Download LAB 09 – Cellular Responses to Stimuli

LAB 09 – Cellular Responses to Stimuli
(Salinity, pH, and Temperature)
March 11-15, 2002
Purpose
Learning Goals
Students will make connections between the cellular
responses witnessed in lab and potential responses of
plants, humans, and other animals to broader stimuli
in their biological communities.
Teaching Goal
Students will explore the structural variability of
cells and relate the structure to the functions
performed by those cells.
Through conscious
thinking about the relationship between form and
function, the consequences of change can be better
understood in the context of environmental impact.
As a foundation for future units (reproduction,
nutrition and diversity), students will become more
aware of the ‘body as a chemistry factory’ from the
cellular to the system level.
Overview (Strategy)
Using single-celled organisms, colonial algae,
and plant tissue samples, students will consider the
role and structure of cells as independent
organisms and as components of something larger,
such as a colony, tissue, or multicellular organism.
Along with descriptions of cellular function and
predictions about form, students will look at
prepared slides of specialized cells (nerve, striated
muscle, bone….) and at the structure of their own
cheek cells.
Students will experiment with responses to
changes in salinity by looking at plant and animal
cells exposed to iso-, hyper-, and hypotonic
solutions.
Students will also use assorted freshwater
organisms (Amoeba, Paramecium, Euglena,
Volvox, Vorticella, Daphnia) to observe organismal
responses to changes in temperature and salinity
Time
170 minutes
Level
Introductory non-majors Biology BIOL102/105
Concurrent Lectures
Humans and other animals: Cells
Comparative reproduction and meiosis
Comparative development
Lab 9 Cellular Responses to Stimuli
B.J. Marshall, P. Verrell, D. Cartwright
Key Concepts and Skills
Concepts
Organisms can be unicellular or multicellular.
Specific structures are the result of cellular
specialization and differentiation
Structural adaptations at all organizational levels
affect an organism’s survival.
Form and function are interrelated and responsive
to change.
Organismal response to environmental stimuli
occurs first at the cellular level. This response or
change at the cellular level will contribute to an
organism’s ability to withstand environmental
stress.
Environmental response is a systemic consequence
of reactions at smaller scales.
Cellular and organismal response is largely
predictable and can be considered in the
development of models for environmental and
health policy issues.
Skills
Observation
Slide preparation
Identification of aquatic organisms and cell types
Data collection and analysis
Scientific discussion / intellectual flexibility
Materials and Tools
Slides and cover slips
Hand Lenses
Dissecting scopes
Compound scopes
Identification guides for algae and pond organisms
Prepared slides of cells to include digestive, nerve,
plant cells, secretory, bone, blood, and other
slides as available and significant
3% NaCl
Distilled water
0.9% NaCl
Cell models (Plant and Animal)
TV and microscope video camera hookup
Pond water, mist bench, and soil infusion samples
Pipettes
Plant and animal specimens as available (Eichornia,
Elodea, red onion, Valisneria, Coleus,
Tradescantia, Carrots, Daphnia)
Flat end toothpicks
Week 9 Page 1
3/6/2002
Background
The cell is the basic unit of life. Groups of cells can
form tissues, then organs and organ systems, or a cell
may be a whole organism such as the single-celled
algae and protists found in ponds.
Because
organisms may be unicellular or multicellular,
differentiation and specialization of cell structure
varies relative to function and may be deleteriously
affected by environmental factors. Critical among the
cellular balancing acts is that of fluid balance; cells
must balance the concentration of salts inside and
outside the cell. You will see the impact of varying
levels of salinity on cells during this lab.
All living things are composed of cells which
are classified as either prokaryotic or eukaryotic.
same components, the plant cell has three significant
differences – the cell wall, central vacuole, and
chloroplasts. The cell wall is located outside the
cell membrane (also called the plasma membrane)
and gives rigidity to the cell. Chloroplasts are the
organelles (small organs) that house chlorophyll and
other pigments used in photosynthesis to convert
light energy to chemical energy.
