Introduction to BIO131 Lab

The labs in this course offer you an opportunity to explore the botanical world on your own, working with other students and with the professor. Much of this is done on your own schedule. Some of you are not used to this level of personal freedom in a course, and may not know when you have 'completed' a lab. You are ready to leave a lab when you have done the following:

  1. Seen all of the structures listed in the lab manual, especially all those that appear in bold face.
  2. Answered all of the questions in the lab manual.
  3. Identified all of the terms in the lab manual, and learned their meaning.
  4. Written down some possible test questions for the material covered. Work with your lab partners to prepare a study guide for the lab.
  5. Most importantly, you have made all the notes, diagrams, photos, or references to pages and figures in your textbook to allow you to study this material after you have left the lab. There will be several weeks between some labs and the test on this material. You need to be able to review the material without returning to the lab. You are welcome to visit the lab outside of your lab period, of course, but you will also need to be able to study on your own.

Today's lab has two parts. The experiment in the second half of the lab, exercise 1C, will take the longest, so get someone in your team to set that up while the others are working on the cyanobacteria mounts.

Cyanobacteria and Plant Cells

Today's lab will introduce you to the cyanobacteria. Like all bacteria, cyanobacteria are prokaryotes. That means they do not have membrane-bound organelles within their cells. Their physical structure is limited to the cell wall, which is made of peptidoglycan, the plasma membrane, which has the same structure as in plants and animals, and inclusions, a form of food storage. However, there is strong evidence to suggest that the organelles found in plants and animals (the mitochondria and chloroplasts) are actually the descendants of bacteria that were engulfed by other cells. Studying cyanobacteria may give us insight into the early evolution of plants, and the photosynthetic process.

In the second part of today's lab, we will start examining true plants. We will learn about the components of individual plant cells. Some of these components, such as the chloroplasts, evolved directly from cyanobacteria. Other components, like the nucleus and vacuole, may have developed from elaborations of the plasma membrane of bacteria-like ancestors.

The materials for today's lab include fresh specimens, available on the side bench, and prepared slides. The slides are found in slide box B.

CAUTION!

The chlorine in tap water will kill cyanobacteria. Do not use fresh tap water when making temporary mounts. Take a few drops of water from the bowl or tube that the cultures are in to make your slides. (Cyanobacteria are very sensitive to water pollution, and are often used as indicators of water quality.)

Cyanobacteria, the Blue-green Algae (Kingdom Eubacteria)

Prokaryotic photosynthesizers that liberate O2 during photosynthesis are called cyanobacteria or Blue-green Algae. Algae is a term used to describe several distantly related groups, including the cyanobacteria, and the photosynthetic protists, which we'll be studying next week. Although the 'Algae' include groups from several different evolutionary branches, the term is useful ecologically. The cyanobacteria and photosynthetic protists together make up the phytoplankton in fresh and salt water environments. The sugars produced by phytoplankton are the foundation of nearly all aquatic communities.

Cyanobacteria algae grow as single cells, simple colonies, or cells arranged end to end, called filaments. Blue-green algae do not form gametes; asexual reproduction is by fragmentation.

Blue-green algae secrete mucilage sheaths that enclose their cells and filaments. The mucilage sheaths sometimes become impregnated with lime, which in time can help form reefs or stromatolites. Since these algae are tolerant of environmental extremes, they often are common in hot springs and polluted streams. Some of them are ecologically important nitrogen fixers.

Representative Cyanobacteria

Gloeocapsa
LIVING
Make a temporary water mount of living Gloeocapsa. Is this organism filamentous or colonial? Can you distinguish the mucilage sheath of each cell? What happens to the mucilage sheath when a cell divides? Note the small size of these cells, and their faint blue-green color.
PRESERVED
Examine slide #1 in slide box B. You may be able to see the mucilage sheaths better on the preserved slide than in the living material.
Oscillatoria

Oscillatoria is a filamentous bacteria that moves by smooth, gliding 'oscillations'. It is common in water troughs, and often forms a mat on damp soil in greenhouses.

LIVING
Make a temporary water mount of a few filaments of living Oscillatoria. You probably will need to use dissecting needles to tease the mass of filaments apart and separate just a few filaments out by themselves. This organism is named for the gliding movement that its living filaments exhibit. Note the extremely small size of the cells, and their color. Even though these organisms are photosynthetic, there are no chloroplasts. Note the filaments and separation disks (formed by a cell dying and gas being trapped by the mucilage sheath). Can you detect the gliding movement that gives this alga its name?
PRESERVED
Examine slide #2 in slide box B. Are the cells of this Oscillatoria species the same size and shape as the living material? Is it easier to find separation disks in the preserved filaments?
Anabaena
LIVING
Make a water squash mount of a living mosquito fern (Azolla), according to directions from your instructor. Anabaena azollae is a blue-green alga that lives symbiotically in special pocket-like structures under this small aquatic fern's leaves. Note the size and color of living Anabaena cells and compare them to the size of the fern cells. Note the heterocysts in the Anabaena filaments.
PRESERVED
(slide #3). Find and identify vegetative cells, heterocysts and akinetes. What is the function of each of these types of cells?

