group. So, since all cellular RNA is removed by HCl hydrolysis, the RNA will not be stain by Feulgen reaction. The application of TCA (trichloroacetic acid) to precipitate proteins at different temperature is to differentiate the time for the protein to clump. Disadvantage of using cold TCA could be that precipitation efficiency is lower at lower temperature because slower Brownian motion of protein molecules means less chance of collision between these molecules and thus slower formation of protein clump. So if using cold TCA, one has to wait for a longer time. The tissue is rinsed with a bisulfate bleaching solution after being treated with Schiffs reagent. This bleaching ensures that the purple coloration is the result of Schiffs reagent having reacted with aldehydes rather than having reverted to fuchsin during staining procedure. The squashing, that is done after the tissue being rinsed was done since each cell in a squash preparation contains all the chromosomes. It also done to flatten the cells so that the chromosomes are equally disperses and is often in the same focal length. Based on the observation, only prophase, anaphase and telophase can be observed. At prophase stage, the chromosomes start to condense. It shows by dark regions in the single cells. At anaphase stage, the chromosomes pairs divide and the sister chromatids migrate to opposite poles of the cell. At telophase stage, the chromosomes at either end of the cell begin to cluster together. The metaphase stage cannot be observed. This maybe occurs because there are too many overlapping chromosomes including centromere regions, so it is impossible to identify centromere positions. The tissue that has been treated shows no chromosome since TCA extracts all cellular nucleic acid, both DNA and RNA. There are some safety precautions that need to be followed when conducting the experiment. The scalpel should be used carefully when cutting the root tip of the onion. Schiffs reagent should be handled with care. It is a strong acid. So, one should not inhale it or contact it with bare hands because it will permanently stain anything it contacts. The Hydrochloric Acid

(HCl) and acetic acid should also be handled with care because it is corrosive and can cause irritation when it came in contact with our skin. Do not inhale or contact it with bare hands. The process of transferring the root tip from the hydrochloric acid must be done using forceps and not using bare hands. . The fragile apparatus such as microscope slide and cover slips should be handled with care. If any of the apparatus broken, report immediately to the staff in charge for the lab. After the experiment was done, ensure all bottle of reagent have been closed tightly. All waste beaker contents should be discarded into the designated container in the lab and glass vials should be emptied and rinsed. The microscopes should be placed where it first is taken according to instructor.

Questions 1) View the prepared slides under the microscope. What are the differences? The difference is the pattern of the chromosomes. During prophase stage, the chromosomes start to condense. It shows by dark regions in the single cells. At anaphase stage, the chromosomes pairs divide and the sister chromatids migrate to opposite poles of the cell. At telophase stage, the chromosomes at either end of the cell begin to cluster together. 2) Can you see the metaphase stage? No. the metaphase stage cannot be seen. This maybe occurs because there are too many overlapping chromosomes including centromere regions, so it is impossible to identify centromere positions.

CONCLUSION As a conclusion, mitotic stage can be observed in the meristemic tissues found in the apical meristem of actively growing roots of plants such as onion. The chromosomes of a cell can be observed under light microscope, and can be made more visible using appropriate staining techniques. Not only that, the chromosome pattern was found to be different in each phases of cell cycle. During prophase stage, the chromosomes start to condense. It shows by dark regions in the single cells. At anaphase stage, the chromosomes pairs divide and the sister chromatids migrate to opposite poles of the cell. At telophase stage, the chromosomes at either end of the cell begin to cluster together.

REFERENCES 1) Edexcel AS Biology , Ann Fullick, Pearson Company, 2008 2) http://staff.jccc.net/pdecell/celldivision/oniontip.html ( 23 May 2012) 3) http://www.csus.edu/indiv/L/LanderholmT/documents/bio02/Lab%209%20Mitosis.pdf (23 May 2012) 4) http://www.wisc-online.com/objects/ViewObject.aspx?ID=ap13604 (23 May 2012) 5) http://www.ncbi.nlm.nih.gov/pubmed/10403113 (23 May 2012)

6) http://ghr.nlm.nih.gov/chromosomes
7) http://en.wikipedia.org/wiki/Chromosome ( 22 May 2012) 8) http://www.microscopy-uk.org.uk/mag/artnov04macro/jronionroot.html ( 20 May 2012) 9) http://employees.csbsju.edu/mcampos/bio114/labmaterials/Lab%207%20mitosis.pdf (23 May 2012)