Figure 3. Eukaryotic cell, Plant
Figure 1. Prokaryotic Cell
Bacteria and archaea are prokaryotic while the
eukaryotic cells are those in all other organisms
including protists, plants, fungi, and animals. In all
eukaryotic cells, most of the genetic material, the
DNA, is located in the membrane bound nucleus.
The membrane allows for separation between the
nucleus with its critical genetic material and the
cytoplasm of the cell. In a prokaryotic cell, the
genetic information is in a region defined as nucleoid
but it is not partitioned by a membrane from the rest
of the cell.
While animal (Figure 2) and plant cells
(Figure 3) are both eukaryotic and share most of the
Figure 2. Eukaryotic Cell, Animal
Lab 9 Cellular Responses to Stimuli
B.J. Marshall, P. Verrell, D. Cartwright
Additional organelles, such as mitochondrion,
ribosomes, and endoplasmic reticulum have specific
roles in cell functioning become more abundant or
less abundant as cells become specialized. For
example, sperm cells require a great deal of energy
to make the long trip up the fallopian tube to
fertilize an egg. As such, sperm cells have a higher
proportion of mitochondria (energy producing
organelle) than would be found in a liver cell.
Ovaries and testes with responsibility for lipid-based
sex hormones have a greater relative proportion of
smooth endoplasmic reticulum. Rough endoplasmic
reticulum is responsible for the production of
secretions such as saliva and mucus. A closer look
at secretory cells would reveal a much higher
relative proportion of this organelle than is typically
shown in generalized cell diagram.
Essential to cellular viability is the proper
functioning of the plasma membrane. Molecules are
allowed to pass into or out of the cell based on the
permeability of this membrane.
The plasma
membrane is considered selectively permeability
since some molecules can pass through easily while
others cannot or only can with an active transport
mechanism. Passive transport is simply diffusion
across the membrane.
Diffusion results as
molecules with a high concentration spread out into
areas of lower concentration. Visualize a package
of Kool-Aid being added to water. As the contents
of the packet reach the water, they very quickly
spread throughout the container. The temperature of
the water, motion, and salinity will all affect the
speed of diffusion. In a cell, no energy needs to be
put forth by the cell itself for diffusion to occur.
Week 9 Page 2
3/6/2002
The plasma membrane serves as a filter to insure
that the right molecules remain inside the cytoplasm
and to prevent large molecules from entering the
cell. This kind of transport is what we experience
during gas exchange as oxygen enters and carbon
dioxide leaves red blood cells. Filtered diffusion or
passive transport of water across a selectively
permeable membrane is referred to as osmosis
(Figure 4).
across the membrane as it seeks equilibrium with
the lower water concentration found outside the cell.
As the water leaves the cell, like air leaving a
balloon, the cell shrivels. When the solute
concentrations are the same on both sides of the
membrane (or inside and outside of the cell) they are
isotonic. (Figure 5).
Figure 5. Impact of salinity concentration on cells
Figure 4. Osmosis and Selective Permeability
Remembering that molecules come in different
sizes, think of a fuel filter that allows fuel and
oxygen molecules to pass but traps contaminants
and keeps them from entering the fuel chamber. If
we use this same idea to discuss water and a
molecule like salt, the side of the filter with the
higher concentration of salt (the solute) is
considered hypertonic. The side of the filter with
the lower concentration of salt (or other solute) is
called hypotonic.
The side with the lower
concentration of solute would have a higher
HYPER = above or more than
HYPO = Below or less than
ISO = Equal or the same
concentration of water. Conversely, the side with
the higher concentration of solute would have a
lower concentration of water. Since the membrane
filters the larger salt molecule but not the water,
water will seek a balanced concentration by
diffusing into the cell across the plasma membrane.
