NANSLO REMOTE LAB
SEMESTER: FALL 2013
TYPE OF LAB: REMOTE LAB
TITLE OF LAB: MEIOSIS AND MITOSIS
Lab format: This lab is a remote lab activity.
Relationship to theory: In this lab you will be examining the underlying processes that make up the cell
cycle.
LEARNING OBJECTIVES
After completing this laboratory experiment, you should be able to do the following things:
1. Describe the cell cycle.
2. Identify the stages of mitosis from prepared slides, this includes: interphase, prophase,
metaphase, anaphase, telophase, and cytokinesis.
3. Describe the stages of meiosis. Understand the difference between the terms haploid and
diploid.
4. Recognize the male testis and female ovaries.
5. Recognize the processes of meiosis and mitosis, compare and contrast the two using the
scientific method.
6. Complete a lab report using the information from objectives #1 - #4. Be able to gather and
interpret information to form a concise and objective laboratory report.
7. Display the ability to use a microscope, calculate simple equations, and capture images.
8. Label the process of mitosis and the organs of meiosis (the ovary and the sperm) with the
captured images and attach them as an appendix A and B to the required lab report.
BACKGROUND INFORMATION
If I asked you “Where do cells come from?”, what would you answer? In modern biology our
understanding of a the cell as the basic building block of life is codified in a set of principles called the
Cell Theory which was first codified by Schleiden and Schwann in 1838-39. The cell theory is second only
to the theory of evolution by natural selection in understanding the relatedness of life. Cell Theory says
the following:
1. All living organisms are composed of one or more cells.
2. Cells are the basic building blocks of all life.
3. All cells are descended from a preexisting cell.
While these may seem like relative simple points it took scientists several centuries to produce the cell
theory.
The development of the cell theory directly follows the development of the microscope. The name
“cell” was coined by Robert Hooke6 in 1665. While observing a piece of cork under his microscope he
thought that the microscopic units that made up the cork looked like the rooms, or “cella” in Latin, that
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NANSLO REMOTE LAB
SEMESTER: FALL 2013
TYPE OF LAB: REMOTE LAB
monks lived in. This was closely followed by the discovery of single celled organisms by Antoni van
Leeuwenhoek3-5 in 1676. Leeuwenhoek discovered motile microscopic particles by examining scrapings
from his teeth under his microscope. In 1838 Metthias Schleiden7 and Theodor Schwann8 presented
evidence that all plants and animals are composed of cells. However, there were still some questions as
to where cells came from, as Schleiden believed cells formed through a process of crystallization. This
theory was simply a variant on the belief of Aristotle that life could come into existence by spontaneous
generation.
It was not until the 1850s that a group of scientist was able to show that new cells were produced from
preexisting cells9. However, most scientists believe the definitive test disproving spontaneous creation
of microbial life was conducted by Louis Pasteur in 186210. In Pasteur’s experiment two flasks were each
set-up with bacterial growing broth (a liquid that is conducive to the growth of bacteria) and
sterilized. Both flasks were left open to the air but in such a way that dust could enter only flask 1 not
flask 2. After a period of time bacteria growth was seen in only flask 1 and not in flask 2. This showed
that dust (bacteria) had to be added to the broth in order for bacteria to grow.
In this lab we will be examining the mechanism underlying the third principle of the cell theory “that all
cells are descended from preexisting cells”. There are two processes involving the production on new
cells, the first process, mitosis, is used for growth and to replace old or dead cells. The second process,
meiosis, is used to produce gametes (egg and sperm) cells that are used for sexual reproduction. An
important distinction to keep in mind is that we describe the type of cell cycle that is occurring based on
what is happening in the nucleus and genetic material.
The mitotic the cell cycle (see figure 1) in the simplest form is composed of two parts Interphase and
Mitosis. However, each of these parts can be further divided. Mitosis can be divided into four parts:
Prophase, Metaphase, Anaphase, and Telophase which will be described below. Mitosis, in fact, means
the division of the nucleus to produce two identical daughter cells. The division of the cell itself is called
cytokinesis and overlaps telophase but is not actually classified as part of it. Interphase (the part of the
cell cycle between actual divisions) is composed of three parts Gap1 (sometimes referred to as growth1)
the cell grows and performs normal cellular functions, Synthesis (s phase) DNA is replicated, and Gap2
(sometimes referred to as growth2) is where the cellular organelles are replicated. There is one
additional phase to the cell cycle Gap0. A cell that has stopped cycling (dividing) either temporarily or
permanently has entered Gap0.
