Its Twins!

Onion Root Tip Mitosis

Difficulty Investment

Objective Molecular Genetics Cell Division Mitosis Introduction Mitosis, cell division which produces two daughter cells both containing a full set of chromosomes, is the natural beginning for a discussion of both cellular reproduction and the molecular basis of heredity. While mitosis itself is not present in all sets of state science standards, the fundamental nature of the concept makes it an important part of any general biology curriculum. An understanding of mitosis is required for a full understanding of meiosis, and meiosis is required for a deep understanding of heredity. Modeling is a valuable way to understand mitosis as a dynamic process. Computer models and animations allow students to watch cell division play out almost exactly as they do in nature. Mitosis is also observable in the lab. Using samples of onion root meristematic tissue, students can freeze cells in the midst of rapid growth and see cells that have been stopped in the middle of mitosis. By sketching the cells that are frozen in different phases of mitosis, students can often intuitively piece together the process of mitosis before they have ever formally discussed it. Procedure

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Materials: Onion roots, acetic acid, hydrochloric acid, aceto-orcein stain, hot plate, forceps, microscope slides, cover slips, d i H 2 O, razor/scalpel, marker Important Aceto-orcein stain takes up to 48 hours to prepare. It is also very light sensitive, so wrap a dark bottle in foil to ensure proper storage. Even wrapped,the stain does not store well, and anything that is not fresh should be filtered two or three times before use. This will ensure crystals do not interfere with visualization. See the stain documentation for more detailed instructions on preparation and storage. Growing Onion Roots 1. Obtain boiling onions (or pearl onions) from a grocery store 2. Insert 3-4 toothpicks into the body of the onion 3. Suspend the onion from the toothpicks into a cup or beaker of water, with the root end down a. The root end should appear flat, possibly with small dried out roots still protruding b. If dead and dry roots are present, remove them before attempting to grow fresh roots 4. Wait 3-4 days for sufficient root growth **See lab pictures for proper set-up

Prepare Fixative Tubes 1. Mix 90 mL of 45% acetic acid and 10 mL of 1M HCL 2. Separate the stock fixative mixture into test tubes, adding about 2 mL of fixative to a test tube 3. Place test tubes in warm water baths at 50C Student Lab Procedure 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Harvest a length of onion root, including the tip, approximately 2 cm long Use a marker to mark the cut end of the root sample Place the root sample in a fixative tube for 6 minutes Dump the test tube contents into a Petri dish and collect the root sample Using a scalpel or razor, cut about 2mm from the tip (non-marked end) and discard the rest of the sample Place the root tip in the center of a microscope slide Add 1 drop of aceto-orcein stain to the root tip Let the stain sit for 90 seconds Rinse excess stain from the sample Repeat steps 7-9 once more Add 1 drop of d i H 2 O and place the cover slip over the sample Press firmly on the cover slip directly over the root sample a. The sample should be flattened and there should be minimal bubbles under the cover slip. View the slide under a microscope

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13. Discussion This lab is unusual because students can successfully complete the lab with very little introduction. The lab procedure should be familiar from their previous experience with creating slides. The students can simply be instructed that they are going to experiment with some different stains to observe the structures that are visible with the new stains. This process will allow the students to use their intuition to guide their work later in the lab. Creating the microscope slides should be a procedure with few complications. When the students are viewing the slides, encourage them to sketch individual cells. If they find (and they should) cells that look significantly different from each other, then they should sketch all of the varieties. With a little guidance, students should have sketches of either four or five cell types. After the students have finished their sketches, discuss what the cells were doing at the tips of the root. Whatever they were doing, we were seeing snapshots of that process. If the roots were growing, then the cells must have been dividing. That means all the cell types in the sketches must be pictures of the cells dividing.

Give the students a chance to attempt to connect their pictures together to describe a dynamic process. Remind the students that their pictures depict still images of a fluid process that occurs in real-time. If the students put their phases up on the board, the groups can vote for which phase is likely first, second, third, etc. The majority of classes will correctly pick each subsequent phase, and in the end they will have independently discovered mitosis. Frequently Encountered Challenges Occasionally While most classes will vote for the correct order of events, there will be some classes that do not. Tell that class that interphase is the starting point (although not by name). Let the class work in groups and vote for which phase comes next. By voting for the correct choice, students usually come up with the correct sequence. Choose your words carefully and allow for some wiggle room if the class vote misplaces a phase. Give the students a fresh vote and continue. Occasionally If the onion roots are not in a period of rapid growth, cells in phases of mitosis may be difficult to find. Ensure the onions have approximately 72 hours to grow roots. This will help minimize the risk, but there is always a chance of having samples with few cells dividing. There are many images online of onion cells dividing, and a Google search for onion root tip mitosis or a similar chain of keywords will return many images that can be provided via projector for students having difficulty finding cells to sketch. Alternatives / Adaptations

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Technology Opportunity If a trinocular microscope is available, students could create a presentation of the different cell types they were able to find in their sample. By attaching a camera to the third ocular lens, students could position each cell type in the center of a frame and take a picture. The pictures could then be loaded into a PowerPoint presentation or other digital product. Image editing and labeling could be done to the images to make the presentations more rigorous for technology savvy classes. Materials Many labs in this book are flexible about the specific lab reagents to be used in the various procedures. This lab, however, requires very specific materials. The fixative, stain, and sample source are all important for the success of the lab. Do not use substitutions.

Its Twins!
Name: Class: Background

Onion Root Tip Mitosis Lab

You have discussed why cells divide; now its time to see how cells divide. If cells are dividing, you should be able to stain the chromosomes in the nucleus and see whats going on. Thats exactly what you will do using onion root tips. Why do you think onion root tips would have a lot of dividing cells? Procedure

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Note: You might recognize this procedure! You did this before when observing cells. This time however, youre going to focus on finding cells that are in the various stages of mitosis. Observing a Plant Cell Dividing Onion Root Tips (Kingdom: Plantae) 1. Harvest a length of onion root, including the tip, approximately 2 cm long 2. Use a marker to mark the cut end of the root sample 3. Place the root sample in a fixative tube for 6 minutes 4. Dump the test tube contents into a Petri dish and collect the root sample 5. Using a scalpel or razor, cut about 2mm from the tip (non-marked end) and discard the rest of the sample 6. Place the root tip in the center of a microscope slide 7. Add 1 drop of aceto-orcein stain to the root tip 8. Let the stain sit for 90 seconds 9. Rinse excess stain from the sample 10. Repeat steps 7-9 once more 11. Add 1 drop of d i H 2 O and place the cover slip over the sample 12. Press firmly on the cover slip directly over the root sample a. The sample should be flattened and there should be minimal bubbles under the cover slip. 13. View the slide under a microscope

Diagram cells that you find that look different from one another try to find 3 or 4.

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Once you have them drawn, put them in sequential order. Place a number under each drawing (i.e. 1 for the first stage, 2 for the second stage and so on). Write a brief explanation to justify your sequencing.