LAB 2
MITOSIS AND MOVEMENT ACROSS CELL MEMBRANES
I CELL DIVISION AND THE CELL CYCLE
At the end of this exercise, you should be able to:
1.
2.
3.
4.
Discuss the stages of the cell cycle.
Explain the importance of mitosis.
Identify the stages of mitosis in slides, models or diagrams.
Describe the events occurring during each stage of mitosis.
PART A: THE CELL CYCLE
Somatic cells reproduce for the purpose of growth, replacement and repair.
Cell
reproduction is a regulated series of events known as the cell cycle. The cell cycle is
divided into two stages:
i)
interphase – the time during which the cell grows in size, carries out its normal
metabolic activities and prepares for cell division,
ii)
cell division – the series of events that lead to the production of two identical cells
often referred to as "daughter cells" from an original "parent cell."
Figure 2.1 The cell cycle.
1. Interphase is divided into three phases:
a) During G1 (1st Gap phase), the cell grows and conducts normal cellular functions.
The cell also prepares for cell division by duplicating most of the organelles and other
cytoplasmic materials.
b) During the S phase (synthesis) of interphase, DNA is replicated.
c) During G2 (2nd Gap phase) Enzymes and other proteins that will aid in mitosis are
produced, and centriole replication is completed.
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The frequency with which cells divide varies depending on the type of tissue. Skin
cells, for example, must be continuously replaced. This replacement is done by
stem cells, cells that continuously divide, giving rise to new cells. You will encounter
other forms of stem cells as the course progresses. Liver cells, on the other hand,
seldom need replacing, and so divide only rarely. Some cells, such as neurons and
muscle cells enter a non-dividing stage known as G0. If these cells die they can
never be replaced.
2. Somatic cell division in eukaryotes (M phase of the cell cycle) consists of a series of
events called mitosis and cytokinesis. Mitosis is the division of the nuclear material
(DNA/chromosomes); cytokinesis is the division of the cytoplasm and organelles
between two daughter cells.
homologous chromosomes
homologous chromosomes
Figure 2.2 Representative chromosomes prior to mitosis
a) Mitosis is subdivided into four major phases:
i)
Prophase
• chromatin fibers condense into visible chromosomes
• the nucleolus disappears
• the nuclear envelope breaks down
• the centrioles are pushed to opposite poles by lengthening microtubules
• spindle fibers form and asters are visible around the centrioles
ii) Metaphase
• the microtubules of the spindle fibers align the centromeres of each pair
of chromatids at the metaphase plate
iii) Anaphase
• the centromeres split and the chromatids separate and move towards
opposite poles.
• the chromatids are now called chromosomes and appear V-shaped
iv) Telophase
• chromosomes are at the poles and begin to uncoil into chromatin
• the nuclear envelope reforms around the chromatin
• the nucleoli reappear
• the spindle breaks down
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b) Cytokinesis begins in late anaphase, with a slight indent in the plasma membrane
called the cleavage furrow. As the microfilaments contract, the plasma membrane
is pulled inward, completing the division of the cell into two separate daughter cells.
When cytokinesis finishes, interphase begins.
By the end of cytokinesis, each daughter cell is genetically identical to the parent cell,
and has half the cytoplasm of the mother cell. The daughter cells grow and carry out
their metabolic processes until it is their turn to divide.
3. Human somatic cells have 23 pairs of chromosomes. One chromosome of each pair is
inherited from each of the parents via the egg and sperm. Each pair of chromosomes
contains similar genes. Genes are segments of DNA that code for an individual's
inherited traits.
Draw and label a dividing cell with six chromosomes during prophase (three maternal and
three paternal).
Draw the six chromosomes to indicate metaphase.
How would the chromosomes appear during anaphase and telophase?
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How many chromosomes are present in this cell during anaphase?
What is the role of microtubules:
a. In prophase?
b. In metaphase?
c. In anaphase?
What is one possible reason that chromatin fibers condense before dividing?
What types of cells enter the G0 phase?
PART B: WHITEFISH MITOSIS
Observe the slide of whitefish blastula. A blastula is an embryonic stage of development
that occurs shortly after fertilization, when the embryo is undergoing rapid cell division.
Scan across a number of blastula slices on the slide to identify cells in interphase and the
phases of mitosis.
Hints:
• For a cell in interphase, look for a distinct nucleus, with no visible chromosomes.
• For prophase, find disorganized but visible chromosomes, with no nuclear membrane.
• In metaphase, the chromosomes will be in a single dark line, lined up on the
metaphase plate.
• For a cell in anaphase, there should be space between two lines of chromosomes;
the chromosomes may appear V-shaped.
• In telophase, there should be a visible cleavage furrow.