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NANSLO REMOTE LAB
SEMESTER: FALL 2013
TYPE OF LAB: REMOTE LAB
The different stages of the cell cycle were identified as morphological changes by Waclaw Mayzel in
18751,2. All these morphological changes can be observed in a compound microscope. During
Interphase (figure 2A) there is a clearly defined nuclear envelope filled with dispersed chromosomes. As
the cell enters Prophase (figure 2B) the chromosomes condense and the mitotic spindle forms. At this
point each chromosome is composed of two sister chromatids joined at the centromere. Additionally,
the nuclear envelope breaks down and the mitotic spindle begins attaching to the chromosomes at the
centromere. (In some texts the breakdown of the nuclear envelope and the attachment of the mitotic
spindle to the chromosomes is listed as an additional stage Prometaphase). In Metaphase (figure 2C)
the chromosomes line up in the middle of the cell forming a structure called the metaphase
plate. During Anaphase (figure 2D) the kinetochore splits and each chromatid now a chromosome is
pulled to opposite sides of the cell. In the last phase Telophase (figure 2E) the chromosomes become
less condensed, two new nuclei form and the mitotic spindle depolymerizes. This officially ends mitosis
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NANSLO REMOTE LAB
SEMESTER: FALL 2013
TYPE OF LAB: REMOTE LAB
which as mentioned before is the replication and division of the nucleus. The cell cycle ends with
cytokinesis, the division of the cytoplasm, which often overlaps late telophase.
The other process, meiosis, is used to produce gametes (sperm and egg in most animals and plants) that
are used to produce the next generation. In meiosis two rounds of cell division occur with only one
round of synthesis. This produces 4 haploid 1n cells (meaing having 23 chromosomes), where mitosis
has diploid 2n (46 chromosomes) . In meiosis S phase occurs as normal in the first round of cell
division. The first difference between mitosis and meiosis occurs in Prophase I (Figure 3) during
Prophase I the homologous chromosomes pair up and exchange genetic material by crossover. This
exchange of genetic material increases the variation in the offspring. The next difference occurs in
anaphase I during anaphase I instead of the kinetochore dividing it stays connected and the homologous
chromosomes are segregated to the opposite poles (figure 3). During Cytokinesis I we see the first
difference between spermatogenesis (sperm formation) and oogenesis (egg formation). During
cytokinesis of the egg the cytoplasm divides unequally with one of the daughter cells getting most of the
cytoplasm, the smaller cell is called a polar body (Figure 3B). The sperm undergo an equal cytokinesis
(Figure 3A). The cells will then enter a second cell cycle without replicating their DNA. This time during
Anaphase II the kinetochore divides and the sister chromatids are pulled to opposite poles of the
cell. Again the egg undergoes unequal cytokinesis producing an oogonia and another polar body, while
the sperm cells divide equally. This produces four 1n cells in the male line and one 1n cell in the
female. These cells then go on to mature in to sperm and egg cells.
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NANSLO REMOTE LAB
SEMESTER: FALL 2013
TYPE OF LAB: REMOTE LAB
References:
1.
2.
3.
4.
5.
6.
Medycyna, czasopismo tygodniowe dla lekarzy (1875; 3(45), 409/0412)
Centralblatt f. die Med. Wissenschaften (1875; 50: 849–852)
Dobell, C. Antony van Leeuwenhoek and His “Little Animals” (Dover, New York, 1960).
Wolpert, L. Curr. Biol. 6, 225–228 (1995).
Singer, S. A Short History of Biology (Clarendon, Oxford, 1931).
Westfall, R. S. Hooke, Robert in Dictionary of Scientific Biography Vol. 7 (ed. Gillespie, C.) 481–
488 (Scribner, New York, 1980).
