LABORATORY: MITOSIS

OVERVIEW

In this laboratory, you will investigate the process of mitosis. You will use prepared slides of onion root tips to study plant mitosis and to calculate the relative duration of the phases of mitosis in an area of a root where rapid cell growth and division is occuring. Prepared slides of the whitefish blastula, time permitting, may be used to study mitosis in animal cells and to compare animal mitosis with plant mitosis.

OBJECTIVES

After doing this laboratory you should:

• understand the different stages of the cell cycle [interphase (G₁, S, G₂)] [M phase (prophase, metaphase, anaphase, telophase)
• view a plant cell under the microscope and determine which stage of the cell cycle the cell is in
• the significance of the events that occur in each of the stages mentioned above
• an understanding of the differences between mitosis in plant cells and in animal cells

INTRODUCTION

All new cells come from previously existing cells. New cells are formed by the process of cell division which involves both replication of the cell's nucleus (Mitosis) and division of the cytoplasm (cytokinesis). It is imperative that the DNA be moved in an extremely efficient manner during division. If the DNA were to be moved in a way that jeopordizes the integrity of it's code the resulting cells would have defective information that would likely lead to the new cells death. Biologists have been amazed by the beauty and accuracy of cell division. The preparations made by the cell in advance of and during division required millions of years to perfect. Without the many intricate steps that occur during division the chance of growth without disease is all but impossible. It has been demonstrated in many experiments that certain chemical substances, both legal as well as illegal, disrupt one or more of these intricate processes and results in either cell death or the formation of defective cells.

There are two types of nuclear division: mitosis and meiosis. In this lab activity you will explore Mitosis. Mitosis typically results in new somatic (body) cells. Formation of an adult organism from a fertilized egg, asexual reproduction, regeneration, growth, and maintenance or repair of body parts are accomplished through mitotic cell division.

Observing Mitosis in Plant and Animal Cells Using Prepared Slides of the Onion Root Tip and Whitefish Blastula

Where does one find cells undergoing mitosis? Plants and animals differ in this respect. In higher plants the process of forming new cells is restricted to special growth regions called meristems. These regions usually occur at the tips of stems or roots (growth in length) and in many is also found in a circular band of the stem allowing secondary growth (growth in width). In animals, cell division occurs anywhere new cells are formed or as new cells replace old ones. However, some tissues in both plants and animals rarely divide once the organism or tissue is mature (ie: fluid conducting tissues and strenghtening tissues in plants, brain neurons and heart muscle in animals).

To study the stages of mitosis, you need to look for tissues where there are many cells rapidly growing and dividing. This restricts your search to the tips of growing plants such as the onion root tip or, in the case of animals, to rapidly dividing tissue such as developing embryos.

Roots consist of different regions. The root cap functions in protection. The apical meristem is the region that contains the highest percentage of cells undergoing mitosis. The region of elongation is the area in which growth occurs. The region of maturation is where root hairs develop and where cells differentiate to become xylem, phloem, and other tissues (Figure 3.1)

Cells of the Apical Meristem Close up.

The whitefish blastula is excellent for the study of cell division in animal tissue. It is a sperical mass of cells that represents a stage in the development of most animals. As soon as fertilization occurs the egg begins to divide, and nuclear division after nuclear division follows. For mammals, what was once just one cell will form a mature individual consisting of Trillions of cells. All of these cells were a consequence of Mitosis. You will be provided with a slide that contains many whitefish blastula which have been sectioned in various planes in relation to the mitotic spindle. A stain has been used to highlight the chromosomes and mitotic spindle apparatus. If time permits, look at the whitefish blastula slide. If you look carefully you will notice that the whitefish cells reveal a couple of features not seen in the onion root tip.

