onion root tip mitosis experiment results

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Onion Root Tip Mitosis Stages, Experiment and Results

Mitosis refers to a type of cell division (cell cycle) through which the cell (parent cell) produces two identical daughter cells. Unlike meiosis, which is also a type of cell division , mitosis results in the production of two diploid daughter cells. The two daughter cells contain the same number of chromosomes as the parent cell.

Given that the process results in the proliferation of cells, it's important for general growth and replacement of damaged cells (e.g. the wound healing process).

In general, mitosis occurs through several stages that include:

• Prophase (divided into prophase and prometaphase)

Because of the rapid rate at which onion root tips grow as a result of rapid cell division, it's possible to observe and identify the different stages of mitosis.

For this section, the experiment will be divided into two main parts.

These include:

• Growing onion root tips
• Sample preparation 

Growing onion roots

*  Growing fresh root tips is recommended given that older or dried roots may produce poor results.

Requirements

• Uncut onions
• Clear glass or plastic jars
• Clean water
• Pour clean water into the clean (and clear) glass/plastic jars - about three-quarters full.
• Carefully place the onion bulbs in the glass/plastic jars so that only the lower surface of the onion comes in contact with the water.
• In a case where the onions are too small, they can be supported using toothpick/splints.
• Let the onions rest on water for 3-4 days - only the lower surface (base from which roots emerge) should be in contact with water.

The following are diagrammatic representations of this setup:

After 3 to 4 days, the onion roots will have grown to a length of about 1 inch.

Sample preparation

·       70 percent alcohol 

·       Aceto-alcohol 

·       Blade 

·       Forceps

·       Glass slides  

·       Water 

·       Glycerin 

·       Stain - Aceto-carmine or Aceto-orcein

·       Hydrochloric acid solution (1N HCL)

·       Onion with freshly grown roots

·       Microscope

·       Stop clock 

·       Water bath

·       Dissecting needles

*  Having a pair of gloves, safety goggles, and safety mask is recommended given that some of the chemical used are hazardous. 

·        Using a blade or scalpel, carefully cut one or several roots from the onion and place them in a petri dish (or any other small clean container)

·        Prepare a water bath (about 55 degrees C) - The temperature should be maintained at 55 degrees C. This may be achieved by using a thermostatically controlled water bath

·        Carefully pour the hydrochloric acid solution into a small bottle and place it in the water bath for about 15 minutes in order to warm the acid

·        Using a pair of forceps, carefully pick one or two of the roots and place them into the bottle of warm hydrochloric acid for about 5 minutes - This serves to break down pectin and calcium pectate as well as other tissues in order to release individual cells

·        Remove the roots from the bottle of acid using the forceps and rinse them in tap water several times and then place them in a clean petri dish 

·        Using a clean blade, sharp scissors or scalpel, cut off the tips of the roots (about 5mm in length)

·        Using the pair of forceps, pick the root tips, and put them into a vial of stain (acetic orcein or aceto-carmine) - Ensure that the root tips are completely immersed in the stain 

·        Place a cap/lid onto the vial (ensure that the cap/lid has a pinprick hole) and place the vial in the water bath (at 55 degrees C) for about 5 minutes - This enhances the staining process

·        Using the forceps, remove the root tips from the vial of stain and place them onto a clean microscope glass slide 

·        Using a dissecting needle, you can gently mash/squash the root tips to spread out the cells on the glass slide - This prevents several layers of cells from overlapping which would otherwise affect the quality of results

·        Cover the sample (root tip) with a coverslip and gently press the coverslip down, then examine the slide under the microscope starting with low magnification 

*  For this experiment, a properly prepared slide should appear light pink due to the stain to almost colorless.

*  Unused roots can be stored in 70 percent alcohol. 

An onion has a total of 8 pairs of chromosomes. This is especially beneficial for this experiment given that fewer chromosomes are slightly easier to see when they condense.

As mentioned, onion root tip cells divide rapidly as the roots elongate to absorb water and various minerals from the soil. 

For this reason, it's possible to identify different stages of cell division by mitosis based on chromosomal distribution.

When viewed under the microscope, a properly prepared slide will yield the following results: 

Under 10X magnification

Under 10X magnification, students will be able to observe several single layers of cells. Depending on how well the slide was prepared, the cells are spread out without any overlapping or with very little overlapping. 

*  Generally, a row of cells (single layer) may consist of between 2 and 5 cells.

The following is a diagrammatic representation of onion root tip cells under 10X magnification: 

Higher magnification (stages of mitosis)

Under high magnification (100X-500X), it becomes possible to identify cells at different stages of mitosis. 