• Any cells that have just completed telophase will be slightly smaller, have a visible
nucleus, and still be very close together.
Sketch what you see in the space below and label all the structures (chromosomes, spindle
fibers, nuclear membrane, cell membrane, asters, nucleoli, chromatin, etc.) that you see.
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Using the materials in the lab and your text, label the structures and stages indicated.
Figure 2.3 Animal mitosis in a cell with four chromosomes.
From Tortora and Derrickson, Principles of
Anatomy and Physiology, 11th Ed., Copyright © 2006 by Biological Sciences Textbooks, Inc. and Bryan Derrickson. This material is used
by permission of John Wiley & Sons, Inc.
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II MOVEMENT ACROSS CELL MEMBRANES
At the end of this exercise, you should be able to:
1.
2.
3.
4.
5.
Compare and contrast the various transport processes that occur across cell
membranes.
Explain Brownian motion and its effect on the distribution of particles in a solution.
Describe the process of diffusion and discuss the variables that can affect the rate of
diffusion.
Describe the process of osmosis and discuss the variables that can affect the rate of
osmosis.
Describe what happens to a red blood cell when placed in an isotonic, hypotonic, or
hypertonic solution.
Cells import a variety of substances such as nutrients, hormones and oxygen from the
extracellular fluid and export wastes and cellular products from the cytosol to the
extracellular fluid. The plasma membrane is a physical barrier that surrounds each cell,
separating it from the environment and regulating which substances pass into and out of
the cell. The plasma membrane is a lipid bilayer with embedded proteins that can form
channels and act as transporters. The plasma membrane is selectively permeable, which
means that it can regulate the passage of materials into and out of the cell. Transport
across the membrane can occur by either passive or active processes.
All molecules have kinetic energy that causes them to be in constant motion. Molecules in
gases and liquids are constantly colliding with adjacent molecules, ricocheting off one
another and changing direction with each collision. This is called Brownian motion, and
can be seen in the movement of small particles suspended in water. Collisions between
molecules cause them to spread out and eventually reach an equal distribution in a
container (equilibrium). Once equilibrium has been reached the molecules will continue to
move spontaneously, but with no net directional change.
Passive processes rely on Brownian movement, and do not require additional energy
expenditure by the cell. Passive processes move molecules along their concentration
gradient, and include filtration, diffusion and osmosis.
Active processes require the use of energy (in the form of adenosine triphosphate, or
ATP). They include active transport (primary and secondary) and vesicular transport.
In active transport, water-soluble substances are moved against their concentration or
electrochemical gradient. Vesicular transport is used to move molecules too large to pass
through membrane channels.
PART A: BROWNIAN MOTION
Demonstration: Observing Brownian motion of ink particles.
Molecules in the cytosol and extracellular fluid are too small for you to see, so we will
demonstrate Brownian motion using fountain pen ink.
1. At the side bench, observe the ink wet mount slide using the 4x objective lens.
You may want to begin by focusing on an air bubble first.
2. Switch to the 10x objective lens, re-focus and move the slide so the air bubble is
just off to one side of the field of view.
3. Move the 40x lens into place and focus on the edge of the air bubble using the
fine focus knob. Once the edge of the air bubble is in focus (a hard, dark curve)
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continue rotating the fine focus knob 1/8th of a turn until you can see the rest of
the visual field filled with tiny particles.
4. Adjust the iris diaphragm to allow a little more or a little less light.
Describe the movement of individual ink particles observed under the microscope.
PART B: DIFFUSION
Diffusion is the movement of molecules from an area of higher concentration of that
molecule to an area of lower concentration of that molecule. The driving force behind
diffusion is kinetic energy. When there is a higher concentration of a substance in one area
the collisions between the molecules will cause the molecules to become evenly distributed,
and equilibrium is eventually reached.
In living systems, examples of simple diffusion include the movement of oxygen from the
lungs into pulmonary capillaries and the movement of urea into the collecting ducts in the
kidneys.
Diffusion of molecules can occur through solids, liquids, gasses, and across cell membranes.
In this section we will see diffusion of molecules through a solid.
Demonstration: Diffusion of dyes through agar.
1. At the side bench are two petri dishes, one set up just prior to your lab, and one
set up 24 hours before your lab. The undersides of the petri dishes have been
marked to divide them into thirds, labeled with S (for safranin O), C (for crystal
violet) or M (for methylene blue).
2. The filter paper discs on the surface of the agar were dipped in 2% safranin O, 2%
methylene blue or 2% crystal violet solutions until saturated.
3. Compare the distance the dyes have diffused: Measure the distance the dye has
diffused using your millimeter ruler and record your findings in Table 2.1.
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Figure 2.4 Results of dye diffusion after 24 hours.
