Looking at Mitosis in Onion Root Tips
You grind up the root tip, squish it under a coverslip, and hope you get a nice spread of cells. That is the short version. The real process involves several stages of mitosis that you can observe if you put in the effort to do the prep correctly. Root tips are useful because the cells there are actively dividing. The meristem region at the very end of the root contains cells that are constantly cycling through mitosis. When you prepare a slide, you should expect to see cells in prophase, metaphase, anaphase, telophase, and interphase. Interphase takes up most of the cells you will find. That is normal. The actual mitotic stages each take a fraction of the total time. I usually fix my root tips in Carnoy's solution for 24 hours before staining. Some people use acetic orcein directly on fresh tissue. Both approaches work. The fixed method gives cleaner chromosomes because the cells are preserved in their current state rather than continuing to divide after you cut them off the onion.
One problem I ran into repeatedly was the root tip splitting open when I mashed it. The cell walls are tough and the tissue tends to tear rather than separate into individual cells. I solved this by macerating the tip in 1M HCl at 60 degrees Celsius for about 10 minutes before squashing. The acid breaks down the middle lamella between cells and everything spreads out much more cleanly. Without that step, I was spending 20 minutes fighting with the slide and getting nothing but thick clumps of tissue under the microscope.
The Preparation Process
Grow your onion roots first. Place the onion over a jar of water so the base touches the surface. Keep it in a dark place for three or four days. The roots grow faster in darkness. You want roots that are about one to two centimeters long. Anything longer and the meristem gets pushed further back from the tip. Shorter roots have less dividing tissue to work with. Cut off the very tip. About five millimeters is enough. Discard the rest of the root. The apical meristem is what matters. That is the region where the cells are actively dividing. Stain the tip. Acetic orcein is the traditional stain. You can buy it prepared or make it yourself by boiling orcein in 45 percent acetic acid. Another option is toluidine blue, which stains nuclei purple and cytoplasm pink. I prefer orcein for chromosome visibility. The dye binds to DNA and gives you contrast against the pale cytoplasm.
Get the Full Details
Place the stained tip on a clean slide. Add a drop of 45 percent acetic acid. Cover with a coverslip. Press down firmly but evenly. I use the eraser end of a pencil and apply steady pressure. Do not rock the pencil back and forth. That just smears the tissue. A firm, vertical press flattens the cells into a single layer without destroying the chromosome structure.
What Each Stage Looks Like
Interphase cells have a clear nucleus with diffuse chromatin. You will see the nucleolus in some cells. The chromosomes are not yet condensed. This is the longest phase and the most common one you will observe. In prophase, the chromatin begins to coil into visible threads. The nuclear envelope starts to break down. By late prophase, the chromosomes appear as distinct, thick lines inside the cell. The spindle fibers are forming but they are too thin to see with a standard light microscope. You are looking for the transition from diffuse grainy material to thick rod-like structures. Metaphase is straightforward. The chromosomes line up along the equatorial plane. They are at their most condensed state. Individual chromosomes are clearly visible with two chromatids joined at the centromere. This is usually the easiest stage to identify because the alignment is so precise.
Anaphase shows the sister chromatids pulling apart toward opposite poles. The V-shape of the chromosomes pointing toward the poles is a useful marker. As the chromatids separate, the cell elongates slightly. This phase is shorter than metaphase so you may see fewer cells in this stage. Telophase has the chromosomes reaching the poles and beginning to decondense. A new nuclear envelope forms around each set. You may see a cell plate starting to form in the center. This is the plant cell version of cytokinesis. The cell plate grows outward until it fuses with the cell wall. Sometimes you will see cells in cytokinesis with a clearly visible cell plate dividing the cell into two. Other times telophase and cytokinesis overlap and it is hard to tell where one ends and the other begins. That is normal. Plant cells do not form cleavage furrows like animal cells do.
Counting and Calculating
Once you have a good spread, you can count the cells in each stage. Find a region where the cells are not overlapping and count at least 500 cells to get reliable numbers. Record how many are in each stage. Divide the count for each stage by the total number of cells. That gives you the relative time spent in each phase. If 60 percent of your cells are in interphase, interphase takes up roughly 60 percent of the cell cycle. This is a standard lab calculation but it depends on having a large enough sample size. Counting 50 cells gives you no meaningful data. The variation is too high. A common mistake is counting cells from the very edge of the root tip. Those cells are differentiating and may no longer be dividing. Stick to the meristematic region, which is the pale, translucent zone about five millimeters from the tip. Cells beyond that point are elongating and preparing for their final differentiation. They will not be in mitosis.
Common Issues
Over-staining is a frequent problem. If you leave the root tip in orcein for too long, the entire cell becomes dark and you cannot distinguish individual chromosomes. Ten minutes is usually sufficient. Longer staining does not improve visibility. It just makes everything black. Under-pressing is another issue. If the cells are still layered on top of each other, you cannot focus on a single plane. Keep pressing until the coverslip slides easily across the slide. The tissue should feel soft, not resistant. If it resists, your HCl treatment was probably insufficient and you need to macerate again next time. Sometimes the chromosomes separate too much during anaphase that they are hard to track. This happens when the cell is over-pressed and the spindle apparatus collapses. Gently release pressure and re-examine. You might find a cell that was only lightly squashed and the chromosomes are still in a more natural arrangement.
Alternatives to Consider
If you are struggling with preparation, commercial slides exist. They are expensive and you get no practice with the technique, but they work. For actual learning, making your own slides is better. The failure rate is high initially but you improve quickly. After five or six attempts, most people can produce readable spreads consistently. Another option is pre-pretreated root tips from biology supply companies. They come fixed and stained. The trade-off is that you lose the ability to adjust timing or staining intensity. For a demonstration, pre-made slides are fine. For understanding the technique, nothing replaces doing it yourself. The whole process from cutting the root tip to having a viewable slide takes about 30 minutes with the HCl pretreatment included. Without it, you can do it in 15 minutes but the results are often poor. The extra 15 minutes is worth it. I once skipped the acid step to save time and spent an hour trying to salvage the slide. It did not work.