Water moves from regions of lower to higher salt
concentrationn. As the water molecules enter the
cell, the interior volume swells with the addition of
water molecules and the cell bursts (Lyses Figure
5b). If, however, the solute concentration is greater
outside the cell, water concentration would be
higher inside causing water to leave the cell. While
the membrane keeps solutes out of the cell, it does
not prevent water inside the cell from diffusing
Lab 9 Cellular Responses to Stimuli
B.J. Marshall, P. Verrell, D. Cartwright
Considering a larger perspective, an aquatic
animal survives because its concentration of solutes
is compatible with survival in the surrounding
water. Without adequate protective and regulatory
mechanisms an organism could become overly
hypotonic to its environment if the surroundings
suddenly became more saline. Higher external
salinity sets up a concentration gradient promoting
water loss. As the cells of the organism begin to lose
water, they shrink. To exercise some control over
passive transport, aquatic organisms have special
adaptive strategies used to regulate water balance
(osmoregulation). Kidneys and contractile vacuoles
are among the strategies used to maintain this
critical balance between water and salt.
For plants the mechanism of osmoregulation
is the same - osmosis or passive transport across a
semi-permeable membrane to maintain balance of
solutes. The ‘ideal’ condition for most plants is to be
hypotonic (lower solute, higher water) to the
environment. Because plants have a cell wall, they
maintain structure better when the plasma
membrane below the cell wall is somewhat
stretched. The fluid content in the cell helps
maintain the turgid (stiff) structure needed by the
plant (Figure 5b). If a plant cell is isotonic to its
environment, solutes are balanced inside and out
causing wilting due to lack of internal support from
water pressure. If the solution or environment in
which the plant lives becomes hypertonic to the
plant (higher solute outside the plant), water loss
will result and the plant cell will shrivel within the
plasma membrane and pull away from the cell wall.
This is called Plasmolysis (Figure 5) and usually
results in cell death.
Week 9 Page 3
3/6/2002
Terrestrial
organisms
also
have
osmoregulatory strategies. A person may sweat
profusely in the hot months of summer. As the body
loses moisture, the person will begin craving water
to maintain fluid balance.
Without sufficient
moisture levels, the body becomes overheated as it
loses its ability to thermoregulate by evaporative
cooling. As we lose moisture through sweating and
by dehydration from the lungs, our cellular
concentration of solute (in this case salt) increases.
This increased salt concentration requires
rehydration (drinking fluids). If the person fails to
get sufficient water, dehydration may result. Heat
cramps, heat exhaustion, and heat stroke are all
potential outcomes of dehydration and require
immediate medical attention to restore fluid balance
and thermoregulatory mechanisms.
Salinity lowers the freezing temperature and
raises the boiling temperature of water. (Think
about salt on an icy sidewalk). It stands to reason
that these interactions of salt and fluid balance also
have impact throughout the temperature range in
which an organism can survive.
Other factors such as pH (a measure of balance
between hydrogen and hydroxide ions) also affect a
cell’s ability to regulate fluid balance in its
environment. With an increase in the potential
acceptance of hydrogen molecules (higher pH) the
solution becomes increasingly basic and pH
measurements will be higher. A decrease in
potential hydrogen (lower pH) indicates increased
acidity in the solution. Figure 6 shows some
common acids and bases. Water is neutral at a pH
Figure 6. pH Scale
Lab 9 Cellular Responses to Stimuli
B.J. Marshall, P. Verrell, D. Cartwright
of 7. This means that an equal quantity of hydrogen
and hydroxide ions are present. Blood has a slightly
higher pH (more hydroxide than hydrogen
molecules) while urine is slightly lower (more
hydrogen than hydroxide) or more acidic. The
gastric juices or stomach acids, are very acidic and
facilitate the breakdown of food for energy. Notice
that milk of magnesium (Tums, Rolaids, Zantac,
etc) are very basic (alkaline) compared to citrus
juices. The basic nature of antacids helps to
neutralize heartburn or an upset or ‘acid’ stomach.
An important consideration for understanding
the impact of pH as a cellular and systemic
influence is the mixing of water in the atmosphere to
form acids. Rain tends to be slightly acidic (low pH)
due to the mixing of carbon dioxide with water to
form a weak acid (carbonic acid). Precipitate (rain
and snow) with pH lower than 5.6 are considered
‘acid rain’. As natural and human produced
emissions mix with water in the atmosphere, the
naturally acidic rain can become even more acidic.
pH ranges about as acidic as citrus fruits have been
recorded in the Eastern U.S. Water with low pH is
able to leach heavy metals out of the soil and carry
them down stream. As acidic water makes its way
to rivers and lakes, it carries with it additional toxins
released from the soil by the acids in the rain.