7. Schleiden, M. J. Arch. Anat. Physiol. Wiss. Med. 13, 137–176 (1838).
8. Schwann, T. Mikroskopische Untersuchungen über die Übereinstimmung in der Struktur und
dem Wachstum der Tiere und Pflanzen (Sander’schen Buchhandlung, Berlin, 1839).
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NANSLO REMOTE LAB
SEMESTER: FALL 2013
TYPE OF LAB: REMOTE LAB
9. Mayr, E. The Growth of the Biological Thought (Belknap, Cambridge, MA, 1982).
10. Pasteur, L. A. Ann. Sci. Nat. (part. zool.) 16, 5–98 (1861).
EQUIPMENT
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Paper
Calculator
Pencil/pen
Slides (in order)
o Whitefish blastula
o Onion Root Tip
o Mammal Graafian Follicles
o Human Testis
Computer (need to use a PC, NOT a Mac)
PREPARING TO USE THE REMOTE WEB_BASED SCIENCE LAB (RWSL):
Follow the instructions provided to you by your instructor to access this lab.
INTRODUCTION TO THE REMOTE EQUIPMENT AND INTERFACE
You are going to be asked to “capture” many images throughout this lab. Prior to beginning the lab, create
a New Folder in the area of your Desktop or Documents if you are using the Microsoft Word Program.
This will allow you to send the images directly to the folder, so that you can access them later. There are
some structures you will need to identify on the captured slides. You can do that AFTER the lab. You are
allowed to hand-write these in, if it makes it easier for you.
EXPERIMENTAL PROCEDURE:
Once you have logged on to the microscope you will perform the following Laboratory procedures:
EXERCISE 1: MITOSIS IN ANIMAL AND PLANT CELLS
New cells are produced in animals and plants by the division of old cells. These new cells can be used for
growth or to replace dead or damaged cells. As stated in the introduction, the cell cycle is divided into
two parts the replication and division of the genetic material (mitosis) and the division of the cytoplasm
(cytokinesis). In this experiment you will use prepared slides of an onion root tip and a whitefish blastula
to identify the stages of mitosis.
The onion root tip is divided into four sections based on the behavior and function of the cells (Figure
4). The first region is the root cap which protects the growing root. The second is the meristeam, a region
of highly mitotically active cells. Then there are the elongation regions where cells are growing, and then
lastly the maturation region where cells become fully mature root cells. At the stage of development you
are looking at, the cells in the white fish blastula are a uniform population.
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NANSLO REMOTE LAB
SEMESTER: FALL 2013
TYPE OF LAB: REMOTE LAB
Procedure: You are required to review two slides. The first is the whitefish blastula (animal cell), and the
second is the onion root tip (plant cell). You will be capturing each phase of mitosis from each specimen.
Remember, this includes prophase, metaphase, anaphase, and telophase. After you complete your data
collection, you will go into your word document and label the slides as instructed in this lab. Begin at the
10x objective focus and then move up to the 60x objective. You will be required to refocus at each stage.
You can use the autofocus button once you have partially focused the image. Attach these captured
images to Appendix A of your report.
1. The team member that has control of the microscope, will ask the technician to select the
prepared slide of the whitefish blastula Locate the blastula, then increase the magnification and
identify each of the stages of mitosis. Use the “capture image” feature on the RWSL control
panel to capture an image of each of the above stages of mitosis. To capture an image you will
click on the capture image button, then click on the “view captured image” towards the bottom
of the screen.
2. After you have completed the lab data collection and captured the pictures, label one cell each
in the stages of mitosis, to include: prophase, metaphase, anaphase, telophase, and cytokinesis.
Use the “picture gallery” in Blackboard under the RWSL homepage icon in your course to help
you to identify the stages. Add to Appendix A of your lab report.
3. Change team members and ask the technician to select the prepared slide of the onion root tip.
4. Locate the onion root tip then increase the magnification and identify the stages of mitosis. Use
the “capture image” feature on the RWSL control panel to capture an image of each of the
above stages of mitosis.
5. After you have completed the lab and captured the pictures, complete the same labeling as in
step 2 and add to appendix A.
