Lab 8. Cell Division

OBJECTIVES

• Understand the processes of mitosis and cytokinesis in plant and animal cells.
• Recognize and observe the stages of cell division in onion root tips.
• Stain onion root tips to visualize the nuclei of mitotic cells.
• Estimate the relative duration of cell cycle phases by observing root tip cells.
• Compare and contrast the processes of mitosis and cytokinesis in animal and plant cells.
• Understand how chromosomes move during mitosis and meiosis.
• Explore how meiosis generates genetic variation.

PRELAB

• Do the practice activity on the Biology Project site (University of Arizona):
  The Biology Project (arizona.edu)
• A data collection table for the onion root tip staging exercise must be in your lab notebook at the beginning of lab.
• Know the definitions of the following terms: cell division, mitosis, meiosis, cytokinesis, cell plate, cleavage furrow, spindle, prophase, metaphase, anaphase, telophase, chromosome, chromatin, centromere, centriole, haploid, diploid. These do NOT have to be in your lab notebook.

INTRODUCTION

Cell division is composed of two processes, mitosis and cytokinesis. Mitosis is the process that leads to the equitable distribution of genetic material into the two nuclei of two daughter cells. Cytokinesis (“cell movement”) is the process that leads to the equitable distribution of cytoplasm to the two daughter cells. In short, mitosis is the division of the nucleus and cytokinesis is the division of the cytoplasm.

In this lab, you will observe these two processes. You will stain the nuclei of onion root tip cells and look for cells in the various phases of mitosis and those undergoing cytokinesis. You will also observe these stages using prepared slides
of onion root tips.

PROCEDURE

Exercise A: Preparation of Onion Root Tips (for Mitotic Study)

Onion bulblets were grown for this activity; actively growing root tips allow you to observe the process of somatic cell reproduction. In this exercise you will prepare your own onion root tip “squash”. You will stain and make a squashed mount of a root tip.

CAUTION: You will be working with strong acids in this lab. You will be required to wear protective eyewear.

MATERIALS

• Onion root tips in Carnoy fixative
• Dropper bottle of 6 Molar HCl
• Forceps
• Dropper bottle of Carnoy fixative
• Razor blade
• Dropper bottle of water
• Watch glass
• Slide
• Toluidine blue
• Spot Plates
• Cover slip
• Kimwipes
• Plastic pipets

1. Onion bulblet roots were grown to 1-2 cm and cut off cleanly with a razor blade. They were then carefully transferred to a small vial of Carnoy fixative. The roots have been in the fixative for at least 4 hours, but no longer than 48 hours.
2.  Using a pair of forceps, obtain one onion root tip and place it in a well of the spot plate.
3. Add a few drops of HCl acid to the root tip, enough to cover it, and let it soak for 4 minutes. BE CAREFUL with the HCl – it is an acid! Wear your goggles!
4. Using forceps, remove the tip and place it in another well of the spot plate.
5. Add a few drops of Carnoy’s Solution, which is in the container that originally held the onion root tips. Again, add enough to cover the root tip. Let it soak for another 4 minutes.
6. Using a pair of forceps, transfer the root tip to a clean slide.
7. With the razor blade, cut off 1-2 mm of the root tip and discard all but the tip.  Use a dissecting microscope to determine which end is the tip! The waste piece can go in the trash, do not put them back in the jar.
8. Cover the tip with a few drops of toluidine blue for 2 minutes.
9. After 2 minutes, gently blot away the stain with a paper towel or Kimwipe®. Be careful not to touch the tip. Throw away the paper towel or Kimwipe®.
10. Add 1 to 2 drops of water to the root tip and gently lower a cover slip onto the tip.
11. Cover the slide with a paper towel and or Kimwipe®, and with eraser end of a pencil, firmly press down on the coverslip to squash the tip. Be very careful not to twist the coverslip! You want to spread the cells into a single layer, but you don’t want to break them apart. Throw away the paper towel or Kimwipe®.
12. Observe your specimen first using the 4X objective to find the tip, then go to progressively higher power to identify cells in different stages of cell division; you will likely find that locating cells in particular phases of mitosis will be easiest under low power. Remember that the tip region will have most such cells.
13. If you find the cells are still too thick, you can try squashing them again.
14. Observe and sketch cells in of each of the following phases of cell division: Interphase, Prophase, Metaphase, Anaphase, and Telophase. Use your book (and the optional Photo Atlas if you have one) as well as the posters and other materials in the lab room as a guide.

Exercise B: Mitosis & Cytokinesis in Plant (Onion Root Tip) Cells

In this exercise you identify the phases of mitosis in the cells of an onion root tip (using a prepared slide). This process is called staging. If you did not find good examples of the stages of mitosis in your squash slide from previous exercise, you may draw the stages of mitosis (in plant cells) from the prepared onion root tip slide. Remember that good drawings include clear representations of structure, have important structures clearly labeled, have a title and overall magnification.