Plant cells

The cyanobacteria are the only prokaryotes we will study in this course. The rest of the taxa we will see are all eukaryotes. Eukaryotes have their chromosomes protected within nuclei, as well as membrane-bound organelles. However, as we will discuss in class, the eukaryotes retain a direct link to the ancestor the share with modern bacteria: their mitochondria and chloroplasts evolved from bacteria cells that were 'swallowed' by another cell. Instead of being digested, they entered into a partnership with the host cell. This means that the cells of our own body, as well as those of all plants and animals, are still partly bacteria.

In the second part of today's lab, we will be examining the basic organization and operation of these 'hybrid' cells. We have three goals: The first is to gain some experience preparing slides and using the compound microscope to examine them. We'll be using the microscope a lot this semester, so this is important. The second goal is to get a deeper understanding of how materials move in and out of plant cells, and what this means and the microscopic and macroscopic (i.e., whole plant) level. We'll do two short experiments to see what happens to plant cells exposed to concentrated solutions, and what the consequences of these cellular level processes on the entire plant. Third, we'll survey some of the main components of plant cells that distinguish them from other groups of organisms.

Exercise 1C will actually take the longest, so set that up first and then go back to 1A. After you set it up, exercise 1B also needs to be left to sit for 10 or 20 minutes, so once you've begun that, you will be able to proceed to exercise 2. But don't forget to check on your slides before too long!

For this part of the lab, you don't need to be able to recognize the taxa that we are studying. The objective here is to understand the way the individual cells function. We aren't yet concerned with which taxa are involved, as what we see in these exercises works basically the same way in all plant cells.

1. Cell shape, organization and structure

A. Cell Structure

We will start by examining living plant cells using the common aquatic plant Elodea.

  1. Use forceps to remove a single leaf from the tip of an Elodea shoot.
  2. Prepare a whole mount: place the leaf in a drop of water on a slide and cover with a cover slip, as described by the lab instructor.
  3. Examine the slide using the 10X lens. You should be able to locate the many green chloroplasts within the cells, the cell wall that surrounds each cell, and the intercellular spaces between some of the cells.
  4. Is there any movement within the cells? There are two processes that make the cell contents move. Brownian motion is caused by the random movements of molecules in the cytoplasm, and causes smaller structures such as the mitochondria to jiggle around. The other kind of movement is more orderly, with chloroplasts and mitochondria winding their way around the cell. This is called cytoplasmic streaming, and should be visible in cells that aren't too cold (or dead!). What part of the cell do the organelles travel through during cytoplasmic streaming? Are there areas that they don't go?
  5. Carefully focus up and down through the cell (this is called through-focusing). What is the three-dimensional shape of the cell, and where are the organelles and cytoplasm arranged? Is there an area of the cell that appear to be empty? What is this?

The central portion of most plant cells is occupied by one or more vacuoles. These are organelles with a single membrane. They aren't usually visible, because they contain mostly water and dissolved salts and sugars: colorless liquid.

B. Water movement in cells

Next, prepare three whole mounts of Elodea leaves.

  1. Place one leaf in distilled water, one in tap water (the same water that the shoots are floating in), and one in the 20% sucrose solution.
  2. Examine the leaf in sucrose solution - what's happening? Compare this leaf to the other two mounts.
  3. After about fifteen minutes, sketch a representative cell from each mount.
  4. Remove the leaf from the sucrose slide and rinse it in distilled water. Make a new mount with this leaf, using a new, clean cover slip and distilled water. Watch this mount for changes over the next 15 minutes.

You should be able to explain what you just saw in terms of osmosis, turgor pressure, water potential, and osmotic potential. What is moving into or out of the cells during this experiment? What substances can cross the plasma membrane?

C. Water movement in plants

How do these microscopic processes effect whole plants?

  1. Get a celery stalk that is about 10cm long, and cut it length-wise into three equal sized pieces.
  2. Place one piece in each of three 50 ml beakers.
  3. Fill the beakers with distilled water, 0.9% sodium chloride, or 10% sodium chloride. Be sure to label the beakers so you'll know what is in each one!
  4. Observe what happens over the next hour - using what you learned in parts A and B, what can you conclude about the structural support of the celery plants? What part of the cell provides the strength to keep the stems upright?
  5. What would happen if you put three pieces of wood in the three different beakers? (If you don't know, try it!). What does this tell you about the part of the cell that gives wood its strength?