Interphase  

While this is not necessarily one of the main stages of mitosis, cells in this state can easily be identified by their prominent nucleoli. 

Diagrammatic representation of an onion root tip cell during interphase:

During interphase, also known as the resting stage, the chromatin is not tightly packed. This allows for DNA to be copied (replication) as the cell prepares for cell division. 

Generally, interphase is divided into three main phases that include:

·      G1 (First gap phase)  - Characterized by cell growth and normal cellular activities

·      S phase (Synthesis)  - This is the phase in which DNA is replicated so that the DNA content is doubled

·      G2 phase (second gap phase)  - During this phase, the cell prepares for division 

During this stage of mitosis, the nucleolus is still visible. However, the nucleus appears grainy due to chromosomal condensation. 

The following is a diagrammatic representation of an onion root tip cell during prophase:

As mentioned, the DNA strands are uncoiled during interphase for transcription to occur. In this state, the DNA is said to be uncondensed. Here, this genetic material is also referred to as chromatin. During prophase, condensed DNA and some proteins form the chromatids which join to become chromosomes (X-shaped). 

Sister chromatids, which contain the same genetic information, are attached at a region known as the centromere which gives the structure an X shape.The kinetochore, which is the site at which microtubules join the chromosomes is also located at the centromere.

*  As the chromatins coil, it becomes increasingly compact which allows the chromosomes to become more visible when viewed under high magnification. 

*  It's also during prophase that spindle microtubules (mitotic spindle) start forming near the nucleus. 

Prometaphase  

Prometaphase is the second stage of mitosis (it has also been referred to as late prophase). This stage is characterized by increased condensation of chromosomes as well as the breakdown of the nuclear envelope (nuclear membrane). 

The nuclear envelope, which consists of an inner and outer membrane, is stabilized by polymerization of lamin proteins (the nuclear lamina). Polymerization of the lamins and the consequent breakdown of filaments into lamin dimers results in the disassembly of the nuclear membrane. 

Apart from increased condensation of the chromosomes and breakdown of the nuclear envelope, this stage is also characterized by the development of kinetochore around the centromere. Moreover, spindle fibers (kinetochore microtubules) have also been shown to start developing during prometaphase and permeate through the disappearing membrane to attach to the chromosomes at the kinetochore. 

*  This stage of mitosis is important because chromosomes (sister chromatids) have to be released from the nuclear membrane in order to be separated in the next stage. 

Metaphase  

In this stage of mitosis, chromosomes align along the equatorial plane of the cell (cell equator) so that the sister chromatids can be separated. During metaphase, chromosomes become more visible because of increased condensation as well as the fact that the nuclear envelope has disappeared. 

Diagrammatic representation of a cell during metaphase: 

Given that the nuclear membrane completely disappears during metaphase, the chromosomes appear in the  cytoplasm . As well, the fully developed spindle fibers originating from the  centrioles , located on opposite poles of the cell, attach to each of the sister chromatids which contribute to their alignment at the cell equator.  

Based on microscopic studies, spindle fibers (microtubules made of proteins) have been shown to be about 25nm in diameter. While they originate from the centrioles and extend to attach to the sister chromatids, they have also been shown to be constantly forming because they are continually broken down. 

As new components (building blocks) are added on one end of the microtubules and removed from the other, it has been suggested that this causes the microtubules to pull the centrioles which in turn contributes to the alignment of the chromosomes. 

*  In late metaphase, the pulling actions of the microtubules, as well as the centrioles, result in the kinetochores (the region at which spindle fibers attach to the chromosomes) facing different directions.

During anaphase, the chromosomes start separating and moving from the equatorial plate of the cell. At the end of this stage (late anaphase), the sister chromatids completely separate and reach the opposite poles of the cell.

In this stage, it's also possible to clearly identify the chromatids given that the process occurs in the cytoplasm.

Diagrammatic representation of a cell during late anaphase: 

Based on microscopic studies, sister chromatids have been shown to separate and move to the opposite poles of the cell at a rate of between 0.2 and 4 um per minute. Initially, polymerization and depolymerization of microtubules was thought to result in the separation and movement of chromatids to the opposite poles of the cell. 

As a result of the depolymerization process, microtubules shorten thus pulling apart the sister chromatids. As well, polymerization of some of the microtubules, those that extend from one pole of the cell to the other without attaching to the chromatids, causes them to grow in length thus pushing poles of the cell apart.

Based on recent studies, however, it has become evident that separation of sister chromatids during anaphase is the result of the actions of enzyme seperase as well as shortening of microtubules. 

Here, the enzyme seperase has been shown to break down cohesin (a component of centromere that links sister chromatids). 