Table 2.1 Diffusion results
DISTANCE DIFFUSED
TIME
(IN MILLIMETERS)
2% SAFRANIN O
2% METHYLENE BLUE
2% CRYSTAL VIOLET
~ 1 hour
~ 24 hours
Look at the plate from the side. Did the dyes diffuse vertically through the agar?
Of the three dyes, which diffused the greatest distance by the end of 24 hours?
Which of the three dyes diffused the shortest distance?
What one variable do you think caused one dye to diffuse a greater or lesser distance
through the agar than the other dyes?
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PART C: OSMOSIS
Osmosis refers to a type of simple diffusion where water moves through a selectively
permeable membrane from an area of higher water concentration to an area of lower water
concentration. Osmosis occurs when the membrane is permeable to water but not to other
substances. Osmosis can also be explained as the net movement of water through a
selectively permeable membrane from an area of lower solute concentration to an area of
higher solute concentration. A solute is any substance that dissolves in another, forming a
solution. The substance the solute dissolves in is known as the solvent.
When comparing two solutions, a solution with a lower concentration of solute is said to be
hypotonic to the solution with the higher concentration of solute. The solution with the
higher concentration of solute is said to be hypertonic to the solution with a lower
concentration of solute. Two solutions with equal solute concentrations are said to be
isotonic.
Demonstration: the Osmometer.
An osmometer demonstration is set up on the side bench, using a thistle tube,
dialysis membrane, and coloured 80% sucrose solution, suspended in a beaker of
water.
Dialysis membrane can be used to demonstrate osmosis because it has very small
holes. Sucrose is a very large molecule and does not fit through the holes, while
water is much smaller and passes freely.
Use the ruler provided to measure the height of the column of solution in the thistle
tube at 15 minute intervals for one hour. Record your measurements in Table 2.2.
Table 2.2 Osmometer results.
TIME
0 minutes
20 minutes
40 minutes
60 minutes
Figure 2.5 Osmometer set-up
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80% SUCROSE
(in mm)
Procedure 1: Osmosis in living cells.
Red blood cells are permeable to water and relatively impermeable to salt. Normally there
is no net movement of water between the cytosol of a RBC and the extracellular fluid
(plasma) because plasma is isotonic to RBCs. In this experiment, you will be observing the
change in appearance of RBCs when they are placed in solutions with different
concentrations of sodium chloride (NaCl).
i.
ii.
iii.
If a RBC is placed in a solution where the cell remains the same size and shape,
then the solution is isotonic to the cell.
When a RBC is placed in a hypotonic solution, water enters the cell, eventually
causing it to burst, a process known as hemolysis.
If a RBC is placed in a hypertonic solution, water will leave the cytosol and enter
the solution outside the cell. The cell crenates and appears shriveled with
spiked edges.
Figure 2.6 Appearance of red blood cells in different salt solutions. From Tortora and
Derrickson, Principles of Anatomy and Physiology, 12th Ed., Copyright © 2009 by Biological Sciences Textbooks, Inc. and Bryan
Derrickson. This material is used by permission of John Wiley & Sons, Inc.
1. Mark three test tubes with a grease pencil to indicate these solutions: 3% NaCl,
0.9% NaCl, and distilled water.
2. At the side bench, place 2 ml of each solution into their respective test tubes.
3. Add one drop of blood to each test tube. Gently rotate each tube to mix.
4. Describe the appearance of the solution in each tube in Table 2.3.
5. Using a new pipette, prepare a wet mount slide of the 3% NaCl blood cells.
Describe the appearance of the RBCs at 40x.
6. Using a new pipette, make a wet mount slide of the 0.9% NaCl blood cells and
observe using a microscope. Describe the appearance of the cells at 40x in Table
2.3.
7. Prepare and observe a wet mount of the blood cells in distilled water.
8. Clean up: Used test tubes should be rinsed out and put in the disposal in the
fume hood. Slides should be rinsed with alcohol and returned to the slide box to
dry. Place used cover slips in the sharps container.
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Table 2.3 Osmosis in RBCs
3% NaCl
0.9% NaCl
appearance
of solution
in test
tube
shape or
appearance
of cells
drawing of
cell shape
Which of the three solutions was hypotonic to the RBCs?
Which of the solutions was hypertonic to the RBCs?
Which of the solutions was isotonic to the RBCs?
Which of the solutions caused the cells to crenate?
Which of the solutions caused the cells to hemolyze?
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DISTILLED WATER
PART D: OSMOSIS AND DIFFUSION ACROSS A SELECTIVELY PERMEABLE
MEMBRANE
In this experiment we will test for the movement of substances through a dialysis
membrane.
Procedure 2: Osmosis and diffusion across dialysis tubing.