2. Cellular components

A. Chromoplasts

In the first part of the lab, we saw chloroplasts. We now move on to the other plastids, starting with the chromoplasts. These organelles don't have any chlorophyll, only carotenoid pigments. Consequently, they are not green, but other colors (we'll see red, orange and yellow today). They are more delicate than the chloroplasts, so you may find that they have split open and spilled their contents into the cells on your slide, so you may have to look around to some that are still intact. Note that carotenoid pigments are not water soluble, so they are found in clumps inside the chromoplasts or in pigment bodies in the cytoplasm.

  1. Prepare a wet mount from one of the flower petals. Tear a piece off the petal with a twisting motion, and then examine the torn edge under the microscope. Where are the pigments within the cell? What shape and color are they?
  2. Now prepare wet mounts of the tomatoes. Squeeze a drop or two of tomato pulp onto a slide and cover it with a cover slip. The pigments are found in crystal-like bodies called pigment bodies.
  3. Using a razor, make very thin longitudinal sections of the carrot root. Examine these under the microscope to identify the chromoplasts and/or pigment bodies, and note their shapes.
B. Leucoplasts

Leucoplasts are non-pigmented plastids found in epidermal cells and in cells not exposed to light. They are more delicate than chloroplasts and chromoplasts, so if you get a clear view of them let the instructor know so we can make sure everyone gets to see them!

  1. Pick a turgid leaf of Zebrina.
  2. Use a razor to remove a small segment from the margin of the leaf and prepare a wet mount.
  3. Examine the outer margin, and find a single, projecting cell. Switch to high power and find the nucleus. The leucoplasts may be visible clustered around the nucleus.

Note the color of these cells. Where is the color found - is it localized in discrete structures, like the carotenoid pigments in the chromoplasts? We'll take another look at this pigment again in exercise 2D.

C. Amyloplasts

Plants use starch as their primary long-term energy storage. We will use potassium iodide stain to identify the starch in the Solanum tuberosum tuber.

  1. Use a razor blade to scrape a small amount of potato into a water drop on a slide, and cover with a cover slip.
  2. Examine the slide under the microscope.
  3. If you place a polarizing filter on the light source, you should be able to see the characteristic crystal like structure of the starch grains.
  4. Place a piece of folded paper along one side of the coverslip while applying a small amount of potassium iodide to the other side. The stain should be drawn under the cover slip and across the potato sample. What does the stain do to the appearance of the starch? What about the rest of the cell?
D. Anthocyanin

Anthocyanin is an important plant pigment, responsible for the reds, blues and purples we see in beets, blueberries, cranberries, red cabbage, and the fall color of red maples.

  1. Take another look at your Zebrina slide, noting the distribution of the color in the cells.
  2. Prepare a section of the red cabbage leaves or beet petioles and compare this to the Zebrina.
  3. Anthocyanin is water soluble, so it can be stored in the vacuole; boiling the cabbage leaves will cause the cells to rupture, releasing the anthocyanins into. Fill two 50ml beakers with the 'cabbage juice' from the demo at the front of the lab.
  4. Observe the color of each beaker. Add a few drops of vinegar to one beaker. What effect does this have?
  5. Now add a few drops of ammonia to the other beaker and compare it to the first.

Ammonia is a base, and vinegar is an acid. What does this experiment tell us about the difference between the taste and appearance of blueberries and cranberries?

E. Crystals

Plants often store excess inorganic material in the vacuole, where they form crystals. Prepare thin sections of the Zebrina stems and Begonia petioles, and try to find the crystals. Which one has raphides (long bundles of needle-like crystals), and which one has druses (compound crystals)?

These crystals are made of calcium oxalate. This compound is very irritating to mucus membranes. If you were to eat plant tissues containing calcium oxalate crystals, it would cause your mouth and throat to itch and swell - what advantage does this provide to the plant that produces the crystals?

Summary

By the end of this lab, you should be able to:

  1. Recognize Gloecapsa, Anabaena, and Oscillatoria as cyanobacteria
  2. Explain the function of the key structures of each organism
  3. Explain the ecological and agricultural significance of Anabaena
  4. Recognize the basic structures of a plant cell: cell wall, plasma membrane, nucleus, mitochondria, chloroplasts, chromoplasts, pigment bodies, leucoplasts, amyloplasts
  5. Understand the differences between carotenoids and anthocyanins
  6. Explain how the single pigment anthocyanin is capable of producing a variety of colors
  7. Know two ways we can identify starch in a plant cell
  8. Recognize two kinds of calcium oxalate crystals and explain their value to the plant

In addition, you should be comfortable with the preparation of slides from fresh tissue and the use of the microscope, using both the regular and oil immersion lenses.