Following the separation of sister chromatids, through the breakdown of the protein cohesin, shortening of microtubules (kinetochore microtubules/spindle) pulls the chromatids to the opposite poles of the cell. 

*  Some of the other factors suggested to contribute to the separation of sister chromatids include the pulling actions of astral microtubules (pulling the poles apart) while interpolar microtubules slide past each other.

Telophase is the fifth stage of mitosis characterized by several key events. These include arrival of the chromosomes at the opposite poles of the cell, gradual breakdown of the spindle fibers as well as development of nuclear envelopes around each set of chromosomes (at the opposite ends/poles of the cells). 

Diagrammatic representation of a cell during telophase (late telophase):

As nuclear envelopes develop around each set of chromosomes located at the opposite poles of the cell, two nuclei are formed in the cell. The DNA also starts to uncondense so that genetic material can be copied later.

Given that the spindle fibers are no longer required, they start to disassemble (break down) in early telophase and continue to do so in late telophase. 

Cytokinesis

Cytokinesis refers to the process through which the cytoplasm separates as the cell divides into two identical daughter cells. Unlike animal cells, plant cells have a rigid cell wall that prevents the cell from easily pinching apart to form two identical daughter cells. 

For this reason, the process of cytokinesis is different in these cells ( plant cells ). During late telophase and early cytokinesis, carbohydrate filled vesicles are released by the Golgi bodies and occupy the equator region of the cell. The vesicles continue fusing to form the cell plate which divided the cell into two. 

Here, the carbohydrates contained in the vesicles form the middle lamella between the membrane of the two cells. The cellulose produced by the two new cells occupies the region between the middle lamella and cell membrane to form the primary cell wall for the two daughter cells. 

Microscope Experiments

Difference between Meiosis and Mitosis

Return to Onion Cells under the Microscope

Return from Onion Root Tip Mitosis to Microscopemaster home

Cooper GM. (2000). The Cell: A Molecular Approach. 2nd edition.

Donald, B. and Richard, J. (2018). Cell Division. An Atlas of Comparative Vertebrate Histology.

Lian, Y. and Chirop, M. (2016). Functional Cell Biology. Encyclopedia of Cell Biology.

Lodish H, Berk A, and Zipursky SL, et al. (2000). Microtubule Dynamics and Motor Proteins during Mitosis: Molecular Cell Biology. 4th edition.

https://employees.csbsju.edu/ssaupe/biol115/cell_division.htm

https://www.yourgenome.org/facts/what-is-mitosis

https://www.nature.com/scitable/topicpage/chromosomes-14121320/#:~:text=During%20interphase%20(1)%2C%20chromatin,mitosis%20(2%2D5) .

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Introduction:

Mitosis has been studied since the early 1880s, Walther Flemming was one of the first to

devote his time to cytology, the study of chromosomes. Much of what we know today about

mitosis originated with Flemming's observations. He saw that chromosomes were "doubled"

when they appeared in prophase, and "solved" the problem of chromosomal partitioning between

mother and daughter cells. This was significant for later work in meiosis and the chromosomal

theory of inheritance. (T.J )

Mitosis is the phase of the cell cycle where chromosomes in the nucleus are evenly

divided between two cells. When the cell division process is complete, two daughter cells with

identical genetic material are produced. (Regina Bailey, 2018)

Mitosis follows G2, the time in which cells separate their duplicated contents and divide.

Division of cells at the end of mitosis yield identical diploid cells. Mitosis involves a five-step

process, and then a final step called cytokinesis. The five steps of mitosis and cytokinesis are

often considered to be two distinct sub-phases within the general cell-cycle phase which is the M

phase. The five steps of mitosis, which includes prophase, metaphase, anaphase, and telophase,

constitute the period in which the cell makes preparations for cell division. The five phases are

differentiated by specific events of preparation for cell division. Cytokinesis refers to the actual

cleavage event, splitting the cell in two.

In this experiment, the root tip of a growing onion cell is observed under the microscope

to study the different stages of mitosis of a growing root tip and the structure of the

chromosomes in each stage. The results are then observed and drawn out.

Objectives:

• To observe the stages of mitosis in a growing root tip of an onion
• To identify the different stages of cell division in a living tissue under the microscope.