• mitotic spindle clearly visible with astral rays
• cleavage furrow during cytokinesis rather than a cell plate

Cells of the Whitefish Blastula close up

PROCEDURE

Obtain a prepared slide of the onion root tip. While observing under scanning power, move the slide to notice that there are three different root tips on this slide. It may be necessary to look at all three tips in order to complete the lab. While one member of the team is following the procedure shown below the other team member should use one of the plastic cups at your lab bench and make 5 small circles on your lab write up which will represent the microscopes field of view. In these circles you will draw an onion root tip cell. One for each of the five stages (interphase, prophase, metaphase, anaphase, telophase)

• Move the meristematic region of one tip into the center of the field of view.
• Switch from scanning power to low power, focus, and move the meristematic region into the center of the field of view again.
• Lower the stage slightly and switch from low power to high power.
• While looking from the side, raise the stage as high as is possible without the objective lens touching the slide. (Be aware these prepared slide cost as much as $10 ea and you will be charged for slides being broken)
• Use the fine adjustment to bring the meristematic tissue into focus.

Drawings & Questions:
Locate a cell revealing the Interphase stage and draw it in one of the circles in your lab write up.
1- What was visible to you that revealed that the cell was in Interphase?

Locate a cell revealing the Prophase stage and draw it in one of the circles in your lab write up.
2- What was visible to you that revealed that the cell was in Prophase?

Locate a cell revealing the Metaphase stage and draw it in one of the circles in your lab write up.
3- What was visible to you that revealed that the cell was in Metaphase?

Locate a cell revealing the Anaphase stage and draw it in one of the circles in your lab write up.
4- What was visible to you that revealed that the cell was in Anaphase?

Locate a cell revealing the Telophase stage and draw it in one of the circles in your lab write up.
5- What was visible to you that revealed that the cell was in Telophase?

6- Explain why the whitefish blastula and onion root tip are selected for a study of mitosis.

7- Why is a cleavage furrow not evident in the onion root tip cells?

Before you begin the next exercise check your ability of identifying onion root tip cells in the various stages of the cell cycle by using the web site shown below
http://www.biology.arizona.edu/cell_bio/activities/cell_cycle/01.html

OVERVIEW

To estimate the relative length of time that a cell spends in the various stages of cell replication, you will examine the mereistematic region of a prepared slide of the onion root tip. The length of the cell cycle is approximately 24 hours or 1440 minutes for cells in actively dividing onion root tips.

PROCEDURE

It is hard to imagine that you can estimate how much time a cell spends in each phase of cell replication from a slide of dead cells yet this is precisely what you will do in this part of the lab. Since you are working with a prepared slide, you cannot get any information about how long it takes a cell to divide. What you can determine is how many cells are in each phase. From this, you can infer the percent of time each cells spends in each phase. BE AWARE THAT BEING ABLE TO ACCURATELY IDENTIFY THE STAGE THAT A CELL IS IN WILL BE NECESSARY TO GET FULL CREDIT FOR THIS PART OF THE LAB ACTIVITY.

• Create a table like the one shown below in your lab write up.
• In an onion root tip slide observe every cell in one high power field of view (40 or more cells) and determine which phase of the cell cycle it is in. This is best done in pairs. The partner observing the slide and moving up and down the rows of cells calls out the phase of each cell viewed while the other partner records a mark in the appropriate part of the table. View the cells in columns and simply count the various stages as you move down the rows. Count at least two full fields of view. If you have not counted at least 120 cells, then count a third field of view.
• It is obvious that all phases of the cell cycle do occur but it is possible that you have moved to a region of the root tip where a particular phase does not seem to be present. make sure that after the third viewing region you have have observed at least one cell in each of the five phases. Record your data in Table 3.1

Table 3.1

8. Calculate the percentage of cells in each phase. Considering that the average complete cycle in onion root tip cells requires approximately 1440 minutes to complete, calculate the amount of time spent in each phase of the cell cycle from the percent of cells in that stage. (ie: 1440 x 25% = 360min)

9. In your lab write up make a Pie Chart that reveals the amount of time the cell spends in each of the five major stages of the cell cycle.

10. Which stage of the cell cycle would you expect to take the longest period of time? Explain your answer!

11. Explain why cells that reach a particular size either divide into two smaller cells or stop growing and remain that size the remainder of their existence.