LAB 09 – Cellular Responses to Stimuli - Washington State... - wsu.eduearthsch/data/lab_9.pdf ·...

Lab 9 Cellular Responses to Stimuli B.J. Marshall, P. Verrell, D. Cartwright Week 9 3/6/2002

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

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


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.

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

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

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.

Figure 1. Prokaryotic Cell

Figure 2. Eukaryotic Cell, Animal


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).

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

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

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

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.

HYPER = above or more than HYPO = Below or less than ISO = Equal or the same


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

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.

Figure 6. pH Scale


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). 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.

Cell Type_______________ Cell Type_______________ Cell Type___________________

Cell Form / Function Relationship




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

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 Animal Cell Solution _________%

Isotonic to Animal Cell Solution _________%

Hypertonic to Animal Cell Solution __________%

Hypotonic to Plant Cell Solution %

Isotonic to Plant Cell Solution %

Hypertonic to Plant Cell Solution %


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.


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

Rapid Bioassessment Protocols (RBPs)

Vocabulary Amoeba Anthocyanins Carotenoids Cell Theory Chilomomas Chlamydomonas

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 ©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.

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