Microscope, bunsen burner, watch glass, coverslips, dropping pipette, filter paper, small

paintbrush, scalpel, 2cm³ acetin orcein stain, 1cm³ molar hydrochloric acid, onion or broad bean

• 10 drops of acetic orcein stain is pipetted into a watch glass and one drop of molar hydrochloric acid was added.
• The terminal tips of 1cm off two onion roots were cut and placed in the watch glass.
• The watch glass was warmed gently over the spirit Bunsen Burner. Liquid was not allowed to boil. The temperature was maintained for five minutes.
• A small paintbrush was used to place the root tips on a clean microscope slide. 2mm nearest the tip was cut off using a scalpel and the rest was discarded.
• 2 drops of acetic orcein stain was added and the cells were gently tease apart to keep them in the same relative positions as far as possible.
• A coverslip was placed over the preparation and covered with several layers of filter papers.
• A little downward pressure was applied to the coverslip using a finger, no lateral movement was allowed.
• The preparations were warmed under the flame for a few seconds and examined it under a microscope, an oil immersion lens was used.
• The slides were examined carefully and any stages of mitosis was identified.

Discussion:

Onion root tips were used in the experiment because the chromosomes in the root tip cells are large and very dark when stained with acetic orcein stain. Cell division occurs rapidly in growing root tips of onion bulbs, hence, many cells in different stages of mitosis can be found.

In our experiment, we failed to obtain a clear view of mitosis in the onion root tips due to some error occured during the experiments. For example, we did not cut the correct part of the onion root tip, which made the mitosis stages cannot be seen under the microscope. We also did not take extra care when pressing the slide onto the the specimen, whereby we did not press it gently causing lateral movement therefore resulting in poor view of mitosis stages in the onion root tip. Moreover, the acetic orcein on the root tip were warmed for too long which was more than few seconds. This caused the observation of the root tip under microscope to appear darker therefore making it harder to distinguish the mitosis stages.

The expected results were obtained under light microscope at 40X which was prepared by the lecturer. From the expected results, all mitosis stages including prophase, metaphase, anaphase, telophase and cytokinesis can be seen and can be distinguished.

Mitosis starts with prophase. Prophase is a stage where chromosomes condense, also known as dark regions meaning they had become compacted and tightly wound. The chromosomes become shorter, thicker and visible under the light microscope. Each chromosome consists of two sister chromatids joined together at the centromere. Then, spindle fibre starts to form without the presence of centrioles as plant cell can form spindle fibres without the presence of centrioles. Each pair of centrioles is then migrates to lie at the opposite poles of the cell. At the end of prophase, the nucleolus disappears and the nuclear membrane disintegrates.

During metaphase, the centromeres of all chromosomes can be seen lined up on the equator of the cell known as the metaphase plate. Spindle fibres are now fully formed and sister chromatids of each chromosomes are attached to each other at the centromere.

Anaphase is the shortest stage of mitosis. During anaphase, the two sister chromatids of each chromosome separate at the centromere. The sister chromatids are then pulled apart to the opposite poles by the shorthening of spindle fibres. The separated sister chromatids are ones referred as daugther chromosomes.

The final stage of mitosis is telophase. It begins when both sets of chromosomes reach the opposite poles of the cell. The chromosomes starts to uncoil and revert to heir extended state (chromatin) again. The nuclear membrane reforms around each set of chromosomes. Process of mitosis is now complete.

Cytokinesis is the final cellular division to form two new cells. In plant cells, membrane- enclosed vesicles were collected at the equator between two nuclei. The vesicles join to form a cell plate. Then, the cell plate grows outwards grows outwards until its edges fuse with the plasma membrane, thus forming new cell wall and plasma membrane. The cell plate is then divided into two daugther cells.

From this experiment, the shape and structure of the onion root cell and its chromosomes can be seen clearly after being stained by acetic orcein stain. Acetic orcein stain turns chromosomes to a purple-red colour which enables us to see it’s structure clearly under a light microscope.

Therefore, we are able to have an idea of precision and the processes that sustain in the life of living organisms. We are able to identify each stage of the mitosis process which is interphase, prophase, metaphase, anaphase and telophase of the growing onion root tip cell by observing the processes between the plant cells. Moreover, it was a learning experience for us to handle the specimens properly in order to get a better field of view of the onion root tips.

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• Biology Article

Study Of Mitosis In Onion Root Tip Cells

Aim of the experiment.

To study and demonstrate mitosis by preparing the mount of an onion root tip cells.