MATERIALS

1. compound microscope
2. onion root tip (prepared slide)
3. Find, observe, and draw examples of interphase, prophase, metaphase, anaphase, and telophase in the prepared onion root tip slide. You only need one sequence of these drawings – drawn either from the “squash” or the prepared slide. You must have your drawings in your lab notebook before you leave the lab session.
4. Remember to begin your observations at using the lowest power objective.
5. Draw your sketches when observing the cells using the high power objective. Label chromosomes, chromatin, nuclear membrane, nucleolus, cell plate and cell wall. Include the total magnification (at which you observed each cell).
6. How to determine the phase of mitosis (staging) of an onion cell:
7. Staging: Determine the stage of mitosis in a block of ~100 onion root tip cells.
   •  You should be observing the cells at high power, with the field of view showing an area where there are many cells undergoing mitosis.
   • Place the pointer on a vertical row of cells.
   • Indicate the stages of each cell as you go down the row by making a check next to that stage in the following data table.
   • Stage all cells that have a visible nucleus.
   • Stage approximately 100 cells. Enter the total number of cells in each stage in the “total” column (in the table below). Compute the percentage of the total number of cells in each stage. The data table should be in your lab notebook.

Duration of Interphase and Mitotic Phases in Onion Root Tip Cells

ANALYSIS

1. Determine the approximate duration of each stage of the cell cycle.

• If onion root tip cells take approximately 16 hours to complete a cycle of cell
  division, calculate the amount of time spent in each stage.
  Time = [(number of cells in each phase)/(total number of cells)] x 16
• Create a pie chart to illustrate the cell cycle in the onion root tip: show the
  amount of time spent in each stage and put each stage in chronological
  order. Why is this the best type of graph to use for this data? You must
  have your pie graph in your lab notebook before you leave the lab
  session.

Post-lab Questions

1. Describe, compare and contrast the appearance of onion root tip cells in interphase, prophase, anaphase, metaphase and telophase. What are the distinguishing features that you could observe in each stage?
2. What structure was visible during cytokinesis, in onion root tip cells? From your reading, describe the main differences between mitosis & cytokinesis in animal and plant cells.
3. Are all of the onion cells in your slide(s) active in cell division? Why did you observe cells from onion root tips to study the phases of mitosis, and not from some other parts of the onion (such as stems or leaves).

EXERCISE C – MODEL SIMULATIONS

Part A – Mitosis

1. Begin with a cell that has 6 beaded chromosomes: two long, different colored chromosomes; 2 medium, different colored chromosomes; and two short, different colored chromosomes. Three chromosomes (one long, one medium, and one short) were present in the sperm and one of each in the egg that fused to form thisindividual. That is why the chromosomes of the same lengths should be different colors. This situationrepresents a diploid cell (2n) where 2n=6. Save the identically colored pipe cleaners for use as replicatedchromosomes.
2. Use the chalk or string to create the cell membrane and nuclear membrane of your cell when it is in interphase, prior to DNA replication (S-phase).
3. Simulate DNA replication by twisting together two identical beaded chromosomes
4. Once all of the chromosomes are replicated, proceed into mitosis.
5. Take your cell through from prophase to telophase, physically moving the beaded chromosomes, erasing or adding in structures as they appear or disappear, and discussing what is occurring with your lab partners. Choose whether this cell was a plant or animal cell, and show cytokinesis. You should end up with two cells that look identical to the one with which you began.
6. In order to get full credit for this activity, you and your partner must demonstrate one round of mitosis to your instructor. You should be able to answer the questions below.
7. Questions to ask yourself at each stage:
   • Are the cells haploid or diploid?
   • Is the DNA of a chromosome replicated or unreplicated?
   • Where are the chromosomes located?
   • How many centromeres are present?
   • How many sister chromatids are present?
   • What are the microtubules doing at this point?

Part B – Meiosis 

1. Now demonstrate meiosis using the same diploid cell (2n) where 2n=6. This means you will need long, medium, and short beaded chromosomes.
2. Use the chalk and/or string as described above
3. Take this cell from interphase through both meiosis I and meiosis II. Discuss what is occurring and be sure that you could label the parts of a drawing of any stage.
4. In order to get full credit for this activity, you and your partner must demonstrate one round of meiosis (meiosis I and II) to your instructor. You should be able to answer the questions below.
5. Questions to ask yourself at each stage:

1. • Could crossing-over occur now?
   • Have the sister chromatids of replicated chromosomes separated from one another?
   • Have the homologous chromosomes separated from one another?

Answer the following questions:

1. All of the trillions of cells in your body trace their ancestry back to a fertilized egg. Further, all of these cells, except for the sperm and eggs in your reproductive organs, arose by mitosis. Based on these observations, are your liver cells genetically different from your nerve cells? Explain in 1-2 sentences.
2. Besides independent assortment of chromosomes, what is one additional way that sexual reproduction can generate genetic diversity?