Before we jump straight in, let’s get the basics down. What is mitosis? In the simplest terms, mitosis is a process where a single (parent) cell divides into two identical (daughter) cells.
Why does this happen? Mitosis happens mainly for growth and to replace any worn-out or damaged cells. So say you fall over and graze or cut your knee, mitosis happens with those injured cells and plays a big part in healing that wound!
Mitosis takes place in stages; prophase, metaphase, anaphase, and telophase.
Using a microscope you can see these different stages in collected cells which can make for a great science experiment for a visual, more hands-on approach to learning about mitosis.
Onion root is a great choice for observing mitosis because root tips grow at a really rapid rate as a result of rapid cell division.
So this experiment is usually broken down into two separate parts. First off, you’ll need to grow your onion roots.
Once you’ve done this, you can move on to the next part which is sample preparation. I’ll break both of these experiments down below so you’ll know exactly how to go about them.
Before we begin, let’s just go over some precautionary measures for either of the experiments. These will ensure that both experiments go smoothly.
- The onion should not be touched or handled by fingers – always use forceps or a similar substitute.
- Be careful when staining – you don’t want it too dark or too light.
- When covering the slide with the coverslip make sure that there are no air bubbles as this will affect the experiment.
- If there is any excess when you put the coverslip on be sure to remove it with blotting paper or a paper towel.
- Be sensible – remember that you are dealing with chemicals such as hydrochloric acid. You should always take care when using chemicals. Be slow and steady in your movements.
- Long hair should be tied back and out of the way.
Experiment One: Growing The Onion Root Tips
It is always best to grow your own onion root tips for this experiment, not only is it more fun to be more involved but older and dried-up roots tend to give worse results than fresh root tips.
Here’s everything that you’re going to need for this part:
- Uncut Onions
- Clear Glass Or Plastic Jars
- Clean Water
Now that you’ve got everything you need, you’re all set to start your experiment. So what are you waiting for, let’s get started!
- First, pour the water into plastic or glass jars. Make sure that the jars are clear and both the water and jars are clean. You’re going to want to pour the water about three-quarters of the way to the top.
- Next, you need to very carefully put the onion bulbs into the jars making sure that it’s only the very lower part of the onion that is touching the water.
- If your onion is too small for your jar, do not panic. They can be supported fairly easily using toothpicks or splints.
- Leave the onions for around three to four days. Remember only the base where the roots emerge should be in the water.
After the three or four days, all having gone well, you should now have roots growing from the bottom of your onion. They should be around an inch long.
If you do, congratulations, you can now move on to the second part of the experiment.
Experiment Two: Sample Preparation
It is always recommended to have gloves, safety goggles, and a safety mask when working with any hazardous chemicals. Although they are not in the requirement list below, you should always have these items in any lab for experimentation.
Here’s everything you need for this part:
- 70 Percent Alcohol
- A Blade
- Glass Slides
- Stain – Aceto-carmine or Aceto-orcein
- Hydrochloric Acid Solution
- Freshly Grown Onion Roots
- A Microscope
- A Stop Clock
- Water Bath
- Dissecting needles
Step By Step Guide
Once again, you’re now ready to start your experiment so let’s jump straight into it!
- First, you’ll need to cut the root (or roots if you have multiple) with a blade knife or scalpel. Once you’ve cut the roots you’ll want to pop them into a petri dish. If you do not have a petri dish any clear container should work fine.
- Next, you need to prepare your water bath. The temperature should be kept at about 55 degrees celsius/ 130 degrees Fahrenheit. You can make sure you do this by using a thermostatically controlled water bath.
- Once you’ve done this, very carefully pour some hydrochloric acid into a smaller bottle and place this smaller bottle into the water bath. Leave this in the bath for around 15 minutes to allow the hydrochloric acid to heat up.
- Once the acid has sat for 15 minutes, use the forceps very carefully to pick up one or two of the roots. Place these roots inside the small bottle of heated hydrochloric acid for about five minutes. The reason you do this is to break down different tissues so that you can release the individual cells.
- Using the forceps carefully remove the roots from the small bottle of hydrochloric acid.
- Next, you’ll need to rinse the roots a few times with tap water. When you’ve done this pop the roots in a new clean petri dish.
- Using a clean blade, you need to cut the tips off of all of your roots. They should be roughly about 5mm in length.
- Get your forceps again and pick up the tips and pop them into a vial of the stain of your choice. The tips need to be fully immersed in the stain for it to work, though.