While some plants do very well in acidic
surroundings such as those found in bogs, most do
not. The inability of plants to tolerate highly acidic
conditions has been associated with serious
environmental challenges from the Black Forests of
Germany to the east coast of the United States. It
has been very complicated to understand the role of
acid rain of plants. We do know that acid rain can
remove nutrients from the soil around plants and
from the leaves themselves.
In addition to the impact on plants, there is
concern that as the acidic rain falls over land and
travels downstream, the heavy metals leached from
the soil may be carried to our water supply making
the water supply not only too acidic to drink but also
contaminated with copper, lead and other metals.
We have briefly explored general cell
classification and the specialized activities of select
organelles in single-celled, multi-cellular and
colonial organisms. We have further explored the
role of diffusion and selective permeability in
osmoregulation and potential environmental impact
from changes in salinity, pH, and temperature. As
we move into the demonstrations and experiments
for this week’s lab, consider the results of your
activities from multiple scales ranging from the
cellular level to ecosystems.
Week 9 Page 4
3/6/2002
What to Do and How to Do It (Students)
Station 1
Look at and draw the cells of the prepared slides and relate cell form to function. (E.g.,
nerve cells have long projections to carry messages).
Cell Type_______________
Cell Form / Function
Relationship
Cell Type_______________
Cell Form / Function
Relationship
Cell Type___________________
Cell Form / Function
Relationship
Station 2 (Adapted from Gunstream, Stanley. 2002. Explorations in Basic Biology. Prentice Hall.)
Wet mount your own epithelial cells
1. Gently scrape the inside of your mouth with the flat side of a toothpick to obtain
epithelial cells
2. Swirl the toothpick (with cells) in a drop of 0.9% NaCl on a clean slide
3. Gently apply a cover slip
4. Observe your cheek cells under 10x and 40x.
5. Identify and draw the cytoplasm, nucleus, and plasma membrane in the cheek cell.
6. Add a drop of methylene blue to the wet mount. As you view the slide again, the
nucleus will be more clearly visible.
Lab 9 Cellular Responses to Stimuli
B.J. Marshall, P. Verrell, D. Cartwright
Week 9 Page 5
3/6/2002
Station 3
1. Apply three separate drops of dilute sheep’s blood to a slide.
2. Add cover slips and view one of the drops first at 10X magnification then at 40X.
**Note that low light enables the best viewing.
3. Add a drop of distilled water to the left drop of blood.
4. Use a torn piece of paper towel positioned against the opposite side of the cover slip
to draw the solution across the blood drop. (TA to demonstrate)
5. Note any changes to the shape of the blood cells.
6. Repeat steps 3-5 substituting 3% and 0.9% solutions to the other drops of blood.
7. Clean the blood slides
Hypotonic to Animal Cell
Solution _________%
Isotonic to Animal Cell
Solution _________%
Hypertonic to Animal Cell
Solution __________%
Station 4
1. Select three small leaves of the aquatic plant Elodea sp. or Anacharis sp. (new
growth works best) blot excess water and position them on three separate slides.
2. Cover each of the three pieces with cover slips.
3. While viewing under 10x magnification, add distilled water by drawing it across the
slide with a torn paper towel and note any changes.
4. One at a time while viewing the slides under low power magnification, add 0.9%
saline solution and 3% saline solution to the remaining leaf pieces.
5. Note any changes
Hypotonic to Plant Cell
Solution
%
Lab 9 Cellular Responses to Stimuli
B.J. Marshall, P. Verrell, D. Cartwright
Isotonic to Plant Cell
Solution
%
Week 9 Page 6
Hypertonic to Plant Cell
Solution
%
3/6/2002
Station 5
1. Put several drops of cold pond water into the reservoir of a plastic dish.
2. Record the temperature and measure the pH of your sample with the pH strips
provided
3. View your microhabitat under low power of a dissecting microscope.
4. Increase magnification as needed to identify organisms (A brief identification guide
has been provided).
5. Repeat the procedure and compare the activity of organisms in warmed pond water
to that of the cooled water
6. Draw and label three of the organisms from your samples and describe any activity
associated with them.