Theory Of The Experiment

For entities to mature, grow, maintain tissues, repair and synthesize new cells, cell division is required. Cell division is of two types:

In mitosis , the nucleus of the Eukaryotic cells divides into two, subsequently resulting in the splitting of the parent cells into two daughter cells. Hence, every cell division involves two chief stages:

• Cytokinesis – Cytoplasm division
• Karyokinesis – Nucleus division

Stages Of Mitosis

The various stages of mitosis are:

1. Prophase

• The process of mitosis is initiated at this stage wherein coiling and thickening of the chromosomes occurs
• Shrinking and hence the disappearance of the nucleolus and nuclear membrane takes place
• The stage reaches its final state when a cluster of fibres organizes to form the spindle fibres

2. Metaphase

• Chromosomes turn thick in this phase. The two chromatids from each of the chromosomes appear distinct
• Each of the chromosomes is fastened to the spindle fibres located on its controller
• Chromosomes align at the centreline of the cell

3. Anaphase

• Each of the chromatid pair detaches from the centromere and approaches the other end of the cell through the spindle fibre
• At this stage, compressing of the cell membrane at the centre takes place

4. Telophase

• Chromatids have reached the other end of the cell
• The disappearance of the spindles
• Chromatin fibres are formed as a result of uncoiling of daughter chromosomes
• The appearance of two daughter nuclei at the opposing ends due to the reformation of the nucleolus and nuclear membrane
• At this phase, splitting of the cell or cytokinesis may also occur

Post mitosis, the next stage is referred to as interphase, which is part of the cell cycle that is non-dividing and between two consecutive cell divisions . A cell spends most of its life in the interphase. It comprises the G1, S and G2 stages.

Why is onion root tip used to demonstrate mitosis in this experiment?

It is because of the meristematic cells that are situated in the tip of the roots that render the most desirable and suitable raw material to study the different stages of mitosis. Onion is a monocot plant. Monocotyledonous plants possess large chromosomes that are clearly visible. Hence, their root tips are used. The period of time taken for mitosis varies as it is dependent on the cell type and type of species.

Is mitosis influenced by any factor? If yes, name them.

Yes, mitosis, the cell cycle is affected by various factors such as time and temperature.

Materials Required

• Compound microscope
• Acetocarmine stain
• N/10 Hydrochloric acid
• Filter paper
• Aceto alcohol (Glacial acetic acid and Ethanol in the ratio 1:3)
• Glass Slide
• Onion root peel
• Watch glass

Procedure Of The Experiment

• Place an onion on a tile
• With the help of a sharp blade, carefully snip the dry roots of the onion
• Place the bulbs in a beaker containing water to grow the root tips
• It may take around 4 to 6 days for the new roots to grow and appear
• Trim around 3 cm of the newly grown roots and place them in a watch glass
• With the help of forceps, shift it to a vial holding freshly prepared aceto-alcohol i.e., a mixture of glacial acetic acid and ethanol in the ratio 1:3
• Allow the root tips to remain in the vial for one complete day
• With the help of forceps, pick one root and set in on a new glass slide
• With the help of a dropper, allow one drop of N/10 HCl to come in contact with the tip of the root. Additionally, add around 2 to 3 drops of the acetocarmine stain
• Heat it lightly on the burner in such a way that the stain does not dry up
• Excessive stain can be carefully treated using filter paper
• The more stained part of the root tip can be trimmed with the help of a blade.
• Discard the lesser stained part while retaining the more stained section
• Add a droplet of water to it
• With the help of a needle, a coverslip can be mounted on it
• Gently tap the coverslip with an unsharpened end of a needle in order for the meristematic tissue of the root tip present under the coverslip to be squashed properly and to be straightened out as a fine cell layer
• The onion root tip cells’ slide is now prepared and ready to be examined for different stages of mitosis
• Observe and study mitosis by placing the slide under the compound microscope. Focus as desired to obtain a distinct and clear image

Observations and Conclusion

• The slide containing the stained root tip cells is placed on the stage of the compound microscope, changes taking place are noted and sketched.
• The different phases of mitosis, such as prophase, metaphase, anaphase and telophase can be observed.

Viva Questions

Q.1. Why is mitosis also referred to as the equational division?

A.1. It is because the chromosome  number present in the daughter cells is the same as the number of chromosomes present in the parent cell.

Q.2. To study mitosis, what is the best time to harvest onion root tips and why?

A.2. Early morning is the best time as the root tips actively undergo cell division in the morning. When such a material is used, all stages of cell division can be observed and studied.

Q.3. Other than an onion, can you suggest any other raw material for the study of mitosis.

A.3. Dividing cells of these can be picked:

• Shoot apex of plants
• Cells from the root tips of any herbaceous plants
• Gills of fish (epithelial cells)
• Tadpole larvae (tail)

Q.4. For the cytological study, why are different parts of monocots preferred?

A.4. Since they possess larger chromosomes that are clearly visible under a light microscope.

Q.5. Why is the stain acetocarmine used in this experiment?

A.5. This stain is used to study chromosomes as it stains them in a deep red tint without staining the cytoplasm.

Q.6. Where does the spindle fibre originate from?

