Onion Root Tip Mitosis Squash Preparation Protocol (Step by Step Chromosome Squash Method)

This post outlines a step by step onion root tip mitosis squash preparation protocol for biology teachers looking to prepare chromosome squash slides.

When you can smell the onions at the end of my hallway, you know it is time to study cell division and make chromosome squash slides. Even after more than 30 years of teaching, this remains one of my favorite biology labs. Students experience everything from frustration when a slide does not turn out to real excitement when they peer into the microscope and realize they made a good one.

What exactly is a chromosome squash?  

A chromosome squash is a slide preparation technique that uses actively dividing cells from an onion root tip to show the different phases of mitosis under a microscope.

How long is this lab?  

This lab takes several days to complete. A short amount of class time is needed to start the onion roots growing. After about five days, students cut the root tips. You should then allow at least two additional class periods for slide preparation and microscopy.

Are there safety precautions that should be followed?  

Yes. This lab requires the use of concentrated hydrochloric acid. It should be conducted in a well ventilated lab with access to a fume hood and an eye wash station. Students must wear safety goggles and lab aprons at all times.

Onion Root Tip Mitosis Squash Preparation Protocol

Begin by purchasing onion sets from a local feed and seed store or a biological supply company. For variety, I typically use red, yellow, and white onions. The color of the onion does not affect the outcome of the lab, but students enjoy growing them, and the lab tables look great when they are full of sprouting onions.

The next step is to "plant" the onions. As you can see in the photo, you will need small plastic cups and toothpicks. A 50-mL beaker works well, also. Students insert three toothpicks into each onion to form a tripod and suspend the onion over a cup filled with water. This setup takes about 20 minutes of class time. Root tips are typically ready for use after about five days.

The root tip is a region of rapid cell division. As the root grows, cells divide continuously, making this tissue ideal for observing mitosis. Once the root tips reach a length of approximately one inch, use a razor blade to cut the roots away from the bulb. Place the root tips in 70 percent isopropyl alcohol. The alcohol kills and preserves the cells, and because many cells are actively dividing, they are often arrested in one of the phases of mitosis. The root tips must soak in the alcohol for at least 24 hours before slide preparation.

Day 5:  Roots are long enough to be cut.

Ready to make the slide?  

It is important to forewarn students that this procedure is not perfect. Several attempts may be required before a slide is clear enough to observe the phases of mitosis. This trial and error is part of the learning process and helps students develop patience and laboratory skills.

To begin slide preparation, remove a root tip from the isopropyl alcohol and place it in a solution of hydrochloric acid and ethyl alcohol. After the appropriate amount of time, transfer the root tip to Carnoy’s solution. Next, place the root tip on a microscope slide and cover it with aceto-orcein stain. While the tissue is in the stain, students should finely chop the root tip using a razor blade. For best results, thorough chopping helps separate the cells.

The final step is the squash technique. Place a cover slip over the chopped root tip and apply gentle, even pressure with your thumb. This spreads the cells into a thin layer suitable for viewing under the microscope.

Place the prepared slide under the microscope and examine it for cells in the different stages of mitosis. Because this technique can be challenging, multiple attempts may be necessary before a clear slide is obtained.

When students finally observe a successful slide, the excitement in the lab is immediate and contagious. I encourage students to take photographs of their slides using their cell phones. While I have never quite mastered this skill myself, students are surprisingly good at capturing clear images.

Have fun teaching!

Frequently Asked Questions: Onion Root Tip Mitosis Squash Preparation

What is an onion root tip mitosis squash?
An onion root tip mitosis squash is a slide preparation technique that uses actively dividing cells from onion root tips to observe the phases of mitosis under a microscope.

What materials are needed for an onion root tip chromosome squash?
This lab requires onion bulbs, water, small cups or beakers, toothpicks, razor blades, microscope slides and cover slips, 70 percent isopropyl alcohol, hydrochloric acid, ethyl alcohol, Carnoy’s solution, and aceto orcein stain.

What stain is used for onion root tip mitosis squashes?
Onion root tip mitosis squashes are commonly stained with aceto orcein or acetocarmine to make chromosomes visible during the stages of mitosis.

What are the steps in the onion root tip mitosis squash preparation protocol?
The procedure includes growing onion roots, cutting and preserving the root tips, treating the tissue with acid and alcohol solutions, staining the cells, chopping the root tip, and gently squashing the tissue under a cover slip to spread the cells.

Why are my chromosome squashes unclear or difficult to see?
Common issues include insufficient chopping of the root tip, too much or too little pressure during the squash, overstaining or understaining, or using root tips that are not actively dividing.