- Make sure that the lid of your vial has a small hole in the top, and then pop the lid on top of the vial. Place this back in the water bath ensuring that it is still at the correct temperature. You’ll do this to enhance the staining process which will make your life a lot easier later on.
- Once it’s been five minutes, use the forceps again to take the root tips out of the vial. Then place them onto a microscope slide – make sure that it’s clean.
- Then with your dissecting needle, squish the root tips so that you spread the cells across the slide. You want to do this so that you don’t have loads of cells overlapping and affecting your results.
- Finally, cover your slide with a coverslip and press down gently. Then slide it under a microscope and use a low magnification setting first to find your cells.
An onion has a total of only eight chromosomes. This makes life a lot easier for us when conducting this experiment as it’s easier to see them when they condense.
Onion root tips actually divide incredibly quickly as the roots grow to absorb water and minerals from soil.
Because onion root does this so fast, it makes it possible for you to identify the varying stages of cell division by mitosis by looking at the roots’ chromosomal distribution.
Under 10x Magnification
Under 10x magnification, you should be able to see several single layers of cells. Because we spread the root earlier in the sample preparation these cells should be separated and distinct and not overlapping one another.
Higher Magnification (Seeing The Phases Of Mitosis)
Once you’ve identified your cells under the microscope and have been able to increase the settings to around 100-500x you should start to be able to identify the different phases.
Different Stages Of Mitosis
Interphase isn’t seen as one of the main stages of mitosis but it is still identifiable under the microscope. Cells in the interphase state should be easily identifiable. You should be able to notice this phase by the prominent nucleoli.
Interphase can sometimes be called the ‘resting’ stage. You’ll notice during this phase that the chromatin isn’t tightly packed together. This means that the DNA can be copied or replicated as the cell begins to prepare to divide.
This is where it can become a little more confusing. Interphase can then be broken down into three more phases.
- G1 – This is the first gap phase and is characterized by both cell growth and normal cellular activities.
- S Phase – This is where the DNA is replicated and so the DNA content is then doubled.
- G2 – This is the second gap phase and is where the cell starts to prepare for division.
Prophase is considered the first step of mitosis. You’ll be able to identify a cell at this stage as the nucleus will still be visible.
However, it will look different from the previous stage. The nucleus will now be more grainy than before – this is due to chromosomal condensation.
In the previous phase, the DNA was uncoiled so that transcription could happen. During this phase, condensed DNA and some proteins form something called the chromatids which then join to become x-shaped chromosomes.
The sister chromatids, which now contain the same genetic information, become attached at the centromere which is what gives it the x-shaped structure.
At the centromere, there is also a kinetochore – this is the site where microtubules join the chromosomes.
Little Tip For Identification – As the chromatins coil, they will become more compact which’ll allow the chromosomes to be more visible under your microscope at a high magnification setting.
It is also during this phase that spindle microtubules will start to form near the nucleus.
Prometaphase is the second stage of mitosis. You may sometimes hear it be called the ‘late prophase’ stage. At this point, there should be an increased condensation of chromosomes.
You should also be able to see the beginning of the breakdown of the nuclear envelope or membrane.
This nuclear envelope should have an inner and an outer membrane. It is stabilized by the polymerization of lamin proteins also known as the nuclear lamina.
This and the breakdown of filaments into lamin dimers means that the nuclear membrane begins to disassemble.
At this stage, there is also a development of kinetochore around the centromere. Spindle fibers which are kinetochore microtubules also begin to develop during the prometaphase.
They then permeate through the membrane which is disappearing and attach to the chromosomes at the kinetochore.
At this point in mitosis, the chromosomes begin to align along the equatorial plane of the cell which is also known as the cell’s equator. This is so the sister chromatids can be separated from one another.
During metaphase, you’ll notice that chromosomes become a lot more visible and this is because of increased condensation.
It should also be really easy to identify this stage because of this, as well as the fact that the nuclear envelope should have completely disappeared by now.
Now that the nuclear membrane is gone, during metaphase, the chromosomes appear in the cytoplasm instead. The spindle fibers should be fully developed now and should be originating from the centrioles.
These are located on opposite poles of the cell and are attached to each of the sister chromatids. This contributes to their alignment at the cell equator.
Spindle fibers are approximately 25nm in diameter. They originate from the centriole and can then extend to attach to the sister chromatids. These will be constantly forming due to the fact that they are continuously broken down.