A.6. The spindle fibres originate from:

• In-plant cells – Cytoplasm
• In animal cells – Centrioles

Q.7. Mention the phase of the cell division in which chromosomes are observed distinctly.

A.7. Metaphase. They are shortest and thickest in this stage and in their condensed form.

Q.8. During metaphase, which chemical can be used to stop the cell division process?

A.8. The spindle fibre formation can be inhibited by Colchicine during this stage.

Q.9. Where is colchicine obtained from?

A.9. Colchicum autumnale plant belonging to the Liliaceae family.

Q.10. Where does mitosis occur?

A.10. This type of cell division takes place in the vegetative cells.

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Mitosis in Onion Root Tips

This classic microscope lab has been used in life science classrooms for decades. It is also a standard part of the AP Biology curriculum as Investigation #7 in the AP Biology lab manual , and can be a great way to apply a basic knowledge of chi-square tests.

Background:

The environment immediately surrounding a cell can have substantial effects on the process of cellular division. Mitosis, one type of cell division, is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. This is the fundamental process that produces most of the cells in multicellular organisms and allows for growth and repair of an organism.

Fungal pathogens in the soil are known to inhibit root growth in important agricultural plants. These fungal pathogens are thought to act through secreting lectin and lectin-like proteins into the soil to promote mitosis in root tips beyond healthy levels. This artificially high level of mitosis is then thought to damage root tissue, eventually slow overall root growth, and weaken the plant.

In this activity, we’ll use lab data to test the hypothesis that lectin promotes mitosis in onion roots. The two variables for the activity are Phase and Treatment. Each row in the dataset is an individual cell observed with a microscope. Phase corresponds to whether the onion cells were observed to be in “Interphase” or “Mitosis” and Treatment corresponds to whether the onion root tips exposed to lectin (“Lectin”) or not (“Control”). 

To test for any statistical effects of lectin on root growth we’re going to use a chi square test of independence. The null hypothesis of this  test is that two (or more) variables are independent of one another. In other words, the null hypothesis assumes that there is no predictive ability of one variable on another. In the case of this lab, this would mean that the proportion of control cells in mitosis are similar to the proportion of lectin-treated cells in mitosis. If we find this to be true, then the phase we observe the onion root tip cell to be in would be independent of the lectin treatment and our data would fail to reject the null hypothesis. However, if the proportions of cells in mitosis are significantly different between the control and lectin-treated samples, then we would reject the null hypothesis and conclude that the lectin treatment does affect the proportion of cells observed in mitosis. 

In the activity, students will use the “Make-a-graph” feature to find the observed counts of cells in different stages of the cell cycle, and use these observed counts to calculate the expected counts for the null hypothesis. They may then either calculate the result by hand and then use the graph-driven hypothesis test to check their results or simply complete the test by hand ( the graph-driven chi-square test for independence is a paid feature, but you can start a 90-day free trial to create classes and give your students access ).

To collect this dataset, both lectin-infused roots and non-exposed lectin root tips (control) were observed using microscopes. While looking through the microscope at a root tip on a stained slide, cells were observed and for each cell it was noted whether that cell was in interphase or undergoing mitosis. Because the cells were preserved in whatever stage of the cell cycle they were in when the slide was prepared, the slide is like a snapshot of what was happening in that root tip at the time of collection.

The null hypothesis is that results will show no difference between the control and treated groups in terms of the time spent in mitosis relative to interphase. The alternative hypothesis is that the lectin treatment will increase the amount of time that cells in the root tip spend in mitosis relative to the time spent in interphase.

Phase: This is a categorical variable with two values. Each observed cell was either in Interphase or Mitosis.

Treatment: This is a categorical variable with two values. The root tip and its cells were either treated with Lectin or a Control (not treated).

1) Use the “ Make a graph ” feature to make a categorical bubble plot (accessible with the scatter plot icon) to compare the relative proportions within each group. Show Treatment on X and Phase on Y. Screenshot your graph below, and answer the question: What does the graph seem to suggest about the impact of lectin on onion root cell metabolism?

2) Given that a p-value of .05 or below indicates a strong evidence that lectin does impact cell growth, give your best estimate for the p-value for a Chi-Square Test of Independence. Without actually doing any calculations, do these results look like a significant deviation from the null expectation for the same proportion of cells in mitosis in both treatment groups?

3) With DataClassroom: Use a graph-driven chi-square test for independence* to have the computer do the calculation. After you have made the graph, just click the Graph Driven Test Button (on the right panel just left of the Appearance button). How close was your estimate of the p-value to the actual calculated value?