Sample #______________________Temperature _____________ _pH________________
Station 6
1. Place a drop of warm pond water on a clean slide and gently add a cover slip.
2. Spend several minutes viewing the slide under a range of magnifications beginning
with low power. You will find a variety of single-celled organisms (See the
identification key provided).
3. Add a drop of 3% saline to the slide by drawing it across the slide with a piece of
torn paper-toweling
4. Observe the slide again throughout your range of magnifications (beginning with
low power) and record your observations in written form.
Lab 9 Cellular Responses to Stimuli
B.J. Marshall, P. Verrell, D. Cartwright
Week 9 Page 7
3/6/2002
Station 7
See Campbell page 108 and CD activity 6D for more information about pigments, their
association with chloroplasts, and their role in photosynthesis.
1. Clip a small piece of one of the available plant samples and place it on a slide with a
drop of the water from the plant’s container.
2. Gently add a cover slip
3. View the sample under low magnification then under higher magnification after you
have located plastids containing pigments (chlorophyll and carotenoids). Other
pigments (anthocyanins) can be found in the cytoplasm.
4. As you repeat the procedure for each of the samples at this station, you may also see
cytoplasmic streaming.
5. List the pigments associated with each of the plant samples in the table below:
Sample A
Sample B
Sample C
Sample D
Sample E
Sample F
Onion
Anthocyanins
Carotenoids
Chlorophyll
Further Investigations
Indicator species
Environmental sentinels
Vocabulary
Amoeba
Anthocyanins
Carotenoids
Cell Theory
Chilomomas
Chlamydomonas
Rapid Bioassessment Protocols (RBPs)
Chlorophyll
Choanoflagellate
Diffusion
Dinoflagellate
Euglena
Giardia
Hypertonic
Hypotonic
Isotonic
Organelle
Osmosis
Paramecium
Passive Transport
Plasmodial Slime mold
Plasmolysis
Vorticella
Suggested Reading
Campbell Pages 60-69, 74-77, 310-322
Web/CD Activities
Apopstosis – Dance of Death
http://www.cellsalive.com/
Algae Image Archive –
http://www.bgsu.edu/departments/biology/algae/html/Image_Archive.html
Essential Biology Place CD
2I - Acids and Bases
4K – Diffusion 4L - Osmosis
14F – Protists
References and Resources
Campbell, Neil A. and Jane B. Reese. 2001. Essential Biology. Benjamin Cummings, New York.
Gibbs, W. Wayt. 2001. Cybernetic Cells. Scientific American. 265(2):53-57.
Stahle, David W., et.al. 2001. Ancient Blue Oaks Reveal Human Impact on San Francisco Bay Salinity. Earth
in Space 13(8): 8-12.
West, Bernadette, Peter M. Sandman and Michael R. Greenberg. 1995. The Reporter’s Environmental
Handbook. Rutgers University Press, New Brunswick, NJ.
Lab 9 Cellular Responses to Stimuli
B.J. Marshall, P. Verrell, D. Cartwright
Week 9 Page 8
3/6/2002
Lab 09 Discussion Questions and Topics
Name______________________
Section_____________________
What advantages do multicellular organisms have over their unicellular neighbors?
What is the value of specialization in multicellular organisms?
Why is it possible to use a simple cheek cell in DNA testing for paternity?
What reaction do you expect when blood is hypotonic to its environment? Hypertonic?
Isotonic?
What reaction do you expect when a plant cell is hypotonic to its environment?
Hypertonic? Isotonic?
What would happen to spinach leaves if you were to quickly submerge them in cold salty
water? Why do you think so?
Discuss the potential impact of drought on a pond ecosystem.
What are advantages of each of the following two approaches?
A. Reduce acid rain by increasing auto emissions standards, installing smokestack
scrubbers, and investing in new energy technologies.
B. Neutralize ponds, lakes and water supplies to mediate the effects of acid rain.
Lab 9 Cellular Responses to Stimuli
B.J. Marshall, P. Verrell, D. Cartwright
Week 9 Page 9
©3/6/2002