As the new building blocks or components are added to one end of the microtubules they are then removed from the other. This is what causes them to pull the centrioles which is a major factor in causing the alignment of the chromosomes.
In the later stages of metaphase, the pulling action of these microtubules makes the kinetochores also face different directions.
Now the chromosomes begin to separate and move from the equatorial plate of the cell. Towards the end of this stage, the sister chromatids will totally separate from one another and reach the opposite poles of the cell.
It’s really easy to identify the chromatids at this stage as the process occurs in the cytoplasm.
Sister chromatids tend to separate at a cell rate of about 0.2 and 4 um per minute. It used to be thought that polymerization and depolymerization of microtubules was the reason that chromatids moved to the opposite ends of the cell.
This was because depolymerization shortened microtubules while polymerization caused microtubules that extended from one pole of the cell to the other without attaching to the chromatids causing them to grow in length and push the poles of the cell apart.
However, it has since been determined that the separation of sister chromatids during anaphase is actually a direct result of the actions of enzyme seperase as well as the shortening of the microtubules.
The enzyme seperase breaks down cohesin which is a component of the centromere that links sister chromatids together.
Once the sister chromatids are separated, the shortening of the microtubules pulls the chromatids apart to opposite sides of the cell.
Telophase is the fifth step in mitosis. Several key events take place here. The chromosomes should now be at opposite ends of the cell and the spindle fibers should gradually break down.
Nuclear envelopes will now also develop around each set of chromosomes at each opposite end of the cells.
While the nuclear envelopes begin to form around each set of chromosomes at each pole of the cell, two nuclei also begin to develop in the cell. The DNA will also begin to uncondense which will allow the genetic material to be copied at a later time.
Now that the spindle fibers are no longer needed they will also begin to break down and disassemble in the early stages of telophase. They will continue to do this throughout this stage into the later telophase stage.
What is Cytokinesis?
It is when the cytoplasm separates as the cell begins to divide into two identical daughter cells.
Plant cells (like the onion you’ll be using in your experiment) have a much more rigid structure to their cell walls and so they can not split apart into two identical daughter cells and so the process of cytokinesis is different in these cells from what you’d expect to see in a human cell.
During the later telophase and earlier cytokinesis stage in plant cells, carbohydrate-filled vesicles are released.
These are released by something called the Golgi bodies and are found at the equator of the cell. These vesicles then continue to fuse to form a cell place which divides the cell into two.
The carbohydrates that are contained in these vesicles then form the middle lamella between the membrane of the two cells.
The cellulose that is produced by the two new cells stays between the middle lamella and the cell membrane and forms the primary cell wall of the two new daughter cells.
Frequently Asked Questions
How Do You Preserve Onion Root Tips For Mitosis?
You want to try and keep the root tips in the fixative for 24 hours at least, and then transfer them into 70% ethanol. This is for preservation and uses in the future. The onion root-tip cells have an approximately 24-hour cycle duration.
Can I Do This Experiment Without Treating The Tips With Hydrochloric Acid?
No. The root tip needs to be heated with the acid to break up the tissues into individual cells.
What Is Fixation Of Root Tips?
Root tips are usually grown and preserved in acetic ethanol fixative. They can be stored for at least two weeks prior to staining if this is done, which can be helpful for class experiments.
Why Does Mitosis Occur In The Tips Of The Root?
For the root to be able to grow in length and bury down in the soil, it needs the tissue region at the root to have active dividing cells. This is why we use the root, as this is where mitosis takes place.
Mitosis has loads of stages to it and it can get really, really confusing to wrap your head around it all so if you’re struggling, don’t be disheartened or discouraged.
There’s a lot of biological jargon to try and understand as well as quite a lot of different processes happening.
Learning about mitosis can seem really difficult, especially when you are simply reading from a page and are staring at a lot of words that seem to make no sense. However, you’ll be pleased to know it’s not actually quite as complicated as it might sound.
Personally, I think that using this onion experiment is a really great way to try and help make mitosis a little less confusing.
It’s a great way for an involved and hands-on approach to learning that lets students actually see what is happening, which in turn will make learning about why it happens easier.
And let’s face it – it’s a lot more fun than reading from a textbook. When learning is fun, it’s also as a rule a whole lot easier to take in. After all distractions and procrastination often come from boredom.
So if you’re looking to learn about mitosis and want to do so in a much more engaging way I would recommend this experiment to any teacher!
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