By Hand: Determine if there is a statistically significant difference between the observed frequency of cells in interphase and mitosis between the two treatment groups. To do so you  will need to do the following:

a) Calculate the expected values assuming independence. For an in-depth explanation of why we calculate independence this way, please see the Teacher’s Note below. (Hint: e1 should be around 71.75)

N = total number of cells in the experiment

e1 = expected value of cells in the control group in interphase = all cells in interphase * all cells in the control group / N.

e2 = expected value of cells in the control group in mitosis = all cells in mitosis * all cells in the control group / N.

e3 = expected value of cells in the treatment group in interphase = all cells in interphase * all cells in the treatment group / N.

e4 = expected value of cells in the treatment group in mitosis = all cells in mitosis * all cells in the treatment group / N.

The table below is a helpful way to organize this information (a modified version of the one provided under Investigation 7 in the AP Biology Lab Manual ):

b) Calculate chi-squared statistic using the following formula:

Χ 2 =Σ(o-e) 2 /e

“o” is the observed count for each category you found in 1), and “e” is the expected count you calculated in a). Make sure each “e” and “o” correspond to the same category. The table below is a helpful way to organize the information. (Hint: the first of the four values you’ll find and add should be 2.089)

c) Compare the chi-squared value to a table of critical values for the chi-squared distribution.

*DataClassroom only has a Chi-Square Test of Independence so we use that here in place of the more appropriate Chi-Square Test of Homogeneity. The difference between these two tests is not in the calculation, but rather in the experimental design and sampling method. Quantitatively and qualitatively the conclusions drawn will be the same.

Teacher’s Note

Chi-square tests can be daunting, especially for high school students without a background in statistics, but they’re really useful for analyzing datasets with categorical variables. Although these are frequently taught by hand, students should understand that these tests are almost never performed by hand in the real world. Scientists rely on computers to perform statistical tests, but ideally have a thorough understanding of how tests work and how their results can be interpreted. 

 All Chi-square tests are looking to see if there is a big enough deviation from expected counts, based on the assumptions of the null-hypothesis , to conclude that those assumptions that gave us the expected counts are not true. If the observed data differ enough from the expected values we reject the null hypothesis. 

A quick breakdown of different types of chi-square tests:

Goodness of fit : used when there is a known, discrete distribution. Rolls of a dice or coin flips are a great example of instances when goodness of fit would be appropriate. 

Test for independence (used in this activity): used to determine whether two different categorical variables are associated with one another.

Test of homogeneity : when data is collected by randomly sampling each subgroup of a categorical variable separately. 

Let’s focus on the tests for independence , since the calculations when we assume independence can be a little tricky. For a these tests, our expected counts are calculated by multiplying the ratio of all observations in the dependent variable category (cell phase, in this case, divided by N , the total number of observations) by the number of elements in the independent variable (treatment) category. If this value is close to the observed number of a dependent variable in an independent variable category, then the impact of the independent variable on the dependent variable would be negligible, and the two variables are thought to be independent.

Chi-square tests have interesting distributions that change with the number of variables studied. Here is a chart of critical values for chi-square distributions with different degrees of freedom. The numbers in the table are chi-squared values for which the probability of the the null hypothesis being true based on the outcomes of a test are the values at the top of each column. For instance, if you conducted a test with 2 degrees of freedom (“df”) and calculated a chi-squared value of 5.991, there’d be a only a 5% chance of obtaining data that differed from the null expectation by that much through random chance . As you can see, when more variables are considered, there needs to be greater deviation from our expected values in order to reject the null hypothesis.

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Arlie's Instruction Manuals

Onion Root Tip Mitosis

Introduction.

In this experiment, we will be using on onion root tip to show the different stages of mitosis. According to the National Human Genome Research Institute, “Mitosis is the process where the cells replicate their chromosomes and then segregates them, producing two identical nuclei in preparation for cell division.” Mitosis is very important to every living organism. Humans use mitosis to create more skin cells, blood cells, muscle cells and more.

Mitosis occurs through different stages. These stages are Prophase, Metaphase, Anaphase and Telophase. The reason why we are using an onion in this experiment is because their root tips grow at a rapid rate due to rapid cell division, which allows us to identify the different stages of mitosis.

In the Results section in the procedure, there will be further explanation on what each stage will look like.

Here are vocabulary terms to be familiar with (click on the word to be taken to the National Human Genome Research Institute website for their definitions):

TECHNICAL BACKGROUND

In order to understand how to do this experiment, you will need to have background on how to work a microscope and how to prepare 1M solution on HCL.