1. Obtain a wet dialysis tube from the dish on the side bench. Fold one end over
twice and clamp.
2. Using a funnel and the labeled graduated cylinders, pour 5 ml of 40% glucose, 5
ml of 10% NaCl and 25 ml of 2% starch into the open end of the dialysis bag.
3. Press any air out of the bag. Fold the edge twice and clamp. Your dialysis bag
should be between 1/4 to 1/2 full.
4. Rinse the bag with tap water and quickly blot dry.
Record the weight of your dialysis bag: ________ grams.
5. Place the dialysis bag in a beaker containing 300 ml of room temperature or
slightly warm tap water (just enough to immerse the bag) and take it back to
your desk. Let stand for 60 minutes.
6. While you are waiting to get results, set up the controls for determining if the
substances crossed the dialysis membrane. A negative control will demonstrate
what happens in the absence of what you are testing for, while a positive control
demonstrates what happens in its presence.
a) Obtain and number 4 test tubes in a test tube rack.
b) At the
o
o
o
o
side bench, pour 2 ml of the solution in the numbered tube:
tube 1 – 2 ml 2% starch
tube 2 – 2 ml tap water
tube 3 – 2 ml 10% NaCl
tube 4 – 2 ml tap water
c) At your desk, add 2 drops of iodine to tubes 1 and 2. Swirl gently and
observe any change in colour. The appearance of a deep blue or black colour
indicates that starch is present, and has formed a starch/iodine complex.
Record your observations in Table 2.4.
d) Add 2 drops of silver nitrate to tubes 3 and 4. Swirl gently and observe any
change in colour. The appearance of a white precipitate or cloudiness
indicates the presence of silver chloride (AgCl), which is formed by the
reaction of silver nitrate (AgNO3) with sodium chloride (NaCl). Record your
observations in Table 2.4.
e) Obtain a glucose test strip from the bottle at the side bench. Compare the
colour of the strip to the control colours on the bottle. Record the colours for
'negative' and ‘positive’ results in Table 2.4.
7. After one hour, remove the bag, rinse, blot dry, and weigh. Return the bag to
your beaker of tap water. Record the weight of your dialysis bag: _______
grams.
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Total change in weight of dialysis bag after one hour: ________ grams.
8. Place the glucose test strip into the beaker for 5 seconds. After 30 seconds use
the colour key on the bottle determine whether there is glucose in the beaker.
Record your results in Table 2.4.
9. Obtain 2 more clean test tubes and one new pipette. Place 2 ml of water from
the test beaker into each test tube. Label one tube "starch" and one tube "Cl-".
Add 2 drops of iodine to the tube labeled "starch" and 2 drops of silver nitrate to
the tube labeled "Cl-". Compare the colour of the tested beaker water to the
colour of the control tubes. Record your results in Table 2.4.
10. Add 5 ml of concentrated iodine to the beaker. Do you observe any colour
change in the beaker or inside the dialysis bag after 2 minutes? Record your
experimental results in Table 2.4.
11. Clean up: Remove the clamps from the dialysis tubing. Empty the beaker and
sac into the sink, and dispose of the dialysis tubing in the garbage. Rinse the
clamps and the beaker and return them to your desk.
Table 2.4 Control and Experimental test results.
SUBSTANCE
starch
chloride ions
(from NaCl)
glucose
CONTROL
TUBE
(COLOUR OBSERVED)
1
positive result
2
negative result
3
positive result
4
negative result
-
positive result
-
negative result
EXPERIMENT
(RESULTS OF TESTING
BEAKER WATER)
dark blue/black
any brown colouring
Did the dialysis bag gain or lose weight?
Which substance(s) net movement caused the change in weight (starch, chloride, glucose,
or water)?
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In Table 2.5 indicate whether the following substances moved across the membrane, and in
which direction. What conclusions can you make about the relative sizes of the substances
used?
Table 2.5 Summary of Osmosis and Diffusion results.
SUBSTANCE
MOVEMENT
(IN, OUT OR NONE)
PROCESS
(OSMOSIS/DIFFUSION)
SIZE (LARGER OR
SMALLER THAN PORES)
starch
chloride ions
glucose
water
iodine
Quiz
A selectively permeable sac containing 5% NaCl, 10% urea and 10% protein is placed in a
beaker of 10% NaCl, 10% urea and 40% protein. Assume that the sac is permeable to all
substances except the protein. State whether each of the following will a) move out of the
sac, b) move into the sac, or c) not move. You may want to diagram the described set-up
and indicate where the substances are at the beginning. Use arrows to indicate the
direction each substance will move.
urea __________
NaCl __________
protein __________
water __________
What would happen in the above example if the sac is only permeable to water?
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