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NANSLO REMOTE LAB
SEMESTER: FALL 2013
TYPE OF LAB: REMOTE LAB
EXERCISE 2: GROWTH IN THE ONION ROOT
In this exercise we are going to study the growth of the onion root. Growth can be effected by both the
number of cells and the size of the cells. You will look at one area of the onion root the tip in the
elongation region. In this region you will determine percentage of cells that are in any stage of mitosis
(often called the mitotic index).
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NANSLO REMOTE LAB
SEMESTER: FALL 2013
TYPE OF LAB: REMOTE LAB
ADDITIONAL RWSL ACTIVITY: CALCULATING THE PERCENT TIME SPENT IN EACH
STAGE OF ONION ROOT TIP MITOSIS
At the time when a slide of an onion root tip was prepared, the cells in the region of cell division were
arrested at their current phase within the cell cycle. Some were fixed at the time of interphase and
others were fixed at some stage of mitosis. The duration of each stage in the cell cycle of the onion root
tip can be estimated by determining the proportion of cells arrested at each stage of mitosis and
interphase.
Let’s assume that you examined a slide and determined the stage at which about 200 cells were
arrested at time of fixation. It is known that onion root tip cells take about 16 hours to complete the cell
cycle. By determining the percentage of cells in each stage of mitosis and in interphase, you can
calculate the amount of time spent in each stage. For example, if twenty cells out of 200 were found to
be in prophase, the percentage of cells is 20/200 x 100 = 10%. This shows that any one of the
hypothetical cells spends 10% of the time in prophase, so they spend 0.10 x 16 hours or 1.6 hr (1 hr and
36 min) in that stage.
Procedure:
Examine the slide of an onion root tip using the 40x objective.
1. Count exactly 50 cells in your field of view.
2. Using those same 50 cells, record the number of cells that are going through each stage of
mitosis. (see the graph below). Multiply each total by 2. (This will give you a representation of
100 cells)
3. Calculate the hours and minutes spent in each stage, assuming the entire cell cycle takes 16
hours (0.1 hr = 6 min).
Example: out of the 100 cells counted (after x 2): 10 are in metaphase. 10 metaphase cells (divided by)
100 total cells = 10%. The complete cell cycle is estimated at 16 hours. 10% of 10 hours is 1.6 hours.
Number of cells in stage = 10; % of total cells in stage = 10%; Hours and minutes of the stage = 1.6
hours
Take a look at Figure 1 in this lab, “The Mitotic Cell Cycle.” This will give you an idea of the time these
cells spend in different stages. Clearly, the majority of your cells will be in interphase.
Complete the data collection as a group. You can choose to do your calculations as a group during the
lab or finish your calculations at home once the data is collected. It may be easier to do at home so that
you can mark the cells you have counted with colored pens.
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NANSLO REMOTE LAB
SEMESTER: FALL 2013
TYPE OF LAB: REMOTE LAB
TABLE 1: DATA ON STAGES IN PLANT CELLS – Elongation Region
Cell Cycle Stage
Number of Cells in
the Stage
% of Total Cells in
the Stage
Interphase
Prophase
Metaphase
Anaphase
Telophase
Total
100
1. Which stage takes the longest? The shortest?
2. How does this compare to the time it takes for meiosis to occur?
Hours and Minutes
in the Stage
16:00
EXERCISE 3: MEIOSIS IN ANIMALS
In most animals meiosis occurs in special tissues, the ovary in females and the testes in males. These
tissues are composed of cells that support the developing germ cells. Additionally, the final cells the egg
and sperm often look different. In this experiment you will identify the support cells in ovaries and identify
the fully differentiated sperm in testes.
Procedure: Once the slides are captured and labeled, add these to Appendix B of your lab report. Use a
40x or 60x objective.
1. Ask the technician to select the prepared slide of the Mammal Graafian Follicles.
2. Capture an image and label the primary follicle, primary oocyte, secondary follicle and
secondary oocyte. Use your “picture gallery” on Blackboard to help you label these structures.
3. Select the prepared slide of the Human Testis. Capture an image and label a mature tailed
sperm. Include in Appendix B.
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