WARNINGS, CAUTIONS, AND DANGERS

• Wear goggles, gloves and lab coat
• Sharp Objects
• Hot surfaces
• This experiment is not edible
• Glass/Plastic Jar
• Blade/Scalpel
• 2 Petri Dish or Small Clean Container
• Medium to Large Glass
• Hot Plate/ Stove
• Small Bottle
• Thermometer
• Vial with Cap/Lid
• Microscope Glass Slide
• Dissection Needle
• 70% Alcohol
• Stain (acetic orcein or aceto-carmine)

Growing Onion Roots:

• Pour water into a clean, clear glass/plastic jar. Fill it about 3/4 full.
• Place the onion bulbs in the jar so that only the lower surface of the onion comes into contact with the water. (If the onion is too small, place toothpick into the onion to keep the only the lower surface in the water and the rest above.)
• Let the onions rest on water for 3-4 days.

Sample Preparation:

• Using a blade or scalpel, carefully cut one or several roots from the onion and place them in petri dish or any small clean container.
• In a medium to large glass container, prepare a water bath.
• If able, set your hot plate to 55 degrees C and place your water bath on it. If you do not have a hot plate, heat your water bath to a stable temperature of 55 degrees C.
• Carefully pour the hydrochloric acid solution into a small bottle and place it in the water bath for about 15 minutes to warm the acid.
• Using the forceps, carefully pick one or two of the roots and place then into the bottle of warm hydrochloric acid for about 5 minuets.
• Remove the roots form the bottle of acid using the forceps and rinse them with tap water several times.
• Place the root tips into a clean petri dish.
• Using a clean blade, cut off the tips of the roots. (about 5 mm in length)
• Using the forceps, pick the root tips and put them in the vial of stain. (acetic orcein or aceto-carmine)
• Make sure the root tips are completely in the stain.
• Place a cap/lid on the vial and make a prick hole through the cap/lid.
• Place the vial in the water bath for about 5 minutes.
• Using the forceps, remove the root tips from the vial of stain and place them onto a clean microscope glass slide.
• Using a dissection needle, gently mash/squash the root tips to spread out the cells on the glass slide.
• Cover the glass slide with a coverslip and gently press the coverslip down.
• Examine the slide under the microscope.

Viewing slides under 10x magnification:

Students should be able to observe several single layers of cells.

• Note: This magnification will not show the stages of mitosis. It will show a row of cells the generally consists of 2 and 5 cells.

Viewing slides under higher magnification (100x-500x)

Students should be able to observe the different stages of mitosis under higher magnification.

The stages that should be visible are:

Interphase is not a part of mitosis, but it is the preparation step. Interphase includes G1, which is characterized by cells growth, S phase, which is where the DNA is replication, and G2, which is where the cells prepares to enter mitosis or M phase.

The way to identify interphase is to notice that the chromatin is not tightly packed.

Prophase is the first step of mitosis. In prophase, the chromatids form together to make the chromosomes. These chromatids that are formed together are called the sister chromatids, which contain the same genetic information and are attached at the centromere. Another important structure that is forming is the mitotic spindle. This structure will be important during a later stage.

During this stage, the nucleolus is still visible, but the nucleus is grainy due to chromosomal condensation.

Prometaphase

This is the second stage of mitosis. During this stage the chromosomes are still condensing, but the nuclear envelope is broken down. Not only are these things happening, but kinetochore start to develop around the centromere.

This is the third stage of mitosis. During this stage, the sister chromatids align along the middle of the cell, preparing for the sister chromatids to be separated. The chromosomes, during this stage, become more visible. Also, the fully developed spindle fibers, located on two different sides of the cell, attach to each of the sister chromatids.

This is the fourth stage of mitosis. During this stage, the two sister chromatids start separating and moving towards the opposite side of the cells.

This is the fifth and final stage of mitosis. During this stage, many different events are taking place. The chromosomes are at the opposite ends of the cell, the breakdown of the spindle fibers begin, the development of the nuclear envelope starts to form around the two sets of chromosomes that are on the opposite ends of the cell, and the DNA starts to de-condense.

Cytokinesis

This step is not a part of the the mitosis phase, but is followed directly after this. During this step, the cytoplasm separates as the cell divides into two identical daughter cells, but since plat cells have cell walls that prevent them from pinching apart, we are not able to see this happening.

Dispose of all solid waste in the trash and all liquid waste can be disposed down the drain with lots of water.

Helpful Video:

“Mitosis.” Genome.Gov , www.genome.gov/genetics-glossary/Mitosis. Accessed 12 May 2024.

Microscope Master. “Onion Root Tip Mitosis.” Microscope Master, Microscope Master, n.d., www.microscopemaster.com/onion-root-tip